JP5289491B2 - Nicotiana nucleic acid molecule and use thereof - Google Patents

Description

  The present invention encodes Nicotiana nucleic acid sequences in Nicotiana plants, such as sequences encoding constitutive or ethylene or senescence inducible polypeptides, particularly cytochrome p450 enzymes (hereinafter referred to as p450 and p450 enzymes). The invention relates to the use of these nucleic acid sequences for modifying the sequences as well as the phenotype of plants.

  During tobacco ripening or drying (curing), the expression of various genes is altered. Such genes can affect metabolic pathways involved in the formation of a number of secondary metabolites including terpenoids, polyphenols and alkaloids that affect the end product's characteristic traits. For example, in many Nicotiana species, biotransformation of nicotine that produces nornicotine occurs during plant senescence and post-harvest or leaf drying stages. Nicotine is the main source of nornicotine. Nornicotine alkaloids are substrates for microorganism-mediated nitrosation that produce tobacco-specific nitrosamine (TSNA) N′-nitronornicotine (NNN) during leaf drying or subsequent leaf storage and processing.

  A gene expressed during ripening or drying of tobacco can be an ethylene-inducible or aging-related gene, such as a gene encoding cytochrome p450. Cytochrome p450 catalyzes the enzymatic reactions of a wide variety of chemically diverse substances including, for example, oxidative, peroxidative and reductive metabolism of endogenous and xenobiotic substances. In plants, p450 is involved in biochemical pathways involving the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, hydrocyanic acid glycosides and glucosinolates being studied (Chappell, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 521-547, 1995 (Non-Patent Document 1)). Cytochrome p450 is also known as p450 heme-thiolate protein and normally acts as a terminal oxidase in a multicomponent electron transport chain called p450-containing monooxygenase system. Specific reactions catalyzed by these enzyme systems include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S- and O-dealkylation, desulfation, deamination and Includes reduction of azo, nitro and N-oxide groups.

  The diverse roles of p450 enzymes in Nicotiana plants have been linked to the production of various plant metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, hydrocyanic acid glycosides and many other compounds. Some p450 enzymes can affect the composition of plant metabolites. For example, it has long been desired to improve the flavor and aroma of certain plants by modifying the plant profile of selected fatty acids by breeding, but with regard to the mechanisms involved in controlling the levels of these leaf components Little is known. Down-regulation or up-regulation of p450 enzymes associated with fatty acid modification may promote the accumulation of desired fatty acids that confer more favorable leaf phenotypic characteristics.

  The functions of p450 enzymes and their broad role in plant components are still being discovered. For example, a special class of p450 enzymes catalyzes the degradation of fatty acids to volatile C6- and C9-aldehydes and β-alcohols, which are the main contributors to the “fresh green” of fruits and vegetables. It was found to be. To improve the quality of leaf components by modifying the lipid composition and related degradation metabolites in Nicotiana leaves, the level of other novel targeted p450s can be altered. Some of these components in leaves are affected by aging that promotes ripening of leaf quality characteristics. Still other reports indicate that the p450 enzyme plays a functional role in the modification of fatty acids involved in plant pathogen interactions and disease resistance.

  Prior to the present invention, their studies on Nicotiana p450 enzymes were very difficult because the forms of p450 enzymes are very diverse and their structures and functions are also varied. Also, for at least one reason, cloning of the p450 enzyme is difficult because the membrane-localized protein of the p450 enzyme is typically present in very small amounts and often becomes unstable during purification. Therefore, there is a need to identify p450 enzymes in plants and the nucleic acid sequences associated with those p450 enzymes. In particular, only a very small number of cytochrome p450 proteins have been reported in Nicotiana. The invention described herein includes the discovery of cytochrome 450 and cytochrome p450 fragments that correspond to several groups of p450 species based on sequence identity.

  In addition to the p450 sequence, the present invention includes the discovery of other components and ethylene or aging-inducible sequences that correspond to the regulatory requirements of metabolic pathways involved in the formation of secondary metabolites that affect tobacco product quality. . These sequences are also useful in developing plant germplasm with desirable traits for use in breeding programs to develop more desirable germplasm and in particular non-GMO (genetically engineered organism) type germplasm.

Chappell, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 521-547, 1995

  The present invention has identified and characterized constitutive and ethylene or senescence-inducing sequences from tobacco (including genomic clones of nicotine demethylase). It also creates plants (eg, transgenic plants) that have the desired trait (eg, nornicotine or N′-nitrosonornicotine (“NNN”) or modifications at both levels relative to the control plant) in the breeding method. The use of these sequences in a method for is also described herein.

  In one embodiment, the present invention relates to a breeding method for producing tobacco plants with reduced expression of a nicotine demethylase gene. The method comprises (a) providing a first tobacco plant having mutant nicotine demethylase gene expression, (b) providing a second tobacco plant containing at least one phenotypic trait, and (c) first tobacco. A plant and a second tobacco plant are crossed to obtain an F1 progeny plant, (d) the F1 progeny seed is collected for mutant nicotine demethylase gene expression, (e) the seed is germinated, Obtaining a tobacco plant with reduced expression of the methylase gene.

  In one embodiment, using the sequences described herein and standard methods known in the art, nicotine demethylase gene expression (eg, level of transcription, post-transcription or translation or level of enzyme activity). Tobacco plants are identified as variants for

  In another embodiment of this method of breeding, the first tobacco plant comprises an endogenous nicotine demethylase gene having a mutation (eg, deletion, substitution, point mutation, translocation, inversion, duplication or insertion). . In another embodiment, the first tobacco plant includes a nicotine demethylase gene having a null mutation, includes a recombinant gene that silences an endogenous nicotine demethylase gene, or reduces or modifies enzyme activity. With accompanying nicotine demethylase. In yet another embodiment, the first tobacco plant nicotine demethylase gene is absent. In yet another embodiment, the first tobacco plant is a transgenic plant.

  Typical first tobacco plants useful in the breeding methods disclosed herein include: Nicotiana africana, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior , Nicotiana exigua, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana hesperis, Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila, Nicotiana noctiflora, Nicotiana noctiflora, Nicosiana noctiflora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens or Nicotiana trigonophylla. Desirably, the first tobacco plant is Nicotiana amplexicaulis, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana debneyi, Nicotiana excelsior, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana hesperis, Nicotiana knightiana, Nicotiana maritima, phonic Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rustica, Nicotiana suaveolens or Nicotiana trigonophylla. Other first tobacco plants include Nicotiana tabacum (or Nicotiana rustica) varieties or related transgenic lines that have been engineered to reduce levels of nicotine emethylase. Other typical first tobacco plants include oriental, dark tobacco, fire-cured or air-cured tobacco, Virginia, or Burley tobacco plants.

  In another embodiment of said breeding method, the second tobacco plant is Nicotiana tabacum. Typical varieties of Nicotiana tabacum include commercial varieties such as: BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800 , CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KT 200, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 160, Little Crittenden, McNair 373, McNair 944, msKY 14 x L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC 606, NC 71, NC 72, NC 810, NC BH 129, OXFORD 207, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H4, RG H51, RGH 4 , RGH 51, RS 1410, SP 168, SP 172, SP 179, SP 210, SP 220, SP G-28, SP G-70, SP H20, SP NF3, TN 86, TN 90, TN 97, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA 309 or VA 359.

  In yet another embodiment, the phenotypic trait of the second tobacco plant includes disease resistance; high yield; high grade index; desiccation; quality of desiccation; mechanical harvestability; holding ability; Quality; height, plant maturity (eg, early ripening, premature to medium, medium, medium to late ripening or late ripening); pattern size (eg, small, moderate or large pattern); or number of leaves per plant ( For example, low (eg, 5-10 leaves), moderate (eg, 11-15 leaves) or high (eg, 16-21 leaves). In yet another embodiment, the method further comprises self-pollination of a male sterile hybrid with a male sterile pollen acceptor, a pollen donor that can be used to produce the hybrid or the plant of step (b) or Including pollination (pollination), or backcrossing or self-pollination of the plant obtained from the germinated seed of step (e).

  In another aspect, the invention relates to a method for breeding a nicotine demethylase deficient trait into a tobacco plant. The method comprises: a) crossing a first tobacco plant having a mutant nicotine demethylase gene expression with a second tobacco plant, b) obtaining a progeny tobacco plant of the cross, and c) extracting a DNA sample from the progeny tobacco plant D) contacting the DNA sample with a marker nucleic acid molecule that hybridizes to a nicotine demethylase gene or fragment thereof, e) performing a marker assisted breeding method for the mutant nicotine demethylase gene expression trait Process. For example, plants are identified as having mutant gene expression of nicotine demethylase, optionally further tested for nicotine demethylase gene expression, or using standard alkaloid profiling or immunoblot analysis. Typically, such marker-assisted breeding methods include amplified fragment length polymorphisms, restriction fragment length polymorphisms, random amplified polymorphism presentations, single nucleotide polymorphisms, microsatellite markers, or targeted inducible localities in the tobacco genome. Includes the use of targeted induced local lesions.

  In yet another aspect, the present invention relates to Nicotiana africana, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua, Nicotiana glutinosa, Nicotiana glutinosa, Nicotiana , Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana otophora, Nicotiana palmeri, Nicotiana palmitiana, foliobag The present invention relates to a method for producing tobacco seeds comprising crossing with a tobacco plant selected from the group consisting of rustica, Nicotiana setchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens and Nicotiana trigonophylla with itself. In one embodiment, the method further comprises a method for producing hybrid tobacco seed comprising crossing a tobacco plant having a mutant nicotine demethylase gene expression with a second different tobacco plant. In yet another embodiment of the invention, the crossing comprises (a) planting seed obtained from crossing a tobacco plant having mutant nicotine demethylase gene expression with a second different tobacco plant, (b) ) Growing a tobacco plant from the seed and flowering the plant; (c) pollinating a tobacco plant having mutant nicotine demethylase gene expression with pollen from a second tobacco plant; And (d) harvesting seeds obtained from the pollination. The method comprises: pollinating the tobacco plant flower with pollen from a tobacco plant flower having a mutant nicotine demethylase gene expression.

  In yet another aspect, the present invention provides (a) preparing a tobacco plant having mutant nicotine demethylase gene expression or a component thereof, and (b) using the tobacco plant breeding technique as a source of breeding material for the plant or The present invention relates to a method for developing tobacco plants in a tobacco breeding program comprising using plant components. Typical plant breeding techniques useful for carrying out this method include bulk selection, backcrossing, self-pollination, introgression crossing, line selection, pure line selection, haploid / doubled haploid breeding or single grain line methods Is included.

  In another embodiment, the invention relates to (a) FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294 Providing a first tobacco plant having a modified attribute comprising a mutant gene expression of a nucleic acid molecule selected from the group consisting of the nucleic acid sequences shown, and (b) providing a second tobacco plant containing at least one phenotypic trait And (c) crossing the first tobacco plant with the second tobacco plant to obtain an F1 progeny plant, (d) collecting the seeds of the F1 progeny having the modified attributes, and (e) germinating the seeds. And a breeding method for producing a tobacco plant having a modified attribute, comprising the step of obtaining a tobacco plant having a modified attribute. In one embodiment, the first tobacco plant comprises the mutations of FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173. -Endogenous nucleic acid molecules selected from the group consisting of the nucleic acid sequence shown in -294. Typical mutations include deletions, substitutions, point mutations, translocations, inversions, duplications or insertions.

  In yet another embodiment, the first tobacco plant of the method comprises a null mutation, wherein FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and An endogenous nucleic acid molecule selected from the group consisting of the nucleic acid sequences shown in FIGS. 173-1 to 173-294 is included. In yet another embodiment, the first tobacco plant is Figure 1, Figures 3-7, Figures 10-158, Figures 162-170, Figures 172-1 to 172-19 and Figures 173-1 to 173-294. A recombinant gene that silences the expression of an endogenous nucleic acid molecule selected from the group consisting of the nucleic acid sequences shown below.

  Optionally, the first tobacco plant encodes a polypeptide having reduced or altered enzymatic activity, as shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and An endogenous nucleic acid molecule selected from the group consisting of the nucleic acid sequences shown in FIGS. 173-1 to 173-294 is included. In yet other embodiments, the first tobacco plant is a transgenic plant.

  Typical first tobacco plants useful in the breeding methods disclosed herein include: Nicotiana africana, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior , Nicotiana exigua, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana hesperis, Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila, Nicotiana noctiflora, Nicotiana noctiflora, Nicosiana noctiflora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens or Nicotiana trigonophylla. Other first tobacco plants include Nicotiana tabacum or Nicotiana rustica varieties. Still other first tobacco plants include oriental, dark tobacco, fire-cured or air-cured tobacco, Virginia, or Burley tobacco plants.

  In another embodiment of said breeding method, the second tobacco plant is Nicotiana tabacum. Typical varieties of Nicotiana tabacum include commercial varieties such as: BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800 , CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KT 200, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 160, Little Crittenden, McNair 373, McNair 944, msKY 14 x L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC 606, NC 71, NC 72, NC 810, NC BH 129, OXFORD 207, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H4, RG H51, RGH 4 , RGH 51, RS 1410, SP 168, SP 172, SP 179, SP 210, SP 220, SP G-28, SP G-70, SP H20, SP NF3, TN 86, TN 90, TN 97, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA 309 or VA 359.

  In yet another embodiment, the phenotypic trait of the second tobacco plant includes disease resistance; high yield; high grade index; desiccation; quality of desiccation; mechanical harvestability; holding ability; Quality; height, plant maturity (eg, early ripening, premature to medium, medium, medium to late ripening or late ripening); pattern size (eg, small, moderate or large pattern); or number of leaves per plant ( For example, low (eg, 5-10 leaves), moderate (eg, 11-15 leaves) or high (eg, 16-21 leaves).

  In yet another embodiment, the method comprises pollinating a male sterile or male sterile hybrid with the plant of step (b), or backcrossing or pollinating a plant obtained from the germinated seed of step (e) Including that. In another aspect, the present invention relates to a method for breeding attributes into tobacco plants. The method is a) selected from the group consisting of the nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294 A first tobacco plant having a modified attribute comprising a variant gene expression of the nucleic acid molecule to be crossed with a second tobacco plant, b) obtaining a progeny tobacco plant of the cross, and c) obtaining a DNA sample from the progeny tobacco plant D) extracting the DNA sample from the nucleic acid sequence shown in FIG. 1, FIGS. 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294 or Contacting with a marker nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of those fragments, and e) performing a marker-assisted breeding method for the modified attribute. Typically, such marker-assisted breeding methods include amplified fragment length polymorphisms, restriction fragment length polymorphisms, random amplified polymorphism presentations, single nucleotide polymorphisms, microsatellite markers, or targeted inducible localities in the tobacco genome. Includes the use of targeted induced local lesions.

  In yet another aspect, the present invention relates to Nicotiana africana, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua, Nicotiana glutinosa, Nicotiana glutinosa, Nicotiana , Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana otophora, Nicotiana palmeri, Nicotiana palmitiana, foliobag Any one of tobacco plants selected from the group consisting of rustica, Nicotiana setchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens and Nicotiana trigonophylla is shown in FIG. 1, FIG. 3-7, FIG. 10-158, FIG. 162-170, FIG. 1 to 172-19 and the group consisting of the nucleic acid sequences shown in FIGS. 173-1 to 173-294 Comprising causing crossed with tobacco plant or a second, different tobacco plants with modified attributes include variants gene expression of a nucleic acid molecule selected et al., It relates to a method of producing the tobacco seeds.

  In yet another embodiment, the crossing comprises (a) planting a seed obtained from crossing a tobacco plant having a modified attribute with a second different tobacco plant, and (b) a tobacco plant from the seed And (c) pollinating tobacco plant flowers with modified attributes with pollen from a second tobacco plant, or modifying flowers of a second tobacco plant And (d) harvesting seeds obtained from the pollination from pollens from tobacco plants having attributes.

  In yet another aspect, the present invention provides (a) FIG. 1, FIGS. 3-7, FIGS. 10-158, FIGS. 162-170, FIGS. 172-1 to 172-19 and FIGS. 173-1 to 173-294 A tobacco plant having a modified attribute including expression of a mutant gene of a nucleic acid molecule selected from the group consisting of the nucleic acid sequences shown in the above or a component thereof, and (b) the plant as a breeding material source using tobacco plant breeding technology Or relates to a method for developing tobacco plants in a tobacco breeding program comprising using plant components. Typical plant breeding techniques useful for carrying out this method include bulk selection, backcrossing, self-pollination, introgression crossing, line selection, pure line selection, haploid / doubled haploid breeding or single grain line methods Is included.

  In a related aspect, the invention relates to a tobacco plant or component thereof produced by any of the above breeding methods. In yet another related aspect, the present invention relates to tissue culture of renewable tobacco cells obtained from any plant that has been bred or produced according to the methods described herein. Such tissue culture regenerates tobacco plants that can express all of the physiological and morphological characteristics of tobacco plants with mutant nicotine demethylase gene expression or modified attributes. Typical renewable cells are embryos, dividing cells, seeds, pollen, leaves, roots, root tips or flowers, or protoplasts or callus derived therefrom.

  In another related aspect, the present invention provides the production of a tobacco product comprising: (a) providing a tobacco plant obtained by any of the above breeding methods; and (b) producing a tobacco product from the tobacco plant. Regarding the method. Typical tobacco products include leaves or stems or both; smokeless tobacco products; wet or dry hookah tobacco; chewing tobacco; cigarette products; cigar products; cigarillos;

  One embodiment of the present invention is directed to the nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294. It relates to an isolated genetic marker comprising a nucleic acid sequence that is substantially identical, desirably at least 70% identical. In a preferred embodiment of this aspect of the invention, the nucleic acid sequence comprises: FIG. 1, FIGS. 3-7, FIGS. 10-158, FIGS. 162-170, FIGS. 172-1 to 172-19 and FIGS. 173-1 to 173 -294 contains a sequence that hybridizes under stringent conditions to the complement of the nucleic acid sequence shown in -294. In other desirable embodiments, the nucleic acid sequence is constitutive or ethylene or senescence-inducible. In addition, the nucleic acid sequence is desirably substantially the same as the amino acid sequence shown in FIGS. 1, 3 and 4, FIGS. 10 to 158, FIGS. 160A to 160E, FIGS. 162 to 170 and FIGS. 172-1 to 172-19. Which encodes a polypeptide which is

  In other desirable embodiments of the invention, the nucleic acid sequence is operably linked to a heterologous gene. Alternatively, the nucleic acid sequence is operably linked to an inducible, constitutive, pathogen or wound-inducible, environmental or developmental regulatory or cell or tissue specific promoter.

  In another embodiment, the invention provides the nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294. And an expression vector capable of directing the expression of the polypeptide encoded by the nucleic acid sequence.

  Other embodiments of the invention include a substance containing the amino acid sequence of the polypeptide shown in FIGS. 1, 3 and 4, FIGS. 10-158, FIGS. 160A-160E, FIGS. 162-170 or FIGS. 172-1 to 172-19. Purely polypeptides as well as antibodies that specifically recognize the polypeptides.

  Another embodiment of the invention is shown in FIG. 1, FIGS. 3-7, FIGS. 10-158, FIGS. 162-170, FIGS. 172-1 to 172-19 and FIGS. 173-1 to 173 expressed in plants or plant components. The plant or plant component containing the isolated nucleic acid sequence shown in -294, or FIGS. 1, 3 and 4, FIGS. 10-158, FIGS. 160A-160E, FIGS. 162-170 and FIGS. 172-1-172 Relates to the plant or plant component containing a nucleic acid sequence encoding a polypeptide substantially identical to the amino acid sequence shown in -19 and expressed in the plant or plant component. Desirably, the plant or plant component is a Nicotiana species, such as the Nicotiana species shown in Table 8. In other desirable embodiments, the plant component is leaves, such as dried tobacco leaves, stems or seeds. A desirable embodiment relates to plants from germinated seeds.

  In another embodiment, the present invention relates to FIG. 1, FIGS. 3-7, FIGS. 10-158, FIGS. 162-170, FIGS. 172-1 to 172-19 and FIGS. 173-1 expressed in plants or plant components. It relates to a tobacco product containing the plant or the plant component containing the isolated nucleic acid sequence shown at ~ 173-294. Desirably, expression of an endogenous gene in the dried tobacco plant or plant component is silenced. In other desirable embodiments, the tobacco products include smokeless tobacco products, wet or dry hookah tobacco, chewing tobacco, cigarette products, cigar products, cigarillos, pipe tobacco or bidi. In particular, the tobacco product of this aspect of the invention can contain dark tobacco, ground tobacco, or can include a flavor component.

  Another aspect of the invention relates to a method for reducing constitutive or ethylene-induced or senescence-induced tobacco polypeptide expression or enzyme activity in plant cells. This method involves reducing the level of endogenous constitutive or ethylene or senescence-induced tobacco polypeptides or enzymatic activity in plant cells. In desirable embodiments, the tobacco polypeptide is p450. In other desirable embodiments, the plant cell is derived from a Nicotiana species, eg, one of the Nicotiana species shown in Table 8.

  In another desirable embodiment, reducing the level of endogenous constitutive or ethylene or aging-inducing tobacco polypeptide is shown in FIGS. 1, 3-7, 10-158, 162-170, 172. -1 to 172-19 and the nucleic acid sequences shown in FIGS. 173-1 to 173-294 or FIG. 1, FIGS. 3 and 4, FIGS. 10 to 158, FIGS. 160A to 160E, FIGS. 162 to 170, and FIGS. Expressing in the plant cell a transgene encoding an antisense nucleic acid molecule of a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in 19. In another desirable embodiment, the transgene encodes a double-stranded RNA molecule of constitutive or ethylene or senescence-induced tobacco nucleic acid or amino acid sequence in the plant cell. In other desirable embodiments, the transgene is expressed, for example, tissue-specific, cell-specific or organ-specific. Also, reducing the level of endogenous constitutive or ethylene or senescence-inducing tobacco polypeptide desirably co-suppresses the constitutive or ethylene or senescence-inducing tobacco polypeptide in the plant cell. including. In another desirable embodiment, reducing the level of endogenous constitutive or ethylene or senescence-inducible tobacco polypeptide comprises expressing a dominant negative gene product in the plant cell. Desirably, endogenous constitutive or ethylene or senescence-inducing tobacco polypeptides are shown in FIGS. 1, 3 and 4, FIGS. 10-158, FIGS. 160A-160E, FIGS. 162-170 and FIGS. Mutations are included in the gene encoding the amino acid sequence shown. In other desirable embodiments, the decrease in expression occurs at the transcriptional, translational or post-translational level.

  Another aspect of the invention relates to a method for increasing constitutive or ethylene or senescence-induced tobacco polypeptide expression or enzyme activity in plant cells. The method includes increasing endogenous constitutive or ethylene or senescence-induced tobacco polypeptide levels or enzyme activity in the plant cell. In desirable embodiments of this aspect of the invention, the plant cell is derived from a Nicotiana species, such as the Nicotiana species shown in Table 8. In another desirable embodiment, increasing the level of a constitutive or ethylene or senescence-inducing tobacco polypeptide is shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1. ~ 172-19 and the nucleic acid sequences shown in Figures 173-1 to 173-294 or in Figures 1, 3 and 4, Figures 10 to 158, Figures 160A to 160E, Figures 162 to 170 and Figures 172-1 to 172-19 Expressing in the plant cell a transgene comprising a nucleic acid sequence encoding a polypeptide containing the amino acid sequence shown. Desirably, increased expression occurs at the transcriptional, translational or post-translational level.

  Another aspect of the invention relates to a method for producing a constitutive or ethylene or aging-inducing tobacco polypeptide. This method comprises (a) a single nucleic acid sequence comprising the nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294. Preparing a cell transformed with the released nucleic acid molecule, (b) culturing the transformed cell under conditions for expression of the isolated nucleic acid molecule, and (c) the constitutive or ethylene or senescence Recovering the inducible tobacco polypeptide. Desirably, the constitutive or ethylene or aging-inducing tobacco polypeptide is p450. In another desirable embodiment, the present invention relates to a recombinant constitutive or ethylene or senescence-inducing tobacco polypeptide produced according to the method of this aspect of the invention.

  In another aspect, the invention relates to a method for isolating a constitutive or ethylene or senescence-inducing tobacco polypeptide or fragment thereof. In this method, the nucleic acid sequence shown in FIG. 1, FIGS. 3 to 7, FIGS. 10 to 158, FIGS. 162 to 170, FIGS. 172-1 to 172-19 and FIGS. 1, at least 70% sequence identity to the nucleic acid sequences shown in FIGS. 3-7, 10-158, 162-170, 172-1 to 172-19 and FIGS. 173-1 to 173-294 Contacting with a nucleic acid preparation from a plant cell under hybridization conditions that result in the detection of the nucleic acid sequence having (b) isolating the hybridizing nucleic acid sequence. Desirably, the constitutive or ethylene or aging-inducing tobacco polypeptide is p450.

  Another aspect of the invention relates to an isolated nucleic acid molecule, such as a DNA sequence, containing a nucleotide sequence encoding nicotine demethylase. In desirable embodiments, the nucleotide sequence of the first aspect is, for example, at least 70% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or nucleotides 2010-2949 and / or 3947- of SEQ ID NO: 4 The nucleotide sequence encoding tobacco nicotine demethylase is substantially identical, such as tobacco nicotine demethylase containing 4562 or containing a nucleotide sequence containing the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. The isolated nucleic acid molecule of the first aspect of the present invention is operably linked to a promoter that is functional in, for example, a plant cell, and is desirably contained in an expression vector. In other desirable embodiments, the expression vector is contained within a cell, eg, a plant cell. Desirably, the plant cells, such as tobacco plant cells, are contained within the plant. In another desirable embodiment, the present invention comprises an isolated nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO: 4 operably linked to a heterologous promoter sequence. It relates to seeds from plants containing the vector, for example tobacco seeds. Furthermore, the present invention relates to a plant derived from a germinated seed containing the expression vector, a leaf (green leaf or dried leaf) derived from the plant, and a product derived from the leaf.

  In another desirable embodiment, the nucleotide sequence is a sequence that hybridizes under stringent conditions to the complement of the nucleotide sequence of SEQ ID NO: 4 and / or SEQ ID NO: 5 or a fragment of SEQ ID NO: 4 or SEQ ID NO: 5 Containing. Desirably, the nucleotide sequence encodes a nicotine demethylase that is substantially identical to the amino acid sequence of SEQ ID NO: 3. In another desirable embodiment of the first aspect of the invention, the nicotine demethylase is altered (eg, reduced) relative to the nicotine demethylase amino acid sequence of SEQ ID NO: 3 or relative to the full-length polypeptide. It has at least 70% amino acid sequence identity to a fragment of nicotine demethylase having enzymatic activity. Desirably, the nicotine demethylase comprises the amino acid sequence of SEQ ID NO: 3.

  In another embodiment, the present invention relates to an isolated nucleic acid molecule containing a promoter that hybridizes under stringent conditions to the sequence of SEQ ID NO: 8 or a fragment thereof and drives transcription. Desirably, the promoter is (i) induced after treatment with ethylene or during senescence, (ii) (a) base pair 1-2009 of SEQ ID NO: 4, or (b) base pair 1- 1 of SEQ ID NO: 4. At least 200 contiguous base pairs identical to 200 contiguous base pairs of the sequence defined by 2009, or (c) 20 contiguous bases of the sequence set forth in base pair 1-2009 of SEQ ID NO: 4 Contains a 20 base pair nucleotide portion that is identical in sequence to the paired portion.

  Another aspect of the invention relates to an isolated nucleic acid promoter containing a nucleotide sequence having 50% or more sequence identity to the sequence of SEQ ID NO: 8. Desirably, the isolated nucleic acid promoter is derived after treatment with ethylene or during senescence and comprises, for example, the sequence of SEQ ID NO: 8. Alternatively, the promoter can comprise a fragment obtainable from SEQ ID NO: 8 that drives transcription of a heterologous gene or reduces or alters nicotine demethylase enzyme activity (eg, silences gene expression). In desirable embodiments, the promoter sequence is operably linked to a heterologous nucleic acid sequence, or may be contained, for example, in an expression vector. In other desirable embodiments, the expression vector is contained within a cell, eg, a plant cell. Desirably, the plant cells, such as tobacco plant cells, are contained within the plant. In another desirable embodiment, the invention comprises an isolated nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO: 8 operably linked to a heterologous nucleic acid sequence. It relates to seeds from plants containing the vector, for example tobacco seeds. Furthermore, the present invention relates to a plant derived from a germinated seed containing the promoter of this aspect of the present invention, a leaf (green leaf or dried leaf) derived from the plant, and a product derived from the leaf.

  Another aspect of the invention relates to a method for expressing a heterologous gene in a plant. This comprises (i) introducing into a plant cell a vector containing a promoter sequence having 50% or more sequence identity to the sequence of SEQ ID NO: 8 operably linked to a heterologous nucleic acid sequence; ii) including regenerating plants from the cells. The method can also include sexually transmitting the vector to progeny, and can further include collecting seed produced by the progeny.

  In yet another aspect, the present invention relates to a method for reducing the expression of nicotine demethylase in tobacco plants. This method comprises (i) introducing into a tobacco plant a vector containing a sequence of SEQ ID NO: 8 or a fragment obtainable from SEQ ID NO: 8 operably linked to a heterologous nucleic acid sequence; Expressing the vector in a plant. In a preferred embodiment of this method, the expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA, or RNA molecules that can induce RNA interference (RNAi).

  In another desirable embodiment, the present invention provides SEQ ID NO: which can reduce or alter nicotine demethylase enzyme activity (eg, silence gene expression) or serve as a molecular marker for identification of nicotine demethylase nucleic acid sequences. It relates to an isolated nucleic acid molecule containing an intron that hybridizes under stringent conditions to a sequence of 7 or a fragment thereof. In a preferred embodiment, the intron is identical to 200 consecutive base pairs of the sequence defined by (a) base pairs 2950-3946 of SEQ ID NO: 4, or (b) base pairs 2950-3946 of SEQ ID NO: 4 At least 200 contiguous base pairs, or (c) a 20 base pair nucleotide portion that is identical in sequence to the contiguous 20 base pair portion of the sequence set forth in base pair 2950-3946 of SEQ ID NO: 4 .

  Another desirable embodiment of the invention is that of SEQ ID NO: 7 that can serve as a molecular marker for reducing or altering nicotine demethylase enzyme activity (eg, silencing gene expression) or for identifying nicotine demethylase nucleic acid sequences. It relates to an isolated nucleic acid intron comprising a nucleotide sequence having 50% or more sequence identity to the sequence or a fragment thereof. Silencing gene expression can include, for example, homologous recombination (eg, using the sequence of SEQ ID NO: 188 or a fragment thereof), or a mutation that results in a gene product that does not have nicotine demethylase activity. In particular, the intron may comprise the sequence of SEQ ID NO: 7 or a fragment obtainable from SEQ ID NO: 7. Desirably, the isolated nucleic acid molecule containing the intron is operably linked to a heterologous nucleic acid sequence, which sequence can desirably be contained within an expression vector. In another embodiment, the expression vector is contained within a cell, eg, a plant cell. In particular, the cell can be a tobacco cell. A plant, such as a plant plant, containing a sequence of SEQ ID NO: 7 or a fragment obtainable from SEQ ID NO: 7 operably linked to a heterologous nucleic acid sequence in an expression vector, such as a tobacco plant, is another aspect of the invention. One preferred embodiment. In addition, seeds from plants, such as tobacco seeds, containing introns that hybridize under stringent conditions to SEQ ID NO: 7 operably linked to heterologous nucleic acid sequences are also desirable. Furthermore, the present invention relates to a plant derived from the germinated seed containing the intron of this aspect of the present invention, a leaf (green leaf or dried leaf) derived from the plant, and a product manufactured from the green leaf or dried leaf.

  Another aspect of the invention relates to a method for expressing an intron in a plant. This method comprises (i) introducing a vector containing a sequence of SEQ ID NO: 7 or a fragment obtainable from SEQ ID NO: 7 operably linked to a heterologous nucleic acid sequence into a plant cell; Including regenerating plants from. In desirable embodiments, the method can include (iii) sexually transferring the vector to progeny and collecting seed produced by the progeny. The method desirably includes, for example, regenerating a plant from germinated seeds, a leaf (green leaf or dried leaf) derived from the plant, and a method for producing a product derived from the leaf.

  In yet another aspect, the present invention relates to a method for reducing the expression of nicotine demethylase in tobacco plants. This method comprises (i) introducing into a tobacco plant a vector containing a sequence of SEQ ID NO: 7 or a fragment obtainable from SEQ ID NO: 7 operably linked to a heterologous nucleic acid sequence; Expressing the vector in a plant. In a preferred embodiment of this method, the expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA, or RNA molecules that can induce RNA interference (RNAi).

  In another desirable embodiment, the present invention can alter the expression pattern of a gene to reduce or alter nicotine demethylase enzyme activity (eg, silence gene expression) or for identification of a nicotine demethylase nucleic acid sequence. It relates to an isolated nucleic acid molecule containing an untranslated region that hybridizes under stringent conditions to the sequence of SEQ ID NO: 9 or a fragment thereof that can be used as a marker. In a preferred embodiment of this aspect of the invention, the untranslated region comprises 200 of the sequence defined by (a) base pairs 4563-6347 of SEQ ID NO: 4, or (b) base pairs 4563-6347 of SEQ ID NO: 4 At least 200 contiguous base pairs identical to the contiguous base pairs of (c), or (c) identical in sequence to a contiguous 20 base pair portion of the sequence set forth in base pairs 4563-6347 of SEQ ID NO: 4 Contains a 20 base pair nucleotide moiety.

  Another desirable embodiment of the present invention relates to an isolated nucleic acid untranslated region containing a nucleotide sequence having 50% or more sequence identity to the sequence of SEQ ID NO: 9 or a fragment thereof. Desirably, the untranslated region comprises the sequence of SEQ ID NO: 9, and the untranslated region alters the expression pattern of the gene to reduce or alter nicotine demethylase enzyme activity (eg, silence gene expression) or nicotine demethylase Includes fragments available from SEQ ID NO: 9 that can be used as markers for identification of nucleic acid sequences. The untranslated region is desirably operably linked to a heterologous nucleic acid sequence and can be contained within an expression vector. Furthermore, the expression vector is desirably contained within a cell, such as a plant cell, such as a tobacco cell. Another desirable embodiment of the present invention is a vector comprising an isolated nucleic acid sequence operably linked to a heterologous nucleic acid sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 9 Plants such as plant cell plants containing, for example, tobacco plants.

  The invention also relates to seeds from plants, such as tobacco seeds, comprising untranslated regions that hybridize under stringent conditions to SEQ ID NO: 9 operably linked to heterologous nucleic acid sequences. Furthermore, the present invention relates to a plant derived from germinated seeds containing the untranslated region of this aspect of the present invention, a leaf (green leaf or dried leaf) derived from the plant, and a product manufactured from the green leaf or dried leaf. About.

  In another aspect, the present invention relates to a method for expressing an untranslated region in a plant. This method comprises: (i) a vector containing an isolated nucleic acid sequence having a sequence identity of 50% or more to the sequence of SEQ ID NO: 9 and operably linked to a heterologous nucleic acid sequence. And (ii) regenerating a plant from the cell. The method can also include (iii) sexually transmitting the vector to progeny, and desirably including an additional step of collecting seed produced by the progeny. . The method desirably includes, for example, regenerating a plant from a germinated seed, a leaf (green leaf or dried leaf) derived from the plant, and a method for producing a product manufactured from the green leaf or dried leaf.

  Furthermore, the present invention relates to a method for reducing or altering the expression of nicotine demethylase in tobacco plants. This method comprises (i) a vector comprising an isolated nucleic acid sequence operably linked to a heterologous nucleic acid sequence having a sequence identity of 50% or more to the sequence of SEQ ID NO: 9 in tobacco plants. And (ii) expressing the vector in the tobacco plant. Desirably, the expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA, or RNA molecules that can induce RNA interference (RNAi).

  Another aspect of the invention relates to an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding nicotine demethylase that can direct expression of the nicotine demethylase encoded by the isolated nucleic acid molecule. Desirably, the vector comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In other desirable embodiments, the present invention provides a plant or plant component, such as a tobacco plant or plant component (e.g., tobacco leaf or plant) comprising a nucleic acid molecule that contains a nucleotide sequence encoding a polypeptide that demethylates nicotine. Stem).

  Another aspect of the present invention relates to a cell containing an isolated nucleic acid molecule comprising a nucleotide sequence encoding nicotine demethylase. Desirably, the cell is a plant cell or a bacterial cell, such as Agrobacterium.

  Another aspect of the invention relates to the plant or plant component (eg, tobacco leaf or stem) containing an isolated nucleic acid molecule encoding nicotine demethylase expressed in the plant or plant component. Desirably, the plant or plant component is an angiosperm, a dicotyledonous plant, a solanaceous plant, or a Nicotiana species. Other desirable embodiments of this aspect are seeds or cells from the plant or plant component, as well as leaves (green leaves or dry leaves) derived from the plants, and articles made therefrom.

  In another embodiment, the present invention relates to a tobacco plant with reduced expression of a nucleic acid sequence encoding a polypeptide that includes, for example, the sequence of SEQ ID NO: 3 and demethylates nicotine. Here, the decreased expression (or decreased enzyme activity) decreases the level of nornicotine in the plant. In desirable embodiments, the tobacco plant comprises a transgenic plant, eg, a transgene that silences gene expression of endogenous tobacco nicotine demethylase when expressed in the transgenic plant.

  In particular, the transgenic plant desirably comprises one or more of the following: a tobacco nicotine demethylase antisense molecule or a transgene expressing an RNA molecule capable of inducing RNA interference (RNAi); A transgene that, when expressed, co-suppresses tobacco nicotine demethylase expression; a dominant negative gene product, eg, a transgene that encodes a variant form of the amino acid sequence of SEQ ID NO: 3; within a gene that encodes the amino acid sequence of SEQ ID NO: 3 Point mutations; deletions in the gene encoding tobacco nicotine demethylase; and insertions in the gene encoding tobacco nicotine demethylase.

  In other desirable embodiments, the reduction in expression of the nucleic acid sequence encoding the polypeptide occurs at the transcriptional, translational or post-translational level.

  Another aspect of the invention relates to tobacco plants containing a recombinant expression cassette that is stably integrated into its genome. Here, the cassette may result in a decrease in nicotine demethylase activity. This tobacco plant seed is the subject in a preferred embodiment. Other desirable embodiments include leaves derived from this plant (green leaves or dry leaves) and articles derived therefrom.

  Another aspect of the invention relates to a method for expressing tobacco nicotine demethylase in a plant. The method includes (i) introducing an expression vector containing a nucleic acid molecule containing a nucleotide sequence encoding nicotine demethylase into a plant cell, and (ii) regenerating the plant from the cell. In desirable embodiments, the method includes sexually transferring the vector to progeny, and desirably also includes an additional step of collecting seed produced by the progeny. Further desirable implementations include plants derived from the germinated seeds, leaves derived from the plants (green leaves or dried leaves), and manufactured articles made from the green leaves or dried leaves.

  Another aspect of the invention relates to substantially pure tobacco nicotine demethylase. Desirably, the tobacco nicotine demethylase comprises an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 3, or comprises the amino acid sequence of SEQ ID NO: 3. In desirable embodiments, the tobacco nicotine demethylase converts nicotine to nornicotine when expressed in plant cells. In other desirable embodiments, the tobacco nicotine demethylase is primarily localized to the leaf when expressed in plant cells, or the tobacco nicotine demethylase is induced by ethylene or expressed during plant senescence. Is done.

  In another embodiment, the present invention relates to a substantially pure antibody that specifically recognizes and binds to tobacco nicotine demethylase. Desirably, the antibody recognizes and binds to a recombinant tobacco nicotine demethylase (eg, one containing the sequence of SEQ ID NO: 3 or a fragment thereof).

  Another embodiment of the present invention relates to a method for producing tobacco nicotine demethylase. This method comprises (a) preparing a cell transformed with an isolated nucleic acid molecule containing a nucleotide sequence encoding a polypeptide that demethylates nicotine, and (b) preparing the isolated nucleic acid molecule Culturing the transformed cell under conditions for expression and (c) recovering tobacco nicotine demethylase. The invention also relates to a recombinant tobacco nicotine demethylase produced by this method.

  In another embodiment, the present invention relates to a method for isolating tobacco nicotine demethylase or a fragment thereof. This method comprises: (a) at least 70% or more of the nucleic acid sequence shown in the nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of SEQ ID NO: 4, 5, 7, 8 or 9 Contacting a nucleic acid molecule of SEQ ID NO: 4, 5, 7, 8 or 9 or a portion thereof with a nucleic acid preparation from a plant cell under hybridization conditions that result in the detection of a nucleic acid sequence having sequence identity; (b) Isolating the hybridizing nucleic acid sequence.

  In another embodiment, the present invention relates to another method for isolating tobacco nicotine demethylase or a fragment thereof. This method comprises (a) preparing a sample of plant cell DNA, and (b) preparing a pair of oligonucleotides having sequence identity to a region of a nucleic acid molecule having a sequence of SEQ ID NO: 4, 5, 7, 8 or 9. (C) contacting the oligonucleotide pair with the plant cell DNA under conditions suitable for DNA amplification by polymerase chain reaction, and (d) isolating the amplified tobacco nicotine demethylase or fragment thereof. Including. In a desirable embodiment of this aspect, the amplification step is performed using a cDNA sample prepared from plant cells. In another desirable embodiment, the tobacco nicotine demethylase encodes a polypeptide that is at least 70% identical to the amino acid sequence of SEQ ID NO: 3.

  Another aspect of the invention relates to a method for reducing the expression of tobacco nicotine demethylase in a plant or plant component. In this method, (a) a transgene encoding tobacco nicotine demethylase operably linked to a functional promoter in a plant cell is introduced into the plant cell to obtain a transformed plant cell; (B) regenerating a plant or plant component from the transformed plant cell, wherein the tobacco nicotine demethylase is expressed in the plant or plant component cell, thereby causing tobacco nicotine depletion in the plant or plant component. Methylase expression is reduced. In particular embodiments of this aspect of the invention, the transgene encoding tobacco nicotine demethylase is inducibly or constitutively expressed in a tissue-specific, cell-specific or organ-specific manner. In another embodiment of this aspect of the invention, the expression of the transgene co-suppresses the expression of endogenous tobacco nicotine demethylase or any other polypeptide described herein.

Another aspect of the invention relates to another method for reducing the expression of tobacco nicotine demethylase or any other polypeptide described herein in a plant or plant component. This method comprises: (a) a transcoding encoding an antisense coding sequence of tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi) operably linked to a functional promoter in plant cells. A gene is introduced into the plant cell to obtain a transformed plant cell, and (b) regenerates a plant or plant component from the transformed plant cell, wherein the coding sequence of tobacco nicotine demethylase RNA molecules capable of inducing antisense or RNA interference (RNAi) are expressed in cells of the plant or plant component, thereby reducing the expression of tobacco nicotine demethylase in the plant or plant component. Desirably, the antisense sequence of tobacco nicotine demethylase or a transgene encoding an RNA molecule capable of inducing RNA interference (RNAi) is inducibly expressed, eg, tissue-specific, cell-specific or organ-specific, or Expressed constitutively. In other desirable embodiments, RNA molecules capable of inducing antisense or RNAi of the coding sequence of tobacco nicotine demethylase are SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9. Contains the complement of SEQ ID NO: 62 , SEQ ID NO: 188 or fragments thereof.

  Another aspect of the invention relates to yet another method for reducing the expression of tobacco nicotine demethylase in a plant or plant component. In this method, (a) a transgene encoding a dominant negative gene product of tobacco nicotine demethylase operably linked to a functional promoter in a plant cell is introduced into the plant cell and transformed. Obtaining a plant cell and (b) regenerating the plant or plant component from the transformed plant cell, wherein the tobacco nicotine demethylase dominant negative gene product is expressed in the plant or plant component cell; Thereby, the expression of tobacco nicotine demethylase in the plant or plant component is reduced. In certain embodiments of this aspect of the invention, the transgene encoding the dominant negative gene product is inducibly expressed, eg, constitutively expressed, eg, tissue specific, cell specific, or organ specific. The

Another aspect of the invention relates to another method for reducing tobacco nicotine demethylase expression or enzyme activity in plant cells. The method includes reducing the level of endogenous tobacco nicotine demethylase in the plant cell. Desirably, the plant cell is derived from a dicotyledonous plant, a solanaceous plant, or a Nicotiana species. In desirable embodiments of this aspect, reducing the level of endogenous tobacco nicotine demethylase comprises transgene encoding an antisense nucleic acid molecule of tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi). Or expressing in a plant cell a transgene encoding a double-stranded RNA molecule of tobacco nicotine demethylase. Desirably, the double-stranded RNA is an RNA corresponding to the sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 62 , SEQ ID NO: 188 or a fragment thereof. Is an array. In another embodiment, reducing the level of endogenous tobacco nicotine demethylase comprises co-suppression of endogenous tobacco nicotine demethylase in the plant cell, or expresses a dominant negative gene product in the plant cell. Including that. In particular, the dominant negative gene product may comprise a gene encoding a variant of the amino acid sequence of SEQ ID NO: 3 or any other amino acid sequence described herein.

  In other desirable embodiments of this aspect of the invention, the endogenous tobacco nicotine demethylase comprises a point mutation in the gene encoding the amino acid sequence of SEQ ID NO: 3. In other desirable embodiments, reducing the level of expression of endogenous tobacco nicotine demethylase involves a deletion in the gene encoding tobacco nicotine demethylase, or within the gene encoding tobacco nicotine demethylase. With insertion. Decreased expression can occur at the transcriptional, translational or post-translational level.

  Another aspect of the invention relates to a method for identifying a compound that alters the expression of tobacco nicotine demethylase in a cell. This method comprises (a) preparing a cell containing a gene encoding tobacco nicotine demethylase, (b) applying a candidate compound to the cell, and (c) expressing the gene encoding the tobacco nicotine demethylase. An increase or decrease in expression relative to an untreated control sample indicates that the compound alters expression of the tobacco nicotine demethylase.

  In a preferred embodiment of this method, the gene of item (a) encodes a tobacco nicotine demethylase having at least 70% identity to the amino acid sequence of SEQ ID NO: 3. Desirably, the compound decreases or increases the expression of the gene encoding the tobacco nicotine demethylase.

  In another embodiment, the present invention relates to another method for identifying a compound that alters the activity of tobacco nicotine demethylase in a cell. This method comprises the steps of (a) preparing a cell expressing a gene encoding tobacco nicotine demethylase, (b) applying a candidate compound to the cell, and (c) measuring the activity of the tobacco nicotine demethylase. Including, where an increase or decrease in activity relative to an untreated control sample indicates that the compound alters the activity of the tobacco nicotine demethylase. In a preferred embodiment of this aspect of the invention, the gene of item (a) encodes a tobacco nicotine demethylase having at least 70% identity to the amino acid sequence of SEQ ID NO: 3. Desirably, the compound reduces or increases the activity of the tobacco nicotine demethylase.

  Another aspect of the invention relates to a dried tobacco plant or plant component containing (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase having altered enzyme activity and a reduced amount of nitrosamine. Desirably, the plant component is tobacco leaf or tobacco stem. In desirable embodiments, the nitrosamine is nornicotine and the content of nornicotine is desirably less than 5 mg / g, less than 4.5 mg / g, less than 4.0 mg / g, less than 3.5 mg / g, less than 3.0 mg / g, More desirably less than 2.5 mg / g, less than 2.0 mg / g, less than 1.5 mg / g, less than 1.0 mg / g, more desirably less than 750 μg / g, less than 500 μg / g, less than 250 μg / g, 100 μg less than 75 μg / g, more preferably less than 50 μg / g, less than 25 μg / g, less than 10 μg / g, less than 7.0 μg / g, less than 5.0 μg / g, less than 4.0 μg / g, Even more desirably, less than 2.0 μg / g, less than 1.0 μg / g, less than 0.5 μg / g, less than 0.4 μg / g, less than 0.2 μg / g, less than 0.1 μg / g, less than 0.05 μg / g or 0.01 μg / g Or the ratio of secondary alkaloids (%) to the total alkaloid content contained therein is less than 90%, less than 70%, less than 50%, less than 30%, less than 10%, desirably 5% Less than 4% 3% Mitsuru, less than 2%, less than 1.5%, less than 1%, more preferably less than 0.75%, less than 0.5%, less than less than 0.25% or 0.1%. In another desirable embodiment, the nitrosamine is N'-nitrosonornicotine (NNN) and the content of N'-NNN is desirably less than 5 mg / g, less than 4.5 mg / g, less than 4.0 mg / g Less than 3.5 mg / g, less than 3.0 mg / g, more preferably less than 2.5 mg / g, less than 2.0 mg / g, less than 1.5 mg / g, less than 1.0 mg / g, more preferably less than 750 μg / g, 500 <μg / g, <250 μg / g, <100 μg / g, even more preferably <75 μg / g, <50 μg / g, <25 μg / g, <10 μg / g, <7.0 μg / g Less than 5.0 μg / g, less than 4.0 μg / g, even more preferably less than 2.0 μg / g, less than 1.0 μg / g, less than 0.5 μg / g, less than 0.4 μg / g, less than 0.2 μg / g, 0.1 μg / g less than g, less than 0.05 μg / g or less than 0.01 μg / g, or where the ratio of secondary alkaloids to the total alkaloid content contained therein is less than 90%, less than 70%, less than 50% Less than 30%, less than 10%, preferably less than 5% , Less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, more preferably less than 0.75%, less than 0.5%, less than less than 0.25% or 0.1%. In a further desirable embodiment of this aspect of the invention, the dried tobacco plant or plant component is dark tobacco, burley tobacco. Examples include fire-cured tobacco, Virginia, air-cured tobacco, oriental tobacco.

  Further, the dried tobacco plant or plant component of the present invention desirably includes a recombinant nicotine demethylase gene, such as one containing the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a fragment thereof. Desirably, expression of an endogenous nicotine demethylase gene or any other nucleic acid sequence described herein in a dried tobacco plant or plant component is silenced.

  Another aspect of the invention is (i) a nicotine demethylase with reduced expression or any other polypeptide described herein or (ii) a nicotine demethylase with altered activity or described herein. To a tobacco product containing a dried tobacco plant or plant component comprising a reduced polypeptide and a reduced amount of nitrosamine. Desirably, the tobacco products include smokeless tobacco products, wet or dry hookah tobacco, chewing tobacco, cigarette products, cigar products, cigarillos, pipe tobacco or bidi. In particular, the tobacco product of this aspect of the invention can contain dark tobacco, ground tobacco, or can include a flavor component.

  The invention also provides for the manufacture of tobacco products, eg, smokeless tobacco products, containing (i) nicotine demethylase with reduced expression or (ii) nicotine demethylase with altered (eg, reduced) enzyme activity and a reduced amount of nitrosamine. Regarding the method. The method comprises preparing a dried tobacco plant or plant component comprising (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase having altered enzyme activity and a reduced amount of nitrosamine, wherein the dried tobacco plant Or producing the tobacco product from plant components.

The definition “enzymatic activity” is demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S- and O-dealkylation, desulfation, deamination and azo, nitro, N-oxide. And other such enzyme reactive chemical group reductions are intended to include (but are not limited to). The altered enzyme activity (change in enzyme activity) is at least 10-20%, preferably at least 25-50%, more preferably at least 55% relative to the activity of a control enzyme (eg, wild-type tobacco plant tobacco nicotine demethylase). Means a decrease in enzyme activity (eg, of tobacco nicotine demethylase) of ~ 95% or more. The activity of an enzyme (eg, nicotine demethylase) can be assayed using standard methods in the art, for example, using the yeast microsome assay described herein.

  The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form or sense or antisense, and unless otherwise limited, hybridizes to nucleic acids in a manner similar to naturally occurring nucleotides. Contains known analogs of natural nucleotides that soy. Unless otherwise indicated, a particular nucleic acid sequence includes its complementary sequence. The terms “operably linked”, “functional combination” and “functional order” refer to a nucleic acid expression control sequence (eg, promoter, signal sequence or series of transcription factor binding sites) and a second nucleic acid. By means of a functional linkage between sequences, the expression control sequence affects the transcription and / or translation of the nucleic acid corresponding to the second sequence. Desirably, a functionally linked nucleic acid sequence refers to a fragment of a gene linked to other sequences of the same gene to form a full-length gene.

  The term “recombinant” when used with respect to a cell indicates that the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, heterologous peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can express a gene or gene fragment in sense or antisense form, or as an RNA molecule that induces RNA interference (RNAi) not found in the natural (non-recombinant) form of the cell. Recombinant cells can also express genes found in the natural form of the cells but modified by artificial means and reintroduced into the cells.

  A “structural gene” is a portion of a gene that includes a DNA segment that encodes a protein, polypeptide, or portion thereof, which does not include, for example, a 5 ′ sequence or 3 ′ UTR that drives transcription initiation. Alternatively, the structural gene can encode a non-translatable product. A structural gene can be one that is normally found in the cell, or one that is not normally found in the cell or cell location (when it is introduced, it is referred to as a “heterologous gene”). A heterologous gene can be derived, in whole or in part, from any source known in the art, including bacterial genomes or episomes, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. A structural gene may contain one or more modifications that may affect the biological activity or its characteristics, the biological activity or chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, one or more nucleotide mutations, insertions, deletions and substitutions.

  A structural gene can constitute an unbroken coding sequence, or it can contain one or more introns flanked by appropriate splice sites. Structural genes can be translatable or non-translatable and can include antisense or RNA molecules that can induce RNA interference (RNAi). A structural gene can be a composite of segments derived from multiple sources and multiple gene sequences (natural or synthetic, where synthetic refers to chemically synthesized DNA).

  An “exon” as used herein with respect to a nucleic acid sequence refers to a portion of a nucleic acid sequence of a gene, where the exon nucleic acid sequence encodes at least one amino acid of the gene product. Exons are typically adjacent to non-coding DNA segments such as introns.

  An “intron” as used herein with respect to a nucleic acid sequence refers to a non-coding region of a gene that flank the coding region. Introns are typically non-coding regions of genes that are transcribed into RNA molecules but then excised by RNA splicing during the production of messenger RNA or other functional structural RNA.

  “3′UTR” as used herein with respect to a nucleic acid sequence means a non-coding nucleic acid sequence close to the stop codon of an exon.

  “Derived” means taken, obtained, received, traced, replicated or transmitted from a source (chemical and / or biological). Derivatives can be made by chemical or biological manipulation of the source, including but not limited to substitution, addition, insertion, deletion, extraction, isolation, mutation and replication.

“Chemical synthesis” with respect to the sequence of DNA means that some of the component nucleotides were constructed in vitro. Manual chemical synthesis of DNA can be accomplished using well-established methods (Caruthers, Methodology of DNA and RNA Sequencing , (1983), Weissman (ed.), Praeger Publishers, New York, Chapter 1). Automated chemical synthesis can be performed using one of a number of commercially available equipment.

  Optimal alignment of sequences for comparison is described, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970 ) Homology alignment algorithms, Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988) similarity search methods, these algorithms (GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Package, This can be done by computerized implementation or scrutiny by Genetics Computer Group, 575 Science Dr., Madison, Wis.

  NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) includes National Center for Biological Information (NCBI, Bethesda, Md.) For use with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Available from several sources or on the Internet. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST. Methods for determining sequence identity using this program are available at http://www.ncbi.nlm.nih.gov/BLAST/blast help.html.

  The terms “substantial amino acid identity” or “substantial amino acid sequence identity” as used herein with respect to amino acid sequences refer to FIGS. 10-159, 160A-160E, 162-170 and 172-1-172- At least 70% sequence identity, preferably 80% amino acid sequence identity, more preferably 90% amino acid sequence identity, most preferably at least 99-100% sequence identity compared to the protein sequence shown in 19. The polypeptide has the property that the peptide contains a sequence having Desirably, in the case of nicotine demethylase, the sequence comparison is performed after the cytochrome p450 motif GXRXCX (G / A) (SEQ ID NO: 2265) of the translated peptide to the stop codon region.

  The terms “substantial nucleic acid identity” or “substantial nucleic acid sequence identity” as used herein with respect to amino acid sequence encode the translated peptide cytochrome p450 motif GXRXCX (G / A) (2265) At least 50%, preferably 60, 65, 70 or 75% sequence identity over the region corresponding to the first nucleic acid after the region to the stop codon, more preferably 81 or 91% A characteristic of the polynucleotide sequence is that the polynucleotide comprises a sequence having nucleic acid sequence identity, most preferably at least 95, 99 or even 100% sequence identity.

  Another indication that the nucleotide sequences are substantially identical is whether the two molecules hybridize to each other under stringent conditions. Stringent conditions vary from sequence to sequence and will vary from situation to situation. Generally, stringent conditions are such that at a constant ionic strength and pH, the temperature is about 5 ° C. to about 22 ° C., usually about 10 ° C. to about 15 ° C. below the thermal melting point (Tm) of the specific sequence. To be elected. Tm is the temperature (at constant ionic strength and pH) at which 50% of the target sequence hybridizes to the match probe. Typically, stringent conditions are those where the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60 ° C. For example, in standard Southern hybridization methods, stringent conditions include initial washing at 42 ° C. in 6 × SSC, followed by at least about 55 ° C., typically about 60 ° C. in 0.2 × SSC. Often it will involve one or more additional washes at a temperature of about 65 ° C.

  Also, for purposes of the present invention, if the nucleotide sequence encodes substantially the same polypeptide and / or protein, the nucleotide sequence is substantially the same. Thus, if one nucleic acid sequence encodes a polypeptide that is substantially the same as a second nucleic acid sequence, the nucleic acid sequences are degenerate (codon degeneracy and genetic) allowed by the genetic code. The description of the cipher is practical even if it does not hybridize under stringent conditions due to Darnell et al. (1990) Molecular Cell Biology, Second Edition Scientific American Books WH Freeman and Company New York) Are identical. Protein purity or homogeneity can be demonstrated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of protein samples followed by visualization after staining. For some purposes, high resolution may be required and HPLC or similar purification means may be used.

  An antibody that “specifically binds” or “specifically recognizes” a particular polypeptide, such as tobacco nicotine demethylase, binds the polypeptide more than an equal amount of any other protein. It means an antibody having a high affinity for it. Desirable antibodies are antibodies that specifically bind to a polypeptide having the amino acid sequence shown in FIGS. 1, 3 and 4, FIGS. 10 to 158, FIGS. 160A to 160E, FIGS. 162 to 170 and FIGS. 172-1 to 172-19. It is. For example, an antibody that specifically binds tobacco nicotine demethylase containing the amino acid sequence of SEQ ID NO: 3 desirably is at least 2-fold, 5-fold, 10-fold, greater than any other equivalent amount of antigen, including related antigens, Has an affinity for that antigen that is 30 or 100 times greater. Antibody binding to an antigen (eg, tobacco nicotine demethylase) can be measured by a number of standard methods, such as Western analysis, ELISA or coprecipitation. Antibodies that specifically bind to a polypeptide (eg, nicotine demethylase) are also useful for purification of the polypeptide.

  As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segments into cells. Vectors can act to replicate DNA and can be independently regenerated in host cells. The term “vehicle” is sometimes used interchangeably with “vector”. As used herein, the term “expression vector” refers to a recombinant DNA molecule containing the appropriate nucleic acid sequence and the desired coding sequence necessary for the expression of a operably linked coding sequence in a particular host organism. Means. The nucleic acid sequences necessary for expression in prokaryotes usually contain a promoter, an operator (optional) and a ribosome binding site, often along with other sequences. Desirably, the promoter comprises the sequence of SEQ ID NO: 8, or a fragment thereof that drives transcription. Also desirable are promoter sequences that drive transcription with at least 50%, 60%, 75%, 80%, 90%, 95% or even 99% sequence identity to the sequence of SEQ ID NO: 8. Eukaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals such as the 3 ′ UTR sequence of SEQ ID NO: 9. In some cases, plant expression vectors have been found to require the presence of a plant-derived intron (eg, an intron having the sequence of SEQ ID NO: 7) for stable expression. Thus, as described in more detail herein, the sequence of SEQ ID NO: 7 or any other intron with an appropriate RNA splice site can be used. Desirable vectors include the nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 or FIGS. 173-1 to 173-294.

  In order to regenerate fully engineered plants with roots, any known in vitro tissue can be obtained by any technique such as in vitro inoculation or to obtain transformed plant cells that can be regenerated into complete plants. Nucleic acids can be inserted into plant cells by culture techniques. Thus, for example, insertion into plant cells can be by in vitro inoculation with pathogenic or non-pathogenic Atumefaciens. Other such tissue culture techniques can also be used.

  "Plant tissue", "plant component" or "plant cell" includes plant differentiated and undifferentiated tissue, including but not limited to roots, shoots, leaves, pollen, seeds, tumor tissue and in culture Various forms of cells, such as single cells, protoplasts, embryos and callus tissues. Plant tissue can be in planta or in organ, tissue or cell culture.

  As used herein, “plant cells” includes plant cells of in planta, as well as plant cells and protoplasts in culture. “CDNA” or “complementary DNA” generally refers to a single-stranded DNA molecule having a nucleotide sequence that is complementary to an unprocessed RNA molecule containing an intron or complementary to a processed mRNA that lacks an intron. means. cDNA is formed by the action of reverse transcriptase on the RNA template.

  As used herein, “tobacco” includes fire-cured, virginia, burley, dark tobacco, oriental, and other types of plants of the genus Nicotiana. Nicotiana seeds are readily available commercially in the form of Nicotiana tabacum.

  “Manufactured product” or “tobacco product” includes products such as wet or dry hookah, chewing tobacco, cigarette products, cigar products, cigarillos, pipe tobacco, bidi and similar tobacco-derived products.

  “Gene silencing” is a gene expression of at least 30-50%, preferably at least 50-80%, more preferably at least 80-95% or more relative to the level in a control plant (eg, a wild-type tobacco plant). It means a reduction in the level of (eg the expression of a gene encoding tobacco nicotine demethylase). Such reduction in expression level is achieved by standard methods known in the art, such as RNA interference, triple-stranded interference, ribozyme, homologous recombination, virus-induced gene silencing, antisense and cosuppression techniques, dominant It can be achieved by expression of negative gene products, generation of mutant genes using standard mutagenesis techniques (eg, those described herein). The level of tobacco nicotine demethylase polypeptide or transcript or both is monitored by any standard technique including, but not limited to, Northern blotting, RNase protection or immunoblotting.

  The “fragment” or “part” of the amino acid sequence is any of the amino acid sequences shown in FIGS. 1, 3, 4, 10 to 158, 160A to 160E, 162 to 170, and 172-1 to 172-19. By means of at least 20, 15, 30, 50, 75, 100, 250, 300, 400 or 500 consecutive amino acids. Exemplary desirable fragments are amino acids 1-313 of the sequence of SEQ ID NO: 3, amino acids 314-517 of the sequence of SEQ ID NO: 3, and sequences of SEQ ID NOs: 2 and 63. Also, with respect to fragments or parts of nucleic acid sequences, desirable fragments are those of the nucleic acid sequences shown in 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294. Any of at least 100, 250, 500, 750, 1000 or 1500 consecutive amino acids. Exemplary desirable fragments are nucleic acids 1-2009, 2010-2949, 2950-3946, 3947-4562, 4563-6347 and 4731-6347 of the sequence of SEQ ID NO: 4.

  By “substantially pure polypeptide” is meant a polypeptide that has been separated from most components that naturally accompany it. However, other proteins found in the microsomal fraction associated with preparations having an enzymatic activity of at least 8.3 pKa / mg protein are also considered to be substantially pure polypeptides. Typically, the polypeptide is substantially pure when it is at least 60% by weight free from the proteins and naturally occurring organic molecules that naturally accompany it. Preferably, the preparation contains at least 75%, more preferably at least 90%, most preferably at least 99% by weight of the desired polypeptide. A substantially pure polypeptide can be obtained, for example, by extraction from a natural source (eg, tobacco plant cells), expression of a recombinant nucleic acid encoding the polypeptide, or chemical synthesis of the protein. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

  By “isolated nucleic acid molecule” is meant a nucleic acid sequence that does not contain a nucleic acid sequence that is naturally adjacent to the sequence of the nucleic acid molecule in the genome of an organism.

  A “transformed cell” is a DNA molecule [eg, a DNA molecule encoding tobacco nicotine demethylase or a nucleic acid sequence disclosed herein (eg, 1, 3-7, 10-158, 162- 170, 172-1 to 172-19 and nucleic acid sequences shown in 173-1 to 173-294)] or the ancestor thereof.

As used herein, “tobacco nicotine demethylase” or “nicotine demethylase” refers to a polypeptide that is substantially identical to the sequence of SEQ ID NO: 3. Desirably, tobacco nicotine demethylase can convert nicotine (also referred to as C ₁₀ H ₁₄ N ₂ , 3- (1-methyl-2-pyrrolidinyl) pyridine) to nornicotine (C ₉ H ₁₂ N ₂ ). The activity of tobacco nicotine demethylase can be assayed using standard methods in the art, for example, by measuring the demethylation of radioactive nicotine by yeast-expressing microsomes as described herein. .

  As used herein, “cytochrome p450” and “p450” are used interchangeably.

  Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the nucleic acid sequence of D35-BG11 (SEQ ID NO: 1) and its translation product (SEQ ID NO: 2).
FIG. 2 is a schematic diagram of the genomic structure of the tobacco nicotine demethylase gene.
FIG. 3 is the genomic tobacco nicotine demethylase nucleic acid sequence (SEQ ID NO: 4) and its translation product (SEQ ID NO: 3).
FIG. 4 shows the nucleic acid sequence (SEQ ID NO: 5) of the coding region of the tobacco nicotine demethylase gene and its translation product (SEQ ID NO: 6).
FIG. 5 is the nucleic acid sequence of an intron (SEQ ID NO: 7) present within the tobacco nicotine demethylase genomic sequence.
FIG. 6 is the nucleic acid sequence (SEQ ID NO: 8) of the tobacco demethylase gene promoter.
FIG. 7 is the nucleic acid sequence of the 3′UTR of the tobacco nicotine demethylase gene (SEQ ID NO: 9).
FIG. 8 is an electrophoresis image showing tobacco line PCR products with Geno full length (“FL”) primer set.
FIG. 9 is an electrophoretic image showing the PCR product of the tobacco line with the primer sets (1), (2), (3) and (4) described in Example 17. The approximate size of the band is 3,500 nucleotides (nt) for FL, 2,600 nt for (1), 1,400 nt for (2), 600 nt for (3) and 1,400 nt for (4) .
FIG. 10 shows the nucleic acid sequence of D58-BG7 (SEQ ID NO: 10) and the amino acid sequence of D58-BG7 (SEQ ID NO: 11) (this is a translation of D58-BG7 (SEQ ID NO: 10)).

FIG. 11 shows the nucleic acid sequence of D58-AB1 (SEQ ID NO: 12) and the amino acid sequence of D58-AB1 (SEQ ID NO: 13) (this is a translation of D58-AB1 (SEQ ID NO: 12)).
FIG. 12 shows the nucleic acid sequence of D186-AH4 (SEQ ID NO: 14) and the amino acid sequence of D186-AH4 (SEQ ID NO: 15) (this is a translation of D186-AH4 (SEQ ID NO: 14)).
FIG. 13 shows the nucleic acid sequence of D58-BE4 (SEQ ID NO: 16) and the amino acid sequence of D58-BE4 (SEQ ID NO: 17) (this is a translation of D58-BE4 (SEQ ID NO: 16)).
FIG. 14 shows the nucleic acid sequence of D56-AH7 (SEQ ID NO: 18) and the amino acid sequence of D56-AH7 (SEQ ID NO: 19) (which is a translation of D56-AH7 (SEQ ID NO: 18)).
FIG. 15 shows the nucleic acid sequence of D13a-5 (SEQ ID NO: 20) and the amino acid sequence of D13a-5 (SEQ ID NO: 21) (this is a translation of D13a-5 (SEQ ID NO: 20)).
FIG. 16 shows the nucleic acid sequence of D56-AG10 (SEQ ID NO: 22) and the amino acid sequence of D56-AG10 (SEQ ID NO: 23) (this is a translation of D56-AG10 (SEQ ID NO: 22)).
FIG. 17 shows the nucleic acid sequence of D35-33 (SEQ ID NO: 24) and the amino acid sequence of D35-33 (SEQ ID NO: 25) (this is a translation of D35-33 (SEQ ID NO: 24)).
FIG. 18 shows the nucleic acid sequence of D34-62 (SEQ ID NO: 26) and the amino acid sequence of D34-62 (SEQ ID NO: 27) (this is a translation of D34-62 (SEQ ID NO: 26)).
FIG. 19 shows the nucleic acid sequence of D56-AA7 (SEQ ID NO: 28) and the amino acid sequence of D56-AA7 (SEQ ID NO: 29) (this is a translation of D56-AA7 (SEQ ID NO: 28)).
FIG. 20 shows the nucleic acid sequence of D56-AE1 (SEQ ID NO: 30) and the amino acid sequence of D56-AE1 (SEQ ID NO: 31) (this is a translation of D56-AE1 (SEQ ID NO: 30)).

FIG. 21 shows the nucleic acid sequence of D35-BB7 (SEQ ID NO: 32) and the amino acid sequence of D35-BB7 (SEQ ID NO: 33) (this is a translation of D35-BB7 (SEQ ID NO: 32)).
FIG. 22 shows the nucleic acid sequence of D177-BA7 (SEQ ID NO: 34) and the amino acid sequence of D177-BA7 (SEQ ID NO: 35) (this is a translation of D177-BA7 (SEQ ID NO: 34)).
FIG. 23 shows the nucleic acid sequence of D56A-AB6 (SEQ ID NO: 36) and the amino acid sequence of D56A-AB6 (SEQ ID NO: 37) (this is a translation of D56A-AB6 (SEQ ID NO: 36)).
FIG. 24 shows the nucleic acid sequence of D144-AE2 (SEQ ID NO: 38) and the amino acid sequence of D144-AE2 (SEQ ID NO: 39) (this is a translation of D144-AE2 (SEQ ID NO: 38)).
FIG. 25 shows the nucleic acid sequence of D56-AG11 (SEQ ID NO: 40) and the amino acid sequence of D56-AG11 (SEQ ID NO: 41) (this is a translation of D56-AG11 (SEQ ID NO: 40)).
FIG. 26 shows the nucleic acid sequence of D179-AA1 (SEQ ID NO: 42) and the amino acid sequence of D179-AA1 (SEQ ID NO: 43) (this is a translation of D179-AA1 (SEQ ID NO: 42)).
FIG. 27 shows the nucleic acid sequence of D56-AC7 (SEQ ID NO: 44) and the amino acid sequence of D56-AC7 (SEQ ID NO: 45) (which is a translation of D56-AC7 (SEQ ID NO: 44)).
FIG. 28 shows the nucleic acid sequence of D144-AD1 (SEQ ID NO: 46) and the amino acid sequence of D144-AD1 (SEQ ID NO: 47) (this is a translation of D144-AD1 (SEQ ID NO: 46)).
FIG. 29 shows the nucleic acid sequence of D144-AB5 (SEQ ID NO: 48) and the amino acid sequence of D144-AB5 (SEQ ID NO: 49) (this is a translation of D144-AB5 (SEQ ID NO: 48)).
FIG. 30 shows the nucleic acid sequence of D181-AB5 (SEQ ID NO: 50) and the amino acid sequence of D181-AB5 (SEQ ID NO: 51) (this is a translation of D181-AB5 (SEQ ID NO: 50)).

FIG. 31 shows the nucleic acid sequence of D73-AC9 (SEQ ID NO: 52) and the amino acid sequence of D73-AC9 (SEQ ID NO: 53) (this is a translation of D73-AC9 (SEQ ID NO: 52)).
FIG. 32 shows the nucleic acid sequence of D56-AC12 (SEQ ID NO: 54) and the amino acid sequence of D56-AC12 (SEQ ID NO: 55) (this is a translation of D56-AC12 (SEQ ID NO: 54)).
FIG. 33 shows the nucleic acid sequence of D58-AB9 (SEQ ID NO: 56) and the amino acid sequence of D58-AB9 (SEQ ID NO: 57) (this is a translation of D58-AB9 (SEQ ID NO: 56)).
FIG. 34 shows the nucleic acid sequence of D56-AG9 (SEQ ID NO: 58) and the amino acid sequence of D56-AG9 (SEQ ID NO: 59) (this is a translation of D56-AG9 (SEQ ID NO: 58)).
FIG. 35 shows the nucleic acid sequence of D56-AG6 (SEQ ID NO: 60) and the amino acid sequence of D56-AG6 (SEQ ID NO: 61) (this is a translation of D56-AG6 (SEQ ID NO: 60)).
FIG. 36 shows the nucleic acid sequence of D35-BG11 (SEQ ID NO: 62) and the amino acid sequence of D35-BG11 (SEQ ID NO: 63) (this is a translation of D35-BG11 (SEQ ID NO: 62)).
FIG. 37 shows the nucleic acid sequence of D35-42 (SEQ ID NO: 64) and the amino acid sequence of D35-42 (SEQ ID NO: 65) (which is a translation of D35-42 (SEQ ID NO: 64)).
FIG. 38 shows the nucleic acid sequence of D35-BA3 (SEQ ID NO: 66) and the amino acid sequence of D35-BA3 (SEQ ID NO: 67) (this is a translation of D35-BA3 (SEQ ID NO: 66)).
FIG. 39 shows the nucleic acid sequence of D34-57 (SEQ ID NO: 68) and the amino acid sequence of D34-57 (SEQ ID NO: 69), which is a translation of D34-57 (SEQ ID NO: 68).
FIG. 40 is the nucleic acid sequence of D34-52 (SEQ ID NO: 70) and the amino acid sequence of D34-52 (SEQ ID NO: 71) (this is a translation of D34-52 (SEQ ID NO: 70)).

FIG. 41 shows the nucleic acid sequence of D34-25 (SEQ ID NO: 72) and the amino acid sequence of D34-25 (SEQ ID NO: 73) (which is a translation of D34-25 (SEQ ID NO: 72)).
FIG. 42 shows the nucleic acid sequence of D56AD10 (SEQ ID NO: 74) and the amino acid sequence of D56AD10 (SEQ ID NO: 75) (which is a translation of D56AD10 (SEQ ID NO: 74)).
FIG. 43 shows the nucleic acid sequence of D56-AA11 (SEQ ID NO: 76) and the amino acid sequence of D56-AA11 (SEQ ID NO: 77) (which is a translation of D56-AA11 (SEQ ID NO: 76)).
FIG. 44 shows the nucleic acid sequence of D177-BD5 (SEQ ID NO: 78) and the amino acid sequence of D177-BD5 (SEQ ID NO: 79) (this is a translation of D177-BD5 (SEQ ID NO: 78)).
FIG. 45 shows the nucleic acid sequence of D56A-AG10 (SEQ ID NO: 80) and the amino acid sequence of D56A-AG10 (SEQ ID NO: 81) (this is a translation of D56A-AG10 (SEQ ID NO: 80)).
FIG. 46 shows the nucleic acid sequence of D58-BC5 (SEQ ID NO: 82) and the amino acid sequence of D58-BC5 (SEQ ID NO: 83) (this is a translation of D58-BC5 (SEQ ID NO: 82)).
FIG. 47 shows the nucleic acid sequence of D58-AD12 (SEQ ID NO: 84) and the amino acid sequence of D58-AD12 (SEQ ID NO: 85) (this is a translation of D58-AD12 (SEQ ID NO: 84)).
FIG. 48 shows the nucleic acid sequence of D56-AC11 (SEQ ID NO: 86) and the amino acid sequence of D56-AC11 (SEQ ID NO: 87) (which is a translation of D56-AC11 (SEQ ID NO: 86)).
FIG. 49 shows the nucleic acid sequence of D35-39 (SEQ ID NO: 88) and the amino acid sequence of D35-39 (SEQ ID NO: 89) (which is a translation of D35-39 (SEQ ID NO: 88)).
FIG. 50 is the nucleic acid sequence of D58-BH4 (SEQ ID NO: 90) and the amino acid sequence of D58-BH4 (SEQ ID NO: 91), which is a translation of D58-BH4 (SEQ ID NO: 90).

FIG. 51 shows the nucleic acid sequence of D177-BD7 (SEQ ID NO: 92) and the amino acid sequence of D177-BD7 (SEQ ID NO: 93) (this is a translation of D177-BD7 (SEQ ID NO: 92)).
FIG. 52 shows the nucleic acid sequence of D176-BF2 (SEQ ID NO: 94) and the amino acid sequence of D176-BF2 (SEQ ID NO: 95) (this is a translation of D176-BF2 (SEQ ID NO: 94)).
FIG. 53 shows the nucleic acid sequence of D56-AD6 (SEQ ID NO: 96) and the amino acid sequence of D56-AD6 (SEQ ID NO: 97) (this is a translation of D56-AD6 (SEQ ID NO: 96)).
FIG. 54 shows the nucleic acid sequence of D73A-AD6 (SEQ ID NO: 98) and the amino acid sequence of D73A-AD6 (SEQ ID NO: 99) (this is a translation of D73A-AD6 (SEQ ID NO: 98)).
FIG. 55 shows the nucleic acid sequence of D70A-BA11 (SEQ ID NO: 100) and the amino acid sequence of D70A-BA11 (SEQ ID NO: 101) (this is a translation of D70A-BA11 (SEQ ID NO: 100)).
FIG. 56 shows the nucleic acid sequence of D70A-BB5 (SEQ ID NO: 102) and the amino acid sequence of D70A-BB5 (SEQ ID NO: 103) (this is a translation of D70A-BB5 (SEQ ID NO: 102)).
FIG. 57 shows the nucleic acid sequence of D70A-AB5 (SEQ ID NO: 104) and the amino acid sequence of D70A-AB5 (SEQ ID NO: 105) (this is a translation of D70A-AB5 (SEQ ID NO: 104)).
FIG. 58 shows the nucleic acid sequence of D70A-AA8 (SEQ ID NO: 106) and the amino acid sequence of D70A-AA8 (SEQ ID NO: 107) (which is a translation of D70A-AA8 (SEQ ID NO: 106)).
FIG. 59 shows the nucleic acid sequence of D70A-AB8 (SEQ ID NO: 108) and the amino acid sequence of D70A-AB8 (SEQ ID NO: 109) (this is a translation of D70A-AB8 (SEQ ID NO: 108)).
FIG. 60 shows the nucleic acid sequence of D70A-BH2 (SEQ ID NO: 110) and the amino acid sequence of D70A-BH2 (SEQ ID NO: 111) (this is a translation of D70A-BH2 (SEQ ID NO: 110)).

FIG. 61 shows the nucleic acid sequence of D70A-AA4 (SEQ ID NO: 112) and the amino acid sequence of D70A-AA4 (SEQ ID NO: 113) (which is a translation of D70A-AA4 (SEQ ID NO: 112)).
FIG. 62 shows the nucleic acid sequence of D70A-BA1 (SEQ ID NO: 114) and the amino acid sequence of D70A-BA1 (SEQ ID NO: 115) (this is a translation of D70A-BA1 (SEQ ID NO: 114)).
FIG. 63 shows the nucleic acid sequence of D70A-BA9 (SEQ ID NO: 116) and the amino acid sequence of D70A-BA9 (SEQ ID NO: 117) (this is a translation of D70A-BA9 (SEQ ID NO: 116)).
FIG. 64 shows the nucleic acid sequence of D70A-BD4 (SEQ ID NO: 118) and the amino acid sequence of D70A-BD4 (SEQ ID NO: 119) (this is a translation of D70A-BD4 (SEQ ID NO: 118)).
FIG. 65 shows the nucleic acid sequence of D181-AC5 (SEQ ID NO: 120) and the amino acid sequence of D181-AC5 (SEQ ID NO: 121) (this is a translation of D181-AC5 (SEQ ID NO: 120)).
FIG. 66 shows the nucleic acid sequence of D144-AH1 (SEQ ID NO: 122) and the amino acid sequence of D144-AH1 (SEQ ID NO: 123) (this is a translation of D144-AH1 (SEQ ID NO: 122)).
FIG. 67 shows the nucleic acid sequence of D34-65 (SEQ ID NO: 124) and the amino acid sequence of D34-65 (SEQ ID NO: 125) (which is a translation of D34-65 (SEQ ID NO: 124)).
FIG. 68 shows the nucleic acid sequence of D35-BG2 (SEQ ID NO: 126) and the amino acid sequence of D35-BG2 (SEQ ID NO: 127) (this is a translation of D35-BG2 (SEQ ID NO: 126)).
FIG. 69 shows the nucleic acid sequence of D73A-AH7 (SEQ ID NO: 128) and the amino acid sequence of D73A-AH7 (SEQ ID NO: 129) (this is a translation of D73A-AH7 (SEQ ID NO: 128)).
FIG. 70 shows the nucleic acid sequence of D58-AA1 (SEQ ID NO: 130) and the amino acid sequence of D58-AA1 (SEQ ID NO: 131) (this is a translation of D58-AA1 (SEQ ID NO: 130)).

FIG. 71 shows the nucleic acid sequence of D73A-AE10 (SEQ ID NO: 132) and the amino acid sequence of D73A-AE10 (SEQ ID NO: 133) (this is a translation of D73A-AE10 (SEQ ID NO: 132)).
FIG. 72 is the nucleic acid sequence of D56A-AC12 (SEQ ID NO: 134) and the amino acid sequence of D56A-AC12 (SEQ ID NO: 135) (this is a translation of D56A-AC12 (SEQ ID NO: 134)).
FIG. 73 shows the nucleic acid sequence of D177-BF7 (SEQ ID NO: 136) and the amino acid sequence of D177-BF7 (SEQ ID NO: 137) (this is a translation of D177-BF7 (SEQ ID NO: 136)).
FIG. 74 shows the nucleic acid sequence of D73A-AG3 (SEQ ID NO: 138) and the amino acid sequence of D73A-AG3 (SEQ ID NO: 139) (which is a translation of D73A-AG3 (SEQ ID NO: 138)).
FIG. 75 shows the nucleic acid sequence of D70A-AA12 (SEQ ID NO: 140) and the amino acid sequence of D70A-AA12 (SEQ ID NO: 141) (this is a translation of D70A-AA12 (SEQ ID NO: 140)).
FIG. 76 shows the nucleic acid sequence of D185-BC1 (SEQ ID NO: 142) and the amino acid sequence of D185-BC1 (SEQ ID NO: 143) (this is a translation of D185-BC1 (SEQ ID NO: 142)).
FIG. 77 shows the nucleic acid sequence of D185-BG2 (SEQ ID NO: 144) and the amino acid sequence of D185-BG2 (SEQ ID NO: 145) (this is a translation of D185-BG2 (SEQ ID NO: 144)).
FIG. 78 shows the nucleic acid sequence of D185-BE1 (SEQ ID NO: 146) and the amino acid sequence of D185-BE1 (SEQ ID NO: 147) (which is a translation of D185-BE1 (SEQ ID NO: 146)).
FIG. 79 shows the nucleic acid sequence of D185-BD2 (SEQ ID NO: 148) and the amino acid sequence of D185-BD2 (SEQ ID NO: 149) (this is a translation of D185-BD2 (SEQ ID NO: 148)).
FIG. 80 shows the nucleic acid sequence of D176-BG2 (SEQ ID NO: 150) and the amino acid sequence of D176-BG2 (SEQ ID NO: 151) (this is a translation of D176-BG2 (SEQ ID NO: 150)).

FIG. 81 shows the nucleic acid sequence of D185-BD3 (SEQ ID NO: 152) and the amino acid sequence of D185-BD3 (SEQ ID NO: 153) (this is a translation of D185-BD3 (SEQ ID NO: 152)).
FIG. 82 is the nucleic acid sequence of D176-BC3 (SEQ ID NO: 154) and the amino acid sequence of D176-BC3 (SEQ ID NO: 155), which is a translation of D176-BC3 (SEQ ID NO: 154).
FIG. 83 shows the nucleic acid sequence of D176-BB3 (SEQ ID NO: 156) and the amino acid sequence of D176-BB3 (SEQ ID NO: 157) (this is a translation of D176-BB3 (SEQ ID NO: 156)).
FIG. 84 shows the nucleic acid sequence of D89-AB1 (SEQ ID NO: 158) and the amino acid sequence of D89-AB1 (SEQ ID NO: 159) (this is a translation of D89-AB1 (SEQ ID NO: 158)).
FIG. 85 shows the nucleic acid sequence of D89-AD2 (SEQ ID NO: 160) and the amino acid sequence of D89-AD2 (SEQ ID NO: 161) (this is a translation of D89-AD2 (SEQ ID NO: 160)).
FIG. 86 shows the nucleic acid sequence of D90A-BB3 (SEQ ID NO: 162) and the amino acid sequence of D90A-BB3 (SEQ ID NO: 163) (which is a translation of D90A-BB3 (SEQ ID NO: 162)).
FIG. 87 shows the nucleic acid sequence of D95-AG1 (SEQ ID NO: 164) and the amino acid sequence of D95-AG1 (SEQ ID NO: 165) (which is a translation of D95-AG1 (SEQ ID NO: 164)).
FIG. 88 is the nucleic acid sequence of D96-AB6 (SEQ ID NO: 166) and the amino acid sequence of D96-AB6 (SEQ ID NO: 167) (this is a translation of D96-AB6 (SEQ ID NO: 166)).
FIG. 89 is the nucleic acid sequence of D96-AC2 (SEQ ID NO: 168) and the amino acid sequence of D96-AC2 (SEQ ID NO: 169) (this is a translation of D96-AC2 (SEQ ID NO: 168)).
FIG. 90 shows the nucleic acid sequence of D98-AA1 (SEQ ID NO: 170) and the amino acid sequence of D98-AA1 (SEQ ID NO: 171) (this is a translation of D98-AA1 (SEQ ID NO: 170)).

FIG. 91 shows the nucleic acid sequence of D98-AG1 (SEQ ID NO: 172) and the amino acid sequence of D98-AG1 (SEQ ID NO: 173) (this is a translation of D98-AG1 (SEQ ID NO: 172)).
FIG. 92 shows the nucleic acid sequence of D100-BE2 (SEQ ID NO: 174) and the amino acid sequence of D100-BE2 (SEQ ID NO: 175) (this is a translation of D100-BE2 (SEQ ID NO: 174)).
FIG. 93 shows the nucleic acid sequence of D100A-AC3 (SEQ ID NO: 176) and the amino acid sequence of D100A-AC3 (SEQ ID NO: 177) (this is a translation of D100A-AC3 (SEQ ID NO: 176)).
FIG. 94 shows the nucleic acid sequence of D104A-AE8 (69,1755) (SEQ ID NO: 178) and the amino acid sequence of D104A-AE8 (69,1755) (SEQ ID NO: 179) (this is D104A-AE8 (69,1755) (sequence No. 178).
FIG. 95 shows the nucleic acid sequence of D105-AD6 (SEQ ID NO: 180) and the amino acid sequence of D105-AD6 (SEQ ID NO: 181) (this is a translation of D105-AD6 (SEQ ID NO: 180)).
FIG. 96 shows the nucleic acid sequence of D109-AH8 (14, 1697) (SEQ ID NO: 182) and the amino acid sequence of D109-AH8 (14, 1697) (SEQ ID NO: 183) (this is D109-AH8 (14, 1697) (sequence No. 182).
FIG. 97 shows the nucleic acid sequence of D110-AF12 (166, 1631) (SEQ ID NO: 184) and the amino acid sequence of D110-AF12 (166, 1631) (SEQ ID NO: 185) (this is D110-AF12 (166, 1631) (sequence No. 184).
FIG. 98 shows the nucleic acid sequence of D112-AA5 (SEQ ID NO: 186) and the amino acid sequence of D112-AA5 (SEQ ID NO: 187) (this is a translation of D112-AA5 (SEQ ID NO: 186)).
FIG. 99 shows the nucleic acid sequence of D120-AH4 (SEQ ID NO: 188) and the amino acid sequence of D120-AH4 (SEQ ID NO: 189), which is a translation of D120-AH4 (SEQ ID NO: 188).
FIG. 100 shows the nucleic acid sequence of D121-AA8 (SEQ ID NO: 190) and the amino acid sequence of D121-AA8 (SEQ ID NO: 191) (this is a translation of D121-AA8 (SEQ ID NO: 190)).

FIG. 101 shows the nucleic acid sequence of D122-AF10 (SEQ ID NO: 192) and the amino acid sequence of D122-AF10 (SEQ ID NO: 193) (this is a translation of D122-AF10 (SEQ ID NO: 192)).
FIG. 102 shows the nucleic acid sequence of D128-AB7 (SEQ ID NO: 194) and the amino acid sequence of D128-AB7 (SEQ ID NO: 195) (this is a translation of D128-AB7 (SEQ ID NO: 194)).
FIG. 103 shows the nucleic acid sequence of D129-AD10 (SEQ ID NO: 196) and the amino acid sequence of D129-AD10 (SEQ ID NO: 197) (this is a translation of D129-AD10 (SEQ ID NO: 196)).
FIG. 104 shows the nucleic acid sequence of D135-AE1 (SEQ ID NO: 198) and the amino acid sequence of D135-AE1 (SEQ ID NO: 199) (this is a translation of D135-AE1 (SEQ ID NO: 198)).
FIG. 105 shows the nucleic acid sequence of D141-AD7 (SEQ ID NO: 200) and the amino acid sequence of D141-AD7 (SEQ ID NO: 201) (this is a translation of D141-AD7 (SEQ ID NO: 200)).
FIG. 106 shows the nucleic acid sequence of D147-AD3 (SEQ ID NO: 202) and the amino acid sequence of D147-AD3 (SEQ ID NO: 203) (this is a translation of D147-AD3 (SEQ ID NO: 202)).
FIG. 107 shows the nucleic acid sequence of D163-AF12 (SEQ ID NO: 204) and the amino acid sequence of D163-AF12 (SEQ ID NO: 205) (this is a translation of D163-AF12 (SEQ ID NO: 204)).
FIG. 108 shows the nucleic acid sequence of D163-AG11 (SEQ ID NO: 206) and the amino acid sequence of D163-AG11 (SEQ ID NO: 207) (this is a translation of D163-AG11 (SEQ ID NO: 206)).
FIG. 109 shows the nucleic acid sequence of D163-AG12 (SEQ ID NO: 208) and the amino acid sequence of D163-AG12 (SEQ ID NO: 209) (this is a translation of D163-AG12 (SEQ ID NO: 208)).
FIG. 110 shows the nucleic acid sequence of D205-BG9 (SEQ ID NO: 210) and the amino acid sequence of D205-BG9 (SEQ ID NO: 211) (this is a translation of D205-BG9 (SEQ ID NO: 210)).

FIG. 111 shows the nucleic acid sequence of D207-AA5 (SEQ ID NO: 212) and the amino acid sequence of D207-AA5 (SEQ ID NO: 213) (this is a translation of D207-AA5 (SEQ ID NO: 212)).
FIG. 112 shows the nucleic acid sequence of D207-AB4 (SEQ ID NO: 214) and the amino acid sequence of D207-AB4 (SEQ ID NO: 215) (this is a translation of D207-AB4 (SEQ ID NO: 214)).
FIG. 113 shows the nucleic acid sequence of D207-AC4 (SEQ ID NO: 216) and the amino acid sequence of D207-AC4 (SEQ ID NO: 217) (this is a translation of D207-AC4 (SEQ ID NO: 216)).
FIG. 114 shows the nucleic acid sequence of D209-AA10 (SEQ ID NO: 218) and the amino acid sequence of D209-AA10 (SEQ ID NO: 219), which is a translation of D209-AA10 (SEQ ID NO: 218).
FIG. 115 shows the nucleic acid sequence of D209-AA12 (SEQ ID NO: 220) and the amino acid sequence of D209-AA12 (SEQ ID NO: 221) (which is a translation of D209-AA12 (SEQ ID NO: 220)).
FIG. 116 shows the nucleic acid sequence of D209-AH10 (SEQ ID NO: 222) and the amino acid sequence of D209-AH10 (SEQ ID NO: 223) (which is a translation of D209-AH10 (SEQ ID NO: 222)).
FIG. 117 shows the nucleic acid sequence of D87A-AF3 (SEQ ID NO: 224) and the amino acid sequence of D87A-AF3 (SEQ ID NO: 225) (which is a translation of D87A-AF3 (SEQ ID NO: 224)).
FIG. 118 is the nucleic acid sequence of D208-AC8 (SEQ ID NO: 226) and the amino acid sequence of D208-AC8 (SEQ ID NO: 227) (this is a translation of D208-AC8 (SEQ ID NO: 226)).
FIG. 119 shows the nucleic acid sequence of D215-AB5 (SEQ ID NO: 228) and the amino acid sequence of D215-AB5 (SEQ ID NO: 229) (this is a translation of D215-AB5 (SEQ ID NO: 228)).
FIG. 120 shows the nucleic acid sequence of D103-AH3 (SEQ ID NO: 230) and the amino acid sequence of D103-AH3 (SEQ ID NO: 231) (which is a translation of D103-AH3 (SEQ ID NO: 230)).

FIG. 121 shows the nucleic acid sequence of D208-AD9 (SEQ ID NO: 232) and the amino acid sequence of D208-AD9 (SEQ ID NO: 233) (which is a translation of D208-AD9 (SEQ ID NO: 232)).
FIG. 122 shows the nucleic acid sequence of D237-AD1 (SEQ ID NO: 234) and the amino acid sequence of D237-AD1 (SEQ ID NO: 235) (this is a translation of D237-AD1 (SEQ ID NO: 234)).
FIG. 123 shows the nucleic acid sequence of D125-AF11 (SEQ ID NO: 236) and the amino acid sequence of D125-AF11 (SEQ ID NO: 237) (this is a translation of D125-AF11 (SEQ ID NO: 236)).
FIG. 124 shows the nucleic acid sequence of D134-AE11 (SEQ ID NO: 238) and the amino acid sequence of D134-AE11 (SEQ ID NO: 239) (this is a translation of D134-AE11 (SEQ ID NO: 238)).
FIG. 125 shows the nucleic acid sequence of D209-AH12 (SEQ ID NO: 240) and the amino acid sequence of D209-AH12 (SEQ ID NO: 241), which is a translation of D209-AH12 (SEQ ID NO: 240).
FIG. 126 shows the nucleic acid sequence of D221-BB8 (SEQ ID NO: 242) and the amino acid sequence of D221-BB8 (SEQ ID NO: 243) (which is a translation of D221-BB8 (SEQ ID NO: 242)).
FIG. 127 shows the nucleic acid sequence of D222-BH4 (SEQ ID NO: 244) and the amino acid sequence of D222-BH4 (SEQ ID NO: 245) (this is a translation of D222-BH4 (SEQ ID NO: 244)).
FIG. 128 shows the nucleic acid sequence of D224-AF10 (SEQ ID NO: 246) and the amino acid sequence of D224-AF10 (SEQ ID NO: 247) (this is a translation of D224-AF10 (SEQ ID NO: 246)).
FIG. 129 shows the nucleic acid sequence of D224-BD11 (SEQ ID NO: 248) and the amino acid sequence of D224-BD11 (SEQ ID NO: 249) (this is a translation of D224-BD11 (SEQ ID NO: 248)).
FIG. 130 shows the nucleic acid sequence of D228-AD7 (SEQ ID NO: 250) and the amino acid sequence of D228-AD7 (SEQ ID NO: 251) (this is a translation of D228-AD7 (SEQ ID NO: 250)).

FIG. 131 shows the nucleic acid sequence of D228-AH8 (SEQ ID NO: 252) and the amino acid sequence of D228-AH8 (SEQ ID NO: 253) (which is a translation of D228-AH8 (SEQ ID NO: 252)).
FIG. 132 shows the nucleic acid sequence of D235-AB1 (SEQ ID NO: 254) and the amino acid sequence of D235-AB1 (SEQ ID NO: 255) (this is a translation of D235-AB1 (SEQ ID NO: 254)).
FIG. 133 shows the nucleic acid sequence of D243-AA2 (SEQ ID NO: 256) and the amino acid sequence of D243-AA2 (SEQ ID NO: 257) (this is a translation of D243-AA2 (SEQ ID NO: 256)).
FIG. 134 shows the nucleic acid sequence of D244-AD4 (SEQ ID NO: 258) and the amino acid sequence of D244-AD4 (SEQ ID NO: 259) (this is a translation of D244-AD4 (SEQ ID NO: 258)).
FIG. 135 shows the nucleic acid sequence of D247-AH1 (SEQ ID NO: 260) and the amino acid sequence of D247-AH1 (SEQ ID NO: 261) (which is a translation of D247-AH1 (SEQ ID NO: 260)).
FIG. 136 shows the nucleic acid sequence of D248-AA6 (SEQ ID NO: 262) and the amino acid sequence of D248-AA6 (SEQ ID NO: 263) (this is a translation of D248-AA6 (SEQ ID NO: 262)).
FIG. 137 shows the nucleic acid sequence of D249-AE8 (SEQ ID NO: 264) and the amino acid sequence of D249-AE8 (SEQ ID NO: 265) (which is a translation of D249-AE8 (SEQ ID NO: 264)).
Figure 138 is the nucleic acid sequence of D250-AC11 (SEQ ID NO: 266) and amino acid sequence of D250-AC11 (SEQ ID NO: 267 and 2298) (These are the translation of D250-AC11 (SEQ ID NO: 266)).
FIG. 139 shows the nucleic acid sequence of D259-AB9 (SEQ ID NO: 268) and the amino acid sequence of D259-AB9 (SEQ ID NO: 269) (this is a translation of D259-AB9 (SEQ ID NO: 268)).
FIG. 140 shows the nucleic acid sequence of D218A-AC2 (SEQ ID NO: 270) and the amino acid sequence of D218A-AC2 (SEQ ID NO: 271) (which is a translation of D218A-AC2 (SEQ ID NO: 270)).

FIG. 141 shows the nucleic acid sequence of D210-BD4 (SEQ ID NO: 272) and the amino acid sequence of D210-BD4 (SEQ ID NO: 273) (this is a translation of D210-BD4 (SEQ ID NO: 272)).
FIG. 142 shows the nucleic acid sequence of D233-AG7 (SEQ ID NO: 274) and the amino acid sequence of D233-AG7 (SEQ ID NO: 275) (this is a translation of D233-AG7 (SEQ ID NO: 274)).
FIG. 143 shows the nucleic acid sequence of D257-AE4 (SEQ ID NO: 276) and the amino acid sequence of D257-AE4 (SEQ ID NO: 277) (which is a translation of D257-AE4 (SEQ ID NO: 276)).
FIG. 144 shows the nucleic acid sequence of D268-AE2 (SEQ ID NO: 278) and the amino acid sequence of D268-AE2 (SEQ ID NO: 279) (which is a translation of D268-AE2 (SEQ ID NO: 278)).
FIG. 145 shows the nucleic acid sequence of D283-AC1 (SEQ ID NO: 280) and the amino acid sequence of D283-AC1 (SEQ ID NO: 281) (which is a translation of D283-AC1 (SEQ ID NO: 280)).
FIG. 146 shows the nucleic acid sequence of D244-AB6 (SEQ ID NO: 282) and the amino acid sequence of D244-AB6 (SEQ ID NO: 283) (which is a translation of D244-AB6 (SEQ ID NO: 282)).
FIG. 147 shows the nucleic acid sequence of D205-BE9 (SEQ ID NO: 284) and the amino acid sequence of D205-BE9 (SEQ ID NO: 285) (this is a translation of D205-BE9 (SEQ ID NO: 284)).
FIG. 148 shows the nucleic acid sequence of D136-AF4 (SEQ ID NO: 286) and the amino acid sequence of D136-AF4 (SEQ ID NO: 287) (this is a translation of D136-AF4 (SEQ ID NO: 286)).
FIG. 149 shows the nucleic acid sequence of D101-BA2 (SEQ ID NO: 288) and the amino acid sequence of D101-BA2 (SEQ ID NO: 289) (this is a translation of D101-BA2 (SEQ ID NO: 288)).
FIG. 150 shows the nucleic acid sequence of D130-AA1 (SEQ ID NO: 290) and the amino acid sequence of D130-AA1 (SEQ ID NO: 291) (this is a translation of D130-AA1 (SEQ ID NO: 290)).

FIG. 151 shows the nucleic acid sequence of D136-AD5 (SEQ ID NO: 292) and the amino acid sequence of D136-AD5 (SEQ ID NO: 293) (this is a translation of D136-AD5 (SEQ ID NO: 292)).
FIG. 152 shows the nucleic acid sequence of D138-AD12 (SEQ ID NO: 294) and the amino acid sequence of D138-AD12 (SEQ ID NO: 295) (this is a translation of D138-AD12 (SEQ ID NO: 294)).
FIG. 153 shows the nucleic acid sequence of D216-AG8 (SEQ ID NO: 296) and the amino acid sequence of D216-AG8 (SEQ ID NO: 297) (which is a translation of D216-AG8 (SEQ ID NO: 296)).
FIG. 154 shows the nucleic acid sequence of D243-AB3 (SEQ ID NO: 298) and the amino acid sequence of D243-AB3 (SEQ ID NO: 299) (which is a translation of D243-AB3 (SEQ ID NO: 298)).
Figure 155 is the nucleic acid sequence of D250-AC11 (SEQ ID NO: 300) and D250-AC11 (SEQ ID NO: 301 and 2298) (These are the translation of D250-AC11 (SEQ ID NO: 300)).
FIG. 156 shows the nucleic acid sequence of D205-AH4 (SEQ ID NO: 302) and the amino acid sequence of D205-AH4 (SEQ ID NO: 303) (this is a translation of D205-AH4 (SEQ ID NO: 302)).
FIG. 157 shows the nucleic acid sequence of D267-AF10 (SEQ ID NO: 304) and the amino acid sequence of D267-AF10 (SEQ ID NO: 305) (this is a translation of D267-AF10 (SEQ ID NO: 304)).
FIG. 158 shows the nucleic acid sequence of D284-AH5 (SEQ ID NO: 306) and the amino acid sequence of D284-AH5 (SEQ ID NO: 307) (which is a translation of D284-AH5 (SEQ ID NO: 306)).
Figure 159A shows the alignment of D58-BG7 (SEQ ID NO: 10), D58-AB1 (SEQ ID NO: 12) and D58-BE4 (SEQ ID NO: 16) and D56-AH7 (SEQ ID NO: 18) and D13a-5 (SEQ ID NO: 20). A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment.
Figure 159B shows the alignment of D56-AG10 (SEQ ID NO: 22), D35-33 (SEQ ID NO: 24) and D34-62 (SEQ ID NO: 26) and D56-AA7 (SEQ ID NO: 28), D56-AE1 (SEQ ID NO: 30). And a nucleic acid sequence alignment set consisting of an alignment of D185-BD3 (SEQ ID NO: 152). The percent identity between the sequences in each alignment is shown under each alignment.
Figure 159C shows alignment of D56A-AB6 (SEQ ID NO: 36), D35-BB7 (SEQ ID NO: 32), D177-BA7 (SEQ ID NO: 34) and D144-AE2 (SEQ ID NO: 38) and D56-AG11 (SEQ ID NO: 40). And a nucleic acid sequence alignment set consisting of an alignment of D179-AA1 (SEQ ID NO: 42). The percent identity between the sequences in each alignment is shown under each alignment.
FIG. 159D shows a set of nucleic acid sequence alignments consisting of an alignment of D56-AC7 (SEQ ID NO: 44) and D144-AD1 (SEQ ID NO: 46) and an alignment of D181-AB5 (SEQ ID NO: 50) and D73-AC9 (SEQ ID NO: 52). It is. The percent identity between the sequences in each alignment is shown under each alignment.
Figure 159E shows the nucleic acid sequences D58-AB9 (SEQ ID NO: 56), D56-AG9 (SEQ ID NO: 58), D35-BG11 (SEQ ID NO: 62), D34-25 (SEQ ID NO: 72), D35-BA3 (SEQ ID NO: 66) , D34-52 (SEQ ID NO: 70), D56-AG6 (SEQ ID NO: 60), D35-42 (SEQ ID NO: 64) and D34-57 (SEQ ID NO: 68). The identity (%) between the sequences of the alignment is shown below the alignment.
FIG. 159F shows an alignment of D177-BD7 (SEQ ID NO: 92) and D177-BD5 (SEQ ID NO: 78) and D56A-AG10 (SEQ ID NO: 80), D58-AD12 (SEQ ID NO: 84) and D58-BC5 (SEQ ID NO: 82). A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment.
FIG.159G shows alignment of D56-AD6 (SEQ ID NO: 96), D56-AC11 (SEQ ID NO: 86), D35-39 (SEQ ID NO: 88) and D58-BH4 (SEQ ID NO: 90) and D73A-AD6 (SEQ ID NO: 98) And a set of nucleic acid sequence alignments consisting of an alignment of D70A-BA11 (SEQ ID NO: 100). The percent identity between the sequences in each alignment is shown under each alignment.
FIG.159H shows alignment of D70A-AB5 (SEQ ID NO: 104) and D70A-AA8 (SEQ ID NO: 106) and D70A-AB8 (SEQ ID NO: 108), D70A-BH2 (SEQ ID NO: 110), D70A-AA4 (SEQ ID NO: 112) A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment.
FIG.159I shows an alignment of D70A-BA1 (SEQ ID NO: 114) and D70A-BA9 (SEQ ID NO: 116) and D144-AH1 (SEQ ID NO: 122), D34-65 (SEQ ID NO: 124) and D181-AC5 (SEQ ID NO: 120) A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment.
FIG. 159J shows the alignment of D58-AA1 (SEQ ID NO: 130), D185-BC1 (SEQ ID NO: 142) and D185-BG2 (SEQ ID NO: 144) and D177-BF7 (SEQ ID NO: 136), D185-BD2 (SEQ ID NO: 148). And a nucleic acid sequence alignment set consisting of an alignment of D185-BE1 (SEQ ID NO: 146). The percent identity between the sequences in each alignment is shown under each alignment.
FIG. 159K is an alignment of nucleic acid sequences D70-AA12 (SEQ ID NO: 140) and D176-BF2 (SEQ ID NO: 94). The identity (%) between the sequences of the alignment is shown below the alignment.
Figure 160A shows D208-AD9 (SEQ ID NO: 2196), D120-AH4 (SEQ ID NO: 2197), D121-AA8 (SEQ ID NO: 2198), D122-AF10 (SEQ ID NO: 2199), D103-AH3 (SEQ ID NO: 2200), D208 -Sequence alignment of AC8 (SEQ ID NO: 2201) and D235-AB1 (SEQ ID NO: 2202); D244-AD4 (SEQ ID NO: 2203), D244-AB6 (SEQ ID NO: 2204), D285-AA8 (SEQ ID NO: 2205), D285-AB9 A set of amino acid sequence alignments consisting of a sequence alignment of (SEQ ID NO: 2206) and D268-AE2 (SEQ ID NO: 2207); and a sequence alignment of D100A-AC3 (SEQ ID NO: 2208) and D100A-BE2 (SEQ ID NO: 2209).
Figure 160B shows the sequence alignment of D205-BG9 (SEQ ID NO: 2210), D205-BE9 (SEQ ID NO: 2211) and D205-AH4 (SEQ ID NO: 2212); D259-AB9 (SEQ ID NO: 2213), D257-AE4 (SEQ ID NO: 2214) ) And D147-AD3 (SEQ ID NO: 2215); D249-AEB (SEQ ID NO: 2216) and D248-AA6 (SEQ ID NO: 2217); D233-AG7 (SEQ ID NO: 2218), D224-BD11 (SEQ ID NO: 2219) and D224-AF10 (SEQ ID NO: 2220); and amino acid sequence alignment consisting of D105-AD6 (SEQ ID NO: 2221), D215-AB5 (SEQ ID NO: 2222) and D135-AE1 (SEQ ID NO: 2223) It is a set of.
Figure 160C shows the sequence alignment of D87A-AF3 (SEQ ID NO: 2224) and D210-BD4 (SEQ ID NO: 2225); D89-AB1 (SEQ ID NO: 2226), D89-AD2 (SEQ ID NO: 2227), D163-AG12 (SEQ ID NO: 2228) ), D163-AG11 (SEQ ID NO: 2229) and D163-AF12 (SEQ ID NO: 2230) sequence alignment; D267-AF10 (SEQ ID NO: 2231), D96-AC2 (SEQ ID NO: 2232), D96-AB6 (SEQ ID NO: 2233), D207-AA5 (SEQ ID NO: 2234), D207-AB4 (SEQ ID NO: 2235) and D207-AC4 (SEQ ID NO: 2236); and D98-AG1 (SEQ ID NO: 2237) and D98-AA1 (SEQ ID NO: 2238) A set of amino acid sequence alignments.
Figure 160D shows the sequence of D209-AA10 (SEQ ID NO: 2239), D209-AA12 (SEQ ID NO: 2240), D209-AH10 (SEQ ID NO: 2241), D209-AH12 (SEQ ID NO: 2242) and D90a-BB3 (SEQ ID NO: 2243). Alignment; Sequence alignment of D129-AD10 (SEQ ID NO: 2244) and D104A-AE8 (SEQ ID NO: 2245); D228-AH8 (SEQ ID NO: 2246), D228-AD7 (SEQ ID NO: 2247), D250-AC11 (SEQ ID NO: 2248) and A set of amino acid sequence alignments consisting of sequence alignment of D247-AH1 (SEQ ID NO: 2249); and D128-AB7 (SEQ ID NO: 2250), D243-AA2 (SEQ ID NO: 2251) and D125-AF11 (SEQ ID NO: 2252) is there.
FIG. 160E is an amino acid sequence alignment of D284-AH5 (SEQ ID NO: 2253) and D110-AF12 (SEQ ID NO: 2254).

FIG. 161 is a schematic diagram showing cloning of a cytochrome p450 cDNA fragment by PCR. The primers used for the cloning are as follows: DM (SEQ ID NO: 2255), DM4 (SEQ ID NO: 2256), DM12 (SEQ ID NO: 2257), DM13 (SEQ ID NO: 2258), DM17 (SEQ ID NO: 2259), OLIGO d (T) (SEQ ID NO: 2260), T7 (SEQ ID NO: 2261) and SP6 (SEQ ID NO: 2262).
FIG. 162 shows the nucleic acid sequence of D425-AB10 (SEQ ID NO: 367) and the amino acid sequence of D425-AB10 (SEQ ID NO: 368) (this is a translation of D425-AB10 (SEQ ID NO: 367)).
FIG. 163 shows the nucleic acid sequence of D425-AB11 (SEQ ID NO: 369) and the amino acid sequence of D425-AB11 (SEQ ID NO: 370) (this is a translation of D425-AB11 (SEQ ID NO: 369)).
FIG. 164 shows the nucleic acid sequence of D425-AC9 (SEQ ID NO: 371) and the amino acid sequence of D425-AC9 (SEQ ID NO: 372) (this is a translation of D425-AC9 (SEQ ID NO: 371)).
FIG. 165 shows the nucleic acid sequence of D425-AC10 (SEQ ID NO: 373) and the amino acid sequence of D425-AC10 (SEQ ID NO: 374) (this is a translation of D425-AC10 (SEQ ID NO: 373)).
FIG. 166 shows the nucleic acid sequence of D425-AC11 (SEQ ID NO: 375) and the amino acid sequence of D425-AC11 (SEQ ID NO: 376), which is a translation of D425-AC11 (SEQ ID NO: 375).
FIG. 167 shows the nucleic acid sequence of D425-AG11 (SEQ ID NO: 377) and the amino acid sequence of D425-AG11 (SEQ ID NO: 378) (this is a translation of D425-AG11 (SEQ ID NO: 377)).
FIG. 168 shows the nucleic acid sequence of D425-AH7 (SEQ ID NO: 379) and the amino acid sequence of D425-AH7 (SEQ ID NO: 380) (this is a translation of D425-AH7 (SEQ ID NO: 379)).
FIG. 169 shows the nucleic acid sequence of D425-AH11 (SEQ ID NO: 381) and the amino acid sequence of D425-AH11 (SEQ ID NO: 382), which is a translation of D425-AH11 (SEQ ID NO: 381).
FIG. 170 shows the nucleic acid sequence of D427-AA5 (SEQ ID NO: 383) and the amino acid sequence of D427-AA5 (SEQ ID NO: 384) (this is a translation of D427-AA5 (SEQ ID NO: 383)).

FIG. 171 shows the probe set sequences (SEQ ID NO: 385 to SEQ ID NO: 445) of the clones on the GeneChip® microarray.
Figure 172-1 shows the nucleic acid sequence of D424-AA4 (SEQ ID NO: 446); the nucleic acid sequence of D424-AF5 (SEQ ID NO: 447) and the amino acid sequence of D424-AF5 (SEQ ID NO: 448) (this is D424-AF5 (SEQ ID NO: 448) 447)); D425-AA11 (SEQ ID NO: 449) nucleic acid sequence and D425-AA11 (SEQ ID NO: 450) And 2299 ) Amino acid sequence (this Et Is a translation of D425-AA11 (SEQ ID NO: 449)); and a nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of D425-AF11 (SEQ ID NO: 451).
FIG. 172-2 shows the amino acid sequence of D425-AF11 (SEQ ID NO: 452) (which is a translation of D425-AF11 (SEQ ID NO: 451)); the nucleic acid sequence of D425-AH10 (SEQ ID NO: 453) and D425-AH10 (SEQ ID NO: 454) amino acid sequence (this is a translation of D425-AH10 (SEQ ID NO: 453)); D426-AA3 (SEQ ID NO: 455) nucleic acid sequence and D426-AA3 (SEQ ID NO: 456) amino acid sequence ( This is a translation of D426-AA3 (SEQ ID NO: 455)); and the nucleic acid sequence of D426-AG1 (SEQ ID NO: 457) and the amino acid sequence of D426-AG1 (SEQ ID NO: 458) (this is D426-AG1 (SEQ ID NO: 458) 457) is a nucleic acid and amino acid sequence group.
Figure 172-3 shows the nucleic acid sequence of D427-AA6 (SEQ ID NO: 459) and the amino acid sequence of D427-AA6 (SEQ ID NO: 460) (this is a translation of D427-AA6 (SEQ ID NO: 459)); D427-AB6 (SEQ ID NO: 461) and D427-AB6 (SEQ ID NO: 462) amino acid sequence (which is a translation of D427-AB6 (SEQ ID NO: 461)); D428-AC9 (SEQ ID NO: 463) nucleic acid sequence and A D428-AC9 (SEQ ID NO: 464) amino acid sequence (which is a translation of D428-AC9 (SEQ ID NO: 463)); and a D428-AH10 (SEQ ID NO: 465) nucleic acid and amino acid sequence group. .
Figure 172-4 shows the amino acid sequence of D428-AH10 (SEQ ID NO: 466) (which is a translation of D428-AH10 (SEQ ID NO: 465)); the nucleic acid sequence of D429-AA1 (SEQ ID NO: 467) and D429-AA1 (SEQ ID NO: 468) amino acid sequence (this is a translation of D429-AA1 (SEQ ID NO: 467)); D430-AA3 (SEQ ID NO: 469) nucleic acid sequence and D430-AA3 (SEQ ID NO: 470) amino acid sequence ( This is a translation of D430-AA3 (SEQ ID NO: 469)); and the nucleic acid sequence of D431-AE6 (SEQ ID NO: 471) and the amino acid sequence of D431-AE6 (SEQ ID NO: 472) (this is D431-AE6 (SEQ ID NO: 472)) 471) is a nucleic acid and amino acid sequence group.
FIG. 172-5 shows the nucleic acid sequence of D113-AE9 (SEQ ID NO: 473) and the amino acid sequence of D113-AE9 (SEQ ID NO: 474) (this is a translation of D113-AE9 (SEQ ID NO: 473)); and D114- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AE12 (SEQ ID NO: 475).
Figure 172-6 shows the amino acid sequence of D114-AE12 (SEQ ID NO: 476) (which is a translation of D114-AE12 (SEQ ID NO: 475)); the nucleic acid sequence of D119-AC3 (SEQ ID NO: 477) and D119-AC3 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of (SEQ ID NO: 478) (this is a translation of D119-AC3 (SEQ ID NO: 477)); and the nucleic acid sequence of D132-AA5 (SEQ ID NO: 479).
Figure 172-7 shows the amino acid sequence of D132-AA5 (SEQ ID NO: 480) (which is a translation of D132-AA5 (SEQ ID NO: 479)); the nucleic acid sequence of D223-BB10 (SEQ ID NO: 481) and D223-BB10 (SEQ ID NO: 482) amino acid sequence (which is a translation of D223-BB10 (SEQ ID NO: 481)); and D245-AA8 (SEQ ID NO: 483) nucleic acid sequence and D245-AA8 (SEQ ID NO: 484) amino acid sequence (This is a nucleic acid and amino acid sequence group consisting of D245-AA8 (SEQ ID NO: 483)).
FIG. 172-8 shows the nucleic acid sequence of D246-AE12 (SEQ ID NO: 485) and the amino acid sequence of D246-AE12 (SEQ ID NO: 486) (which is a translation of D246-AE12 (SEQ ID NO: 485)); and D279- A nucleic acid and amino acid sequence group consisting of a nucleic acid sequence of AD1 (SEQ ID NO: 487) and an amino acid sequence of D279-AD1 (SEQ ID NO: 488) (which is a translation of D279-AD1 (SEQ ID NO: 487)).
Figure 172-9 shows the nucleic acid sequence of D282-AA10 (SEQ ID NO: 489) and the amino acid sequence of D282-AA10 (SEQ ID NO: 490), which is a translation of D282-AA10 (SEQ ID NO: 489); and D295- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AA1 (SEQ ID NO: 491).
FIG. 172-10 shows the amino acid sequence of D295-AA1 (SEQ ID NO: 492), which is a translation of D295-AA1 (SEQ ID NO: 491); the nucleic acid sequence of D101A-AE2 (SEQ ID NO: 493) and D101A-AE2 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of (SEQ ID NO: 494) (this is a translation of D101A-AE2 (SEQ ID NO: 493)); and the nucleic acid sequence of D108-AA4 (SEQ ID NO: 495).
Figure 172-11 shows the amino acid sequence of D108-AA4 (SEQ ID NO: 496) (this is a translation of D108-AA4 (SEQ ID NO: 495)); the nucleic acid sequence of D124-AC5 (5 ') (SEQ ID NO: 497) And the amino acid sequence of D124-AC5 (5 ′) (SEQ ID NO: 498), which is a translation of D124-AC5 (5 ′) (SEQ ID NO: 497); D124-AC5 (3 ′) (SEQ ID NO: 499) And the amino acid sequence of D124-AC5 (3 ′) (SEQ ID NO: 500), which is a translation of D124-AC5 (3 ′) (SEQ ID NO: 499); and D141-AD7 (SEQ ID NO: 501) A nucleic acid and amino acid sequence group consisting of the nucleic acid sequences.
FIG. 172-12 shows the amino acid sequence of D141-AD7 (SEQ ID NO: 502), which is a translation of D141-AD7 (SEQ ID NO: 501); and the nucleic acid sequence of D148-AD1 (SEQ ID NO: 503) and D148 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of -AD1 (SEQ ID NO: 504) (this is a translation of D148-AD1 (SEQ ID NO: 503)).
FIG. 172-13 shows the nucleic acid sequence of D212-BC11 (SEQ ID NO: 505) and the amino acid sequence of D212-BC11 (SEQ ID NO: 506) (which is a translation of D212-BC11 (SEQ ID NO: 505)); and D217- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AB10 (SEQ ID NO: 507) and the amino acid sequence of D217-AB10 (SEQ ID NO: 508) (which is a translation of D217-AB10 (SEQ ID NO: 507)).
Figure 172-14 shows the nucleic acid sequence of D220-BC6 (SEQ ID NO: 509) and the amino acid sequence of D220-BC6 (SEQ ID NO: 510) (this is a translation of D220-BC6 (SEQ ID NO: 509)); D225-AG9 (SEQ ID NO: 511) and D225-AG9 (SEQ ID NO: 512) amino acid sequence (which is a translation of D225-AG9 (SEQ ID NO: 511)); D231-AF1 (SEQ ID NO: 513) nucleic acid sequence and A nucleic acid and amino acid sequence group consisting of the amino acid sequence of D231-AF1 (SEQ ID NO: 514) (this is a translation of D231-AF1 (SEQ ID NO: 513)); and the nucleic acid sequence of D232-AH5 (SEQ ID NO: 515) .
Figure 172-15 shows the amino acid sequence of D232-AH5 (SEQ ID NO: 516) (which is a translation of D232-AH5 (SEQ ID NO: 515)); the nucleic acid sequence of D240-BB8 (SEQ ID NO: 517) and D240-BB8 (SEQ ID NO: 518) amino acid sequence (this is a translation of D240-BB8 (SEQ ID NO: 517)); D280-AA6 (SEQ ID NO: 519) nucleic acid sequence and D280-AA6 (SEQ ID NO: 520) amino acid sequence ( This is a translation of D280-AA6 (SEQ ID NO: 519)); and the nucleic acid sequence of D285-AD7 (SEQ ID NO: 521) and the amino acid sequence of D285-AD7 (SEQ ID NO: 522) (this is D285-AD7 (SEQ ID NO: 521)) 521) is a nucleic acid and amino acid sequence group.
Figure 172-16 shows the nucleic acid sequence of D285-AH9 (SEQ ID NO: 523) and the amino acid sequence of D285-AH9 (SEQ ID NO: 524) (this is a translation of D285-AH9 (SEQ ID NO: 523)); D99-AB3 (SEQ ID NO: 525) and D99-AB3 (SEQ ID NO: 526) amino acid sequence (which is a translation of D99-AB3 (SEQ ID NO: 525)); and D99-AC2 (SEQ ID NO: 527) nucleic acid sequence And a group of nucleic acid and amino acid sequences consisting of the amino acid sequence of D99-AC2 (SEQ ID NO: 528) (which is a translation of D99-AC2 (SEQ ID NO: 527)).
Figure 172-17 shows the nucleic acid sequence of D99-AF11 (SEQ ID NO: 529) and the amino acid sequence of D99-AF11 (SEQ ID NO: 530) (this is a translation of D99-AF11 (SEQ ID NO: 529)); D99-AH4 (SEQ ID NO: 531) and D99-AH4 (SEQ ID NO: 532) amino acid sequence (which is a translation of D99-AH4 (SEQ ID NO: 531)); D99-AH7 (SEQ ID NO: 533) nucleic acid sequence and D99-AH7 (SEQ ID NO: 534) amino acid sequence (this is a translation of D99-AH7 (SEQ ID NO: 533)); D99-DB4 (SEQ ID NO: 535) nucleic acid sequence and D99-DB4 (SEQ ID NO: 536) A nucleic acid and amino acid sequence group consisting of an amino acid sequence (which is a translation of D99-DB4 (SEQ ID NO: 535)).
Figure 172-18 shows the nucleic acid sequence of D99-DG4 (SEQ ID NO: 537) and the amino acid sequence of D99-DG4 (SEQ ID NO: 538) (this is a translation of D99-DG4 (SEQ ID NO: 537)); D40-2 (SEQ ID NO: 539) and D40-2 (SEQ ID NO: 540) amino acid sequence (which is a translation of D40-2 (SEQ ID NO: 539)); D301-EE11 (SEQ ID NO: 541) nucleic acid sequence and A nucleic acid and amino acid sequence group consisting of the amino acid sequence of D301-EE11 (SEQ ID NO: 542) (this is a translation of D301-EE11 (SEQ ID NO: 541)); and the nucleic acid sequence of D302-AE10 (SEQ ID NO: 543) .
Figure 172-19 shows the amino acid sequence of D302-AE10 (SEQ ID NO: 544) (which is a translation of D302-AE10 (SEQ ID NO: 543)); the nucleic acid sequence of D303-AC6 (SEQ ID NO: 545) and D303-AC6 (SEQ ID NO: 546) amino acid sequence (this is a translation of D303-AC6 (SEQ ID NO: 545)); and D303-AC11 (SEQ ID NO: 547) nucleic acid sequence and D303-AC11 (SEQ ID NO: 548) amino acid sequence This is a nucleic acid and amino acid sequence group consisting of (which is a translation of D303-AC11 (SEQ ID NO: 547)).
Figure 173-1 shows 40-17 (SEQ ID NO: 549), 40-20 (SEQ ID NO: 550), 40-23 (SEQ ID NO: 551), 40-26 (SEQ ID NO: 552), D40-27 (SEQ ID NO: 553) , 40-28 (SEQ ID NO: 554) and 40-78 (SEQ ID NO: 555).
Figure 173-2 shows 40-83 (SEQ ID NO: 556), D40-86 (SEQ ID NO: 557), D40-97 (SEQ ID NO: 558), 40-100 (SEQ ID NO: 559), 40-107 (SEQ ID NO: 560) , D41-41 (SEQ ID NO: 561) and D41-60 (SEQ ID NO: 562).
Figure 173-3 shows D41-65 (SEQ ID NO: 563), D41-67 (SEQ ID NO: 564), D41-69 (SEQ ID NO: 565), D41-99 (SEQ ID NO: 566), D42-AA3 (SEQ ID NO: 567) , D42-AA7 (SEQ ID NO: 568) and D42-AC3 (SEQ ID NO: 569).
Figure 173-4 shows D42-AC7 (SEQ ID NO: 570), D42-AC8 (SEQ ID NO: 571), D42-AD3 (SEQ ID NO: 572), D42-AD12 (SEQ ID NO: 573), D42-AF3 (SEQ ID NO: 574) And the nucleic acid sequence of D42-AH3 (SEQ ID NO: 575).
Figure 173-5 shows D42-BA2 (SEQ ID NO: 576), D42-BB11 (SEQ ID NO: 577), D42-KC9 (SEQ ID NO: 578), D42-KC10 (SEQ ID NO: 579), D42-LB2 (SEQ ID NO: 580) , D42-LC7 (SEQ ID NO: 581), D42-TG7 (SEQ ID NO: 582) and D42-UG8 (SEQ ID NO: 583).
Figure 173-6 shows D42-ZB1 (SEQ ID NO: 584), D300-AA7 (SEQ ID NO: 585), D300-AB3 (SEQ ID NO: 586), D300-AB7 (SEQ ID NO: 587), D300-AB10 (SEQ ID NO: 588) , D300-AB11 (SEQ ID NO: 589) and D300-AC2 (SEQ ID NO: 590).
Figure 173-7 shows D300-AC6 (SEQ ID NO: 591), D300-AC7 (SEQ ID NO: 592), D300-AD4 (SEQ ID NO: 593), D300-AD6 (SEQ ID NO: 594), D300-AD10 (SEQ ID NO: 595) , D300-AE3 (SEQ ID NO: 596) and D300-AE9 (SEQ ID NO: 597).
Figure 173-8 shows D300-AE11 (SEQ ID NO: 598), D300-AF4 (SEQ ID NO: 599), D300-AF5 (SEQ ID NO: 600), D300-AF7 (SEQ ID NO: 601), D300-AF8 (SEQ ID NO: 602) , D300-AF9 (SEQ ID NO: 603) and D300-AF11 (SEQ ID NO: 604) nucleic acid sequences.
Figure 173-9 shows D300-AG1 (SEQ ID NO: 605), D300-AG2 (SEQ ID NO: 606), D300-AG3 (SEQ ID NO: 607), D300-AG10 (SEQ ID NO: 608), D300-AH6 (SEQ ID NO: 609) , D300-AH9 (SEQ ID NO: 610), D300-AH10 (SEQ ID NO: 611) and D300-AH11 (SEQ ID NO: 612).
Figure 173-10 shows D300-BA5 (SEQ ID NO: 613), D300-BA6 (SEQ ID NO: 614), D300-BA7 (SEQ ID NO: 615), D300-BA12 (SEQ ID NO: 616), D300-BB6 (SEQ ID NO: 617) , D300-BB7 (SEQ ID NO: 618) and D300-BC1 (SEQ ID NO: 619).
Figure 173-11 shows D300-BC4 (SEQ ID NO: 620), D300-BC7 (SEQ ID NO: 621), D300-BC8 (SEQ ID NO: 622), D300-BC9 (SEQ ID NO: 623), D300-BD2 (SEQ ID NO: 624) , D300-BD7 (SEQ ID NO: 625) and D300-BE1 (SEQ ID NO: 626).
Figure 173-12 shows D300-BE2 (SEQ ID NO: 627), D300-BE7 (SEQ ID NO: 628), D300-BE8 (SEQ ID NO: 629), D300-BE9 (SEQ ID NO: 630), D300-BE12 (SEQ ID NO: 631) , D300-BF7 (SEQ ID NO: 632), D300-BF11 (SEQ ID NO: 633) and D300-BG1 (SEQ ID NO: 634).
Figure 173-13 shows D300-BG2 (SEQ ID NO: 635), D300-BG5 (SEQ ID NO: 636), D300-BH6 (SEQ ID NO: 637), D300-BH9 (SEQ ID NO: 638), D300-CA1 (SEQ ID NO: 639) , D300-CA6 (SEQ ID NO: 640) and D300-CB1 (SEQ ID NO: 641) nucleic acid sequences.
Figure 173-14 shows D300-CB12 (SEQ ID NO: 642), D300-CC2 (SEQ ID NO: 643), D300-CD2 (SEQ ID NO: 644), D300-CD3 (SEQ ID NO: 645), D300-CD4 (SEQ ID NO: 646) , D300-CD5 (SEQ ID NO: 647) and D300-CE3 (SEQ ID NO: 648).
Figure 173-15 shows D300-CE7 (SEQ ID NO: 649), D300-CF1 (SEQ ID NO: 650), D300-CF2 (SEQ ID NO: 651), D300-CF11 (SEQ ID NO: 652), D300-CG2 (SEQ ID NO: 653) , D300-CG7 (SEQ ID NO: 654), D300-CH1 (SEQ ID NO: 655) and D300-CH3 (SEQ ID NO: 656).
Figure 173-16 shows D300-CH5 (SEQ ID NO: 657), D300-CH7 (SEQ ID NO: 658), D300-DA1 (SEQ ID NO: 659), D300-DA4 (SEQ ID NO: 660), D300-DB2 (SEQ ID NO: 661) , D300-DB4 (SEQ ID NO: 662), D300-DB6 (SEQ ID NO: 663) and D300-DB7-c (SEQ ID NO: 664).
Figure 173-17 shows D300-DB8 (SEQ ID NO: 665), D300-DC2 (SEQ ID NO: 666), D300-DC4 (SEQ ID NO: 667), D300-DC7 (SEQ ID NO: 668), D300-DC10 (SEQ ID NO: 669) , D300-DC11 (SEQ ID NO: 670) and D300-DD5 (SEQ ID NO: 671).
Figure 173-18 shows D300-DD6 (SEQ ID NO: 672), D300-DE2 (SEQ ID NO: 673), D300-DE9 (SEQ ID NO: 674), D300-DE10 (SEQ ID NO: 675), D300-DF5 (SEQ ID NO: 676) , D300-DF6 (SEQ ID NO: 677), D300-DF8 (SEQ ID NO: 678) and D300-DF12 (SEQ ID NO: 679).
Figure 173-19 shows D300-DG1 (SEQ ID NO: 680), D300-DG3 (SEQ ID NO: 681), D300-DG9 (SEQ ID NO: 682), D300-DG11 (SEQ ID NO: 683), D300-DH10 (SEQ ID NO: 684) , D301-AB10 (SEQ ID NO: 685) and D301-AC5 (SEQ ID NO: 686).
Figure 173-20 shows D301-AC7 (SEQ ID NO: 687), D301-AC9 (SEQ ID NO: 688), D301-AD1 (SEQ ID NO: 689), D301-AD2 (SEQ ID NO: 690), D301-AE4c (SEQ ID NO: 691) , D301-AF12 (SEQ ID NO: 692), D301-AH6 (SEQ ID NO: 693) and D301-AH12 (SEQ ID NO: 694).
Figure 173-21 shows D301-BB8 (SEQ ID NO: 695), D301-BB9 (SEQ ID NO: 696), D301-BC1 (SEQ ID NO: 697), D301-BC8 (SEQ ID NO: 698), D301-BD1 (SEQ ID NO: 699) , D301-BD2 (SEQ ID NO: 700) and D301-BF3 (SEQ ID NO: 701).
Figure 173-22 shows D301-DA4 (SEQ ID NO: 702), D301-DA8 (SEQ ID NO: 703), D301-DB5 (SEQ ID NO: 704), D301-DC6 (SEQ ID NO: 705), D301-DD4 (SEQ ID NO: 706) , D301-DG1 (SEQ ID NO: 707), D301-DG4c (SEQ ID NO: 708) and D301-EA5 (SEQ ID NO: 709).
Figure 173-23 shows D301-EA6 (SEQ ID NO: 710), D301-EA7 (SEQ ID NO: 711), D301-EC9 (SEQ ID NO: 712), D301-EC10 (SEQ ID NO: 713), D301-ED5 (SEQ ID NO: 714) , D301-ED7 (SEQ ID NO: 715) and D301-EE7 (SEQ ID NO: 716).
Figure 173-24 shows D301-EF2 (SEQ ID NO: 717), D301-EF5 (SEQ ID NO: 718), D301-EF10 (SEQ ID NO: 719), D301-EG12 (SEQ ID NO: 720), D302-AB8 (SEQ ID NO: 721) , D302-AB9 (SEQ ID NO: 722), D302-AB12 (SEQ ID NO: 723) and D302-AC1 (SEQ ID NO: 724).
Figure 173-25 shows D302-AC5 (SEQ ID NO: 725), D302-AC6 (SEQ ID NO: 726), D302-AC7 (SEQ ID NO: 727), D302-AC12 (SEQ ID NO: 728), D302-AD4 (SEQ ID NO: 729) , D302-AD7 (SEQ ID NO: 730) and D302-AE7 (SEQ ID NO: 731) nucleic acid sequences.
Figure 173-26 shows D302-AF6 (SEQ ID NO: 732), D302-AF8 (SEQ ID NO: 733), D302-AG2 (SEQ ID NO: 734), D302-AH10 (SEQ ID NO: 735), D302-BA2 (SEQ ID NO: 736) , D302-BA6 (SEQ ID NO: 737), D302-BC3 (SEQ ID NO: 738) and D302-BC6 (SEQ ID NO: 739).
Figure 173-27 shows D302-BC9 (SEQ ID NO: 740), D302-BD3 (SEQ ID NO: 741), D302-BD7 (SEQ ID NO: 742), D302-BD9 (SEQ ID NO: 743), D302-BE1 (SEQ ID NO: 744) And the nucleic acid sequence of D302-BE2 (SEQ ID NO: 745).
Figure 173-28 shows D302-BE6 (SEQ ID NO: 746), D302-BE7 (SEQ ID NO: 747), D302-BF1 (SEQ ID NO: 748), D302-BF5 (SEQ ID NO: 749), D302-BG1 (SEQ ID NO: 750) , D302-bg3 (SEQ ID NO: 751) and D302-BG7 (SEQ ID NO: 752).
Figure 173-29 shows D302-Bg9 (SEQ ID NO: 753), D302-BH1 (SEQ ID NO: 754), D302-bh9 (SEQ ID NO: 755), D302-CA5 (SEQ ID NO: 756), D302-CB7 (SEQ ID NO: 757) , D302-CC1 (SEQ ID NO: 758), D302-CC3 (SEQ ID NO: 759) and D302-CD2 (SEQ ID NO: 760).
Figure 173-30 shows D302-CE3 (SEQ ID NO: 761), D302-CE9 (SEQ ID NO: 762), D302-CF2 (SEQ ID NO: 763), D302-CF5 (SEQ ID NO: 764), D302-CF6 (SEQ ID NO: 765) , D302-CH1 (SEQ ID NO: 766) and D302-CH4 (SEQ ID NO: 767).
Figure 173-31 shows D302-CH5 (SEQ ID NO: 768), D302-CH6 (SEQ ID NO: 769), D302-DA2 (SEQ ID NO: 770), D302-DA5 (SEQ ID NO: 771), D302-DA9 (SEQ ID NO: 772) , D302-DC4 (SEQ ID NO: 773) and D302-DC7 (SEQ ID NO: 774).
Figure 173-32 shows D302-DC8 (SEQ ID NO: 775), D302-DC11 (SEQ ID NO: 776), D302-DD3 (SEQ ID NO: 777), D302-DF5 (SEQ ID NO: 778), D302-DF7 (SEQ ID NO: 779) , D302-DG9 (SEQ ID NO: 780) and D302-DH8 (SEQ ID NO: 781).
Figures 173-33 show D303-AA9 (SEQ ID NO: 782), D303-AA10 (SEQ ID NO: 783), D303-AB11 (SEQ ID NO: 784), D303-AC4 (SEQ ID NO: 785), D303-AD3 (SEQ ID NO: 786) , D303-AD11 (SEQ ID NO: 787), D303-AE1 (SEQ ID NO: 788) and D303-ae2 (SEQ ID NO: 789).
Figure 173-34 shows D303-ae6 (SEQ ID NO: 790), D303-AF5 (SEQ ID NO: 791), D303-AF12 (SEQ ID NO: 792), D303-AG1 (SEQ ID NO: 793), D303-AG8 (SEQ ID NO: 794) , D303-AG9 (SEQ ID NO: 795), D303-AH5 (SEQ ID NO: 796) and D303-BA1 (SEQ ID NO: 797).
Figure 173-35 shows D303-BA5 (SEQ ID NO: 798), D303-BB6 (SEQ ID NO: 799), D303-BC1 (SEQ ID NO: 800), D303-BC2 (SEQ ID NO: 801), D303-BC8 (SEQ ID NO: 802) , D303-BC11 (SEQ ID NO: 803), D303-bd2 (SEQ ID NO: 804) and D303-BD6 (SEQ ID NO: 805).
Figure 173-36 shows D303-BD9 (SEQ ID NO: 806), D303-BE3 (SEQ ID NO: 807), D303-BE4 (SEQ ID NO: 808), D303-BE7 (SEQ ID NO: 809), D303-BF1 (SEQ ID NO: 810) , D303-BF6 (SEQ ID NO: 811), D303-BG2 (SEQ ID NO: 812) and D303-BG11 (SEQ ID NO: 813).
Figures 173-37 show D303-BH1 (SEQ ID NO: 814), D303-BH6 (SEQ ID NO: 815), D414-AG2 (SEQ ID NO: 816), D35-33 (SEQ ID NO: 817), D35-34 (SEQ ID NO: 818) And the nucleic acid sequence of D35-35 (SEQ ID NO: 819).
FIGS. 173-38 are the nucleic acid sequences of D35-41 (SEQ ID NO: 820), D35-43 (SEQ ID NO: 821), D35-51 (SEQ ID NO: 822) and D35-AC4 (SEQ ID NO: 823).
Figure 173-39 shows D35-AC12 (SEQ ID NO: 824), D35-AE2 (SEQ ID NO: 825), D35-AE6 (SEQ ID NO: 826), D35-AF1 (SEQ ID NO: 827), D35-AF3 (SEQ ID NO: 828) And the nucleic acid sequence of D35-AG3 (SEQ ID NO: 829).
Figure 173-40 shows D35-BA5 (SEQ ID NO: 830), D35-BA12 (SEQ ID NO: 831), D35-BB1 (SEQ ID NO: 832), D35-BB3 (SEQ ID NO: 833) and D35-BB10 (SEQ ID NO: 834) The nucleic acid sequence of
Figures 173-41 shows D35-BB12 (SEQ ID NO: 835), D55-AA8 (SEQ ID NO: 836), D55-AB1 (SEQ ID NO: 837), D55-AB5 (SEQ ID NO: 838) and D55-AB6 (SEQ ID NO: 839) The nucleic acid sequence of
Figures 173-42 show D55-AB7 (SEQ ID NO: 840), D55-AB10 (SEQ ID NO: 841), D55-AA2 (SEQ ID NO: 842), D55-AC2 (SEQ ID NO: 843) and D55-AC5 (SEQ ID NO: 844) The nucleic acid sequence of
Figure 173-43 shows D55-AC7 (SEQ ID NO: 845), D55-BA7 (SEQ ID NO: 846), D55-BA8 (SEQ ID NO: 847), D55-BA10 (SEQ ID NO: 848), D55-BA11 (SEQ ID NO: 849) And the nucleic acid sequence of D55-BB9 (SEQ ID NO: 850).
Figure 173-44 shows D55-BB10 (SEQ ID NO: 851), D55-BB12 (SEQ ID NO: 852), D55-BC4 (SEQ ID NO: 853), D55-BC5 (SEQ ID NO: 854), D55-AA6 (SEQ ID NO: 855) And the nucleic acid sequence of D56-AE5 (SEQ ID NO: 856).
Figure 173-45 shows D56-AA2 (SEQ ID NO: 857), D56-AA4 (SEQ ID NO: 858), D56-AA8 (SEQ ID NO: 859), D56-AA9 (SEQ ID NO: 860), D56-AB1 (SEQ ID NO: 861) And the nucleic acid sequence of D56-AB7 (SEQ ID NO: 862).
Figures 173-46 show D56-AB9 (SEQ ID NO: 863), D56-AC9 (SEQ ID NO: 864), D56-AD3 (SEQ ID NO: 865), D56-AG8 (SEQ ID NO: 866) and D56-AH1 (SEQ ID NO: 867) The nucleic acid sequence of
Figures 173-47 show D56-AH10 (SEQ ID NO: 868), D56-AE2 (SEQ ID NO: 869), D57-AB2 (SEQ ID NO: 870), D57-AB5 (SEQ ID NO: 871) and D57-AB6 (SEQ ID NO: 872) The nucleic acid sequence of
Figure 173-48 shows D57-AB8 (SEQ ID NO: 873), D57-AB11 (SEQ ID NO: 874), D57-AB12 (SEQ ID NO: 875), D57-AC1 (SEQ ID NO: 876), D57-AC4 (SEQ ID NO: 877) And the nucleic acid sequence of D57-AC12 (SEQ ID NO: 878).
Figure 173-49 shows D57-AD5 (SEQ ID NO: 879), D57-AD8 (SEQ ID NO: 880), D57-AD9 (SEQ ID NO: 881), D57-AE1 (SEQ ID NO: 882), D57-AE2 (SEQ ID NO: 883) And the nucleic acid sequence of D57-AE7 (SEQ ID NO: 884).
Figure 173-50 shows D57-AF2 (SEQ ID NO: 885), D57-AE12 (SEQ ID NO: 886), D57-AF3 (SEQ ID NO: 887), D57-AF5 (SEQ ID NO: 888) and D57-AF9 (SEQ ID NO: 889) The nucleic acid sequence of
FIGS. 173-51 are the nucleic acid sequences of D57-AG5 (SEQ ID NO: 890), D57-AG7 (SEQ ID NO: 891), D57-AG11 (SEQ ID NO: 892) and D57-AH10 (SEQ ID NO: 893).
Figures 173-52 show D58-AA3 (SEQ ID NO: 894), D58-AA5 (SEQ ID NO: 895), D58-AB3 (SEQ ID NO: 896), D58-AB5 (SEQ ID NO: 897) and D58-AC1 (SEQ ID NO: 898) The nucleic acid sequence of
FIGS. 173-53 are the nucleic acid sequences of D58-AC9 (SEQ ID NO: 899), D58-AC11 (SEQ ID NO: 900), D58-AD2 (SEQ ID NO: 901) and D58-AD5 (SEQ ID NO: 902).
Figures 173-54 show D58-AD7 (SEQ ID NO: 903), D58-AD8 (SEQ ID NO: 904), D58-AD10 (SEQ ID NO: 905), D58-AE1 (SEQ ID NO: 906) and D58-AE2 (SEQ ID NO: 907) The nucleic acid sequence of
Figures 173-55 show D58-AE5 (SEQ ID NO: 908), D58-AE9 (SEQ ID NO: 909), D58-AE10 (SEQ ID NO: 910), D58-AE11 (SEQ ID NO: 911) and D58-AF3 (SEQ ID NO: 912) The nucleic acid sequence of
Figures 173-56 show D58-AF6 (SEQ ID NO: 913), D58-AH4 (SEQ ID NO: 914), D58-AH7 (SEQ ID NO: 915), D58-AH9 (SEQ ID NO: 916) and D58-AH10 (SEQ ID NO: 917) The nucleic acid sequence of
Figure 173-57 shows D58-BA5 (SEQ ID NO: 918), D58-BA7 (SEQ ID NO: 919), D58-BB7 (SEQ ID NO: 920), D58-BC5 (SEQ ID NO: 921), D58-BD3 (SEQ ID NO: 922) And the nucleic acid sequence of D58-BD7 (SEQ ID NO: 923).
Figures 173-58 show D58-BD8 (SEQ ID NO: 924), D58-BD10 (SEQ ID NO: 925), D58-BE2 (SEQ ID NO: 926), D58-BE11 (SEQ ID NO: 927) and D58-BF1 (SEQ ID NO: 928) The nucleic acid sequence of
Figure 173-59 shows D58-BF2 (SEQ ID NO: 929), D58-BG3 (SEQ ID NO: 930), D58-BG5 (SEQ ID NO: 931), D58-BG8 (SEQ ID NO: 932), D58-BG10 (SEQ ID NO: 933) And the nucleic acid sequence of D58-BG12 (SEQ ID NO: 934).
Figure 173-60 shows D58-BH10 (SEQ ID NO: 935), D58-BH8 (SEQ ID NO: 936), D58-BH2 (SEQ ID NO: 937), D60-AA1 (SEQ ID NO: 938), D60-AA2 (SEQ ID NO: 939) And the nucleic acid sequence of D60-AA3 (SEQ ID NO: 940).
Figure 173-61 shows D60-AA5 (SEQ ID NO: 941), D60-AA6 (SEQ ID NO: 942), D60-AA7 (SEQ ID NO: 943), D60-AA8 (SEQ ID NO: 944), D60-AA10 (SEQ ID NO: 945) And the nucleic acid sequence of D60-AA11 (SEQ ID NO: 946).
Figure 173-62 shows D60-AA12 (SEQ ID NO: 947), D60-AB3 (SEQ ID NO: 948), D60-AB5 (SEQ ID NO: 949), D60-AB6 (SEQ ID NO: 950), D60-AB7 (SEQ ID NO: 951) And the nucleic acid sequence of D60-AB8 (SEQ ID NO: 952).
Figure 173-63 shows D60-AB11 (SEQ ID NO: 953), D60-AC3 (SEQ ID NO: 954), D60-AC5 (SEQ ID NO: 955), D60-AC7 (SEQ ID NO: 956), D60-AC8 (SEQ ID NO: 957) And the nucleic acid sequence of D60-AC11 (SEQ ID NO: 958).
Figure 173-64 shows D60-AC12 (SEQ ID NO: 959), D60-AD3 (SEQ ID NO: 960), D60-AD4 (SEQ ID NO: 961), D60-AD5 (SEQ ID NO: 962), D60-AD7 (SEQ ID NO: 963) And the nucleic acid sequence of D60-AE1 (SEQ ID NO: 964).
Figure 173-65 shows D60-AE4 (SEQ ID NO: 965), D60-AE6 (SEQ ID NO: 966), D60-AE8 (SEQ ID NO: 967), D60-AE12 (SEQ ID NO: 968), D60-AF5 (SEQ ID NO: 969) And the nucleic acid sequence of D60-AF8 (SEQ ID NO: 970).
Figure 173-66 shows D60-AF11 (SEQ ID NO: 971), D60-AG1 (SEQ ID NO: 972), D60-AG10 (SEQ ID NO: 973), D60-AG12 (SEQ ID NO: 974), D60-AH1 (SEQ ID NO: 975) And the nucleic acid sequence of D60-AH5 (SEQ ID NO: 976).
Figures 173-67 show D60-AH6 (SEQ ID NO: 977), D60-AH9 (SEQ ID NO: 978), D60-AH10 (SEQ ID NO: 979), D60-AH11 (SEQ ID NO: 980) and D63-AA12 (SEQ ID NO: 981) The nucleic acid sequence of
Figures 173-68 show D64-4 (SEQ ID NO: 982), D64-AB4 (SEQ ID NO: 983), D64-AB10 (SEQ ID NO: 984), D65-AA6 (SEQ ID NO: 985) and D65-AA7 (SEQ ID NO: 986). The nucleic acid sequence of
Figure 173-69 shows D65-AA9 (SEQ ID NO: 987), D65-AB4 (SEQ ID NO: 988), D65-AB5 (SEQ ID NO: 989), D65-AB8 (SEQ ID NO: 990), D65-AB9 (SEQ ID NO: 991) And the nucleic acid sequence of D65-AB11 (SEQ ID NO: 992).
Figure 173-70 shows D65-AC1 (SEQ ID NO: 993), D65-AC6 (SEQ ID NO: 994), D65-AC10 (SEQ ID NO: 995), D65-AC11 (SEQ ID NO: 996), D65-AD3 (SEQ ID NO: 997) And the nucleic acid sequence of D65-AE1 (SEQ ID NO: 998).
Figure 173-71 shows D65-AE2 (SEQ ID NO: 999), D65-AE10 (SEQ ID NO: 1000), D65-AE11 (SEQ ID NO: 1001), D65-AE12 (SEQ ID NO: 1002), D65-AF1 (SEQ ID NO: 1003) And the nucleic acid sequence of D65-AF5 (SEQ ID NO: 1004).
Figure 173-72 shows D65-AF7 (SEQ ID NO: 1005), D65-AG11 (SEQ ID NO: 1006), D65-CE1 (SEQ ID NO: 1007), D65-CE2 (SEQ ID NO: 1008), D65-CE7 (SEQ ID NO: 1009) And the nucleic acid sequence of D65-CE9 (SEQ ID NO: 1010).
Figure 173-73 shows D65-CE12 (SEQ ID NO: 1011), D65-CF3 (SEQ ID NO: 1012), D65-CF10 (SEQ ID NO: 1013), D65-CF12 (SEQ ID NO: 1014), D65-CG2 (SEQ ID NO: 1015) , D65-CG5 (SEQ ID NO: 1016) and D65-CH3 (SEQ ID NO: 1017).
Figure 173-74 shows D65-CH4 (SEQ ID NO: 1018), D65-CH6 (SEQ ID NO: 1019), D65-CH8 (SEQ ID NO: 1020), D65-CH9 (SEQ ID NO: 1021), D65-CH11 (SEQ ID NO: 1022) And the nucleic acid sequence of D65-CH12 (SEQ ID NO: 1023).
Figures 173-75 show D66-AA4 (SEQ ID NO: 1024), D66-AA5 (SEQ ID NO: 1025), D66-AA6 (SEQ ID NO: 1026), D66-AA9 (SEQ ID NO: 1027) and D66-AB5 (SEQ ID NO: 1028) The nucleic acid sequence of
Figures 173-76 show D66-AB1 (SEQ ID NO: 1029), D66-AB7 (SEQ ID NO: 1030), D66-AC1 (SEQ ID NO: 1031), D66-AC2 (SEQ ID NO: 1032) and D66-AC4 (SEQ ID NO: 1033) The nucleic acid sequence of
FIGS. 173-77 are the nucleic acid sequences of D66-AC7 (SEQ ID NO: 1034), D66-AD1 (SEQ ID NO: 1035), D66-AD3 (SEQ ID NO: 1036) and D66-AD6 (SEQ ID NO: 1037).
Figure 173-78 shows D66-AD8 (SEQ ID NO: 1038), D66-AE1 (SEQ ID NO: 1039), D66-AE3 (SEQ ID NO: 1040), D66-AE6 (SEQ ID NO: 1041), D66-AE8 (SEQ ID NO: 1042) And the nucleic acid sequence of D66-AF3 (SEQ ID NO: 2263).
FIGS. 173-79 are the nucleic acid sequences of D66-AF5 (SEQ ID NO: 1043), D66-AF8 (SEQ ID NO: 1044), D66-AF9 (SEQ ID NO: 1045) and D66-AG1 (SEQ ID NO: 1046).
Figure 173-80 shows D66-AG4 (SEQ ID NO: 1047), D66-AG5 (SEQ ID NO: 1048), D66-AH4 (SEQ ID NO: 1049), D66-AH5 (SEQ ID NO: 1050), D66-AH7 (SEQ ID NO: 1051) And the nucleic acid sequence of D66-AH8 (SEQ ID NO: 1052).
FIGS. 173-81 are the nucleic acid sequences of D66-AH9 (SEQ ID NO: 1053), D66-BA3 (SEQ ID NO: 1054), D66-BA8 (SEQ ID NO: 1055) and D66-BB1 (SEQ ID NO: 1056).
FIGS. 173-82 are the nucleic acid sequences of D66-BB2 (SEQ ID NO: 1057), D66-BB3 (SEQ ID NO: 1058), D66-BB5 (SEQ ID NO: 1059) and D66-BB7 (SEQ ID NO: 1060).
Figure 173-83 shows D66-BB9 (SEQ ID NO: 1061), D66-BB10 (SEQ ID NO: 1062), D66-BC6 (SEQ ID NO: 1063), D66-BD1 (SEQ ID NO: 1064) and D66-BD2 (SEQ ID NO: 1065) The nucleic acid sequence of
Figures 173-84 show D66-BD9 (SEQ ID NO: 1066), D67-AA2 (SEQ ID NO: 1067), D67-AA3 (SEQ ID NO: 1068), D67-AB1 (SEQ ID NO: 1069) and D67-AC5 (SEQ ID NO: 1070) The nucleic acid sequence of
Figures 173-85 show D67-AD1 (SEQ ID NO: 1071), D67-AD4 (SEQ ID NO: 1072), D67-AD6 (SEQ ID NO: 1073), D67-AE2 (SEQ ID NO: 1074) and D67-AE6 (SEQ ID NO: 1075) The nucleic acid sequence of
Figures 173-86 show D67-AF6 (SEQ ID NO: 1076), D67-AG1 (SEQ ID NO: 1077), D67-AG3 (SEQ ID NO: 1078), D67-AG5 (SEQ ID NO: 1079) and D67-AG6 (SEQ ID NO: 1080) The nucleic acid sequence of
Figures 173-87 show D69-AA5 (SEQ ID NO: 1081), D69-AB1 (SEQ ID NO: 1082), D69-AB8 (SEQ ID NO: 1083), D69-AB9 (SEQ ID NO: 1084) and D69-AC4 (SEQ ID NO: 1085) The nucleic acid sequence of
Figure 173-88 shows D70A-AB10 (SEQ ID NO: 1086), D70A-AC2 (SEQ ID NO: 1087), D70A-AC3 (SEQ ID NO: 1088), D70A-AD3 (SEQ ID NO: 1089), D70A-AD5 (SEQ ID NO: 1090) And the nucleic acid sequence of D70A-AD6 (SEQ ID NO: 1091).
Figure 173-89 shows D70A-AD11 (SEQ ID NO: 1092), D70A-AE10 (SEQ ID NO: 1093), D70A-AF6 (SEQ ID NO: 1094), D70A-AF8 (SEQ ID NO: 1095), D70A-AF10 (SEQ ID NO: 1096) , D70A-AH5 (SEQ ID NO: 1097) and D70A-AH8 (SEQ ID NO: 1098).
Figure 173-90 shows D70A-BA3 (SEQ ID NO: 1099), D70A-BA6 (SEQ ID NO: 1100), D70A-BA10 (SEQ ID NO: 1101), D70A-BB8 (SEQ ID NO: 1102) and D70A-BB11 (SEQ ID NO: 1103) The nucleic acid sequence of
Figure 173-91 shows D70A-BC7 (SEQ ID NO: 1104), D70A-BD8 (SEQ ID NO: 1105), D70A-BE1 (SEQ ID NO: 1106), D70A-BE5 (SEQ ID NO: 1107), D70A-BE6 (SEQ ID NO: 1108) And the nucleic acid sequence of D70A-BE8 (SEQ ID NO: 1109).
Figure 173-92 shows D70A-BF2 (SEQ ID NO: 1110), D70A-BF3 (SEQ ID NO: 1111), D70A-BF10 (SEQ ID NO: 1112), D70A-BG9 (SEQ ID NO: 1113), D70A-BH8 (SEQ ID NO: 1114) And the nucleic acid sequence of D70-AE2 (SEQ ID NO: 1115).
Figure 173-93 shows D70-AE6 (SEQ ID NO: 1116), D70-AF3 (SEQ ID NO: 1117), D70-AG10 (SEQ ID NO: 1118), D70-AH7 (SEQ ID NO: 1119), D70-BE6 (SEQ ID NO: 1120) And the nucleic acid sequence of D70-BE7 (SEQ ID NO: 1121).
Figure 173-94 shows D70A-BC12 (SEQ ID NO: 1122), D72-AB2 (SEQ ID NO: 1123), D72-AB6 (SEQ ID NO: 1124), D73A-AA12 (SEQ ID NO: 1125) and D73A-AB4 (SEQ ID NO: 1126) The nucleic acid sequence of
Figures 173-95 show D73A-AB9 (SEQ ID NO: 1127), D73A-AB11 (SEQ ID NO: 1128), D73A-AC1 (SEQ ID NO: 1129), D73A-AC3 (SEQ ID NO: 1130) and D73A-AC8 (SEQ ID NO: 1131) The nucleic acid sequence of
Figure 173-96 shows D73A-AD3 (SEQ ID NO: 1132), D73A-AE1 (SEQ ID NO: 1133), D73A-AE3 (SEQ ID NO: 1134), D73A-AE4 (SEQ ID NO: 1135), D73A-AE5 (SEQ ID NO: 1136) And the nucleic acid sequence of D73A-AE9 (SEQ ID NO: 1137).
Figure 173-97 shows D73A-AE10 (SEQ ID NO: 1138), D73A-AF3 (SEQ ID NO: 1139), D73A-AF4 (SEQ ID NO: 1140), D73A-AF7 (SEQ ID NO: 1141), D73A-AF8 (SEQ ID NO: 1142) And the nucleic acid sequence of D73A-AG11 (SEQ ID NO: 1143).
Figure 173-98 shows D73A-AH1 (SEQ ID NO: 1144), D73A-AH5 (SEQ ID NO: 1145), D73A-AH9 (SEQ ID NO: 1146), D73A-AH12 (SEQ ID NO: 1147), D73A-BA9 (SEQ ID NO: 1148) And the nucleic acid sequence of D73A-BB3 (SEQ ID NO: 1149).
Figures 173-99 show D73A-BB9 (SEQ ID NO: 1150), D73A-BE1 (SEQ ID NO: 1151), D73A-BF3 (SEQ ID NO: 1152), D73A-BG1 (SEQ ID NO: 1153) and D73A-BG3 (SEQ ID NO: 1154) The nucleic acid sequence of
Figure 173-100 shows D73A-BG5 (SEQ ID NO: 1155), D73A-BH1 (SEQ ID NO: 1156), D73-AC1 (SEQ ID NO: 1157), D73-AD8 (SEQ ID NO: 1158) and D73-AD12 (SEQ ID NO: 1159) The nucleic acid sequence of
Figure 173-101 shows D80-AB2 (SEQ ID NO: 1160), D81-AA5 (SEQ ID NO: 1161), D81-AB4 (SEQ ID NO: 1162), D81-AB6 (SEQ ID NO: 1163) and D81-AC5 (SEQ ID NO: 1164) The nucleic acid sequence of
FIGS. 173-102 are the nucleic acid sequences of D82-AA8 (SEQ ID NO: 1165), D82-AC9 (SEQ ID NO: 1166), D82-AH9 (SEQ ID NO: 1167) and D83-AD10 (SEQ ID NO: 1168).
Figure 173-103 shows D83-AG10 (SEQ ID NO: 1169), D84-AD3 (SEQ ID NO: 1170), D84-AE1 (SEQ ID NO: 1171), D84-AF4 (SEQ ID NO: 1172), D84-AG1 (SEQ ID NO: 1173) And the nucleic acid sequence of D87-AA1 (SEQ ID NO: 1174).
Figure 173-104 shows D87-AA3 (SEQ ID NO: 1175), D87A-AA1 (SEQ ID NO: 1176), D87A-AB1 (SEQ ID NO: 1177), D87A-AB3 (SEQ ID NO: 1178), D87A-AC1 (SEQ ID NO: 1179) And the nucleic acid sequence of D87A-AC3 (SEQ ID NO: 1180).
Figure 173-105 shows D87A-AF2 (SEQ ID NO: 1181), D87A-AG1 (SEQ ID NO: 1182), D87A-AH1 (SEQ ID NO: 1183), D87A-AH3 (SEQ ID NO: 1184), D87-AB2 (SEQ ID NO: 1185) And the nucleic acid sequence of D88-AA10 (SEQ ID NO: 1186).
Figures 173-106 show D88-AB3 (SEQ ID NO: 1187), D88-AB6 (SEQ ID NO: 1188), D88-AB7 (SEQ ID NO: 1189), D88-AB8 (SEQ ID NO: 1190), D88-AC5 (SEQ ID NO: 1191) And the nucleic acid sequence of D88-AC9 (SEQ ID NO: 1192).
Figures 173-107 show D88-AD8 (SEQ ID NO: 1193), D88-AE5 (SEQ ID NO: 1194), D88-AE6 (SEQ ID NO: 1195), D88-AE8 (SEQ ID NO: 1196) and D91-AA4 (SEQ ID NO: 1197) The nucleic acid sequence of
Figure 173-108 shows D92-AD9 (SEQ ID NO: 1198), D92-AE9 (SEQ ID NO: 1199), D92-AF8 (SEQ ID NO: 1200), D92-AG10 (SEQ ID NO: 1201), D92-AH9 (SEQ ID NO: 1202) And the nucleic acid sequence of D93-AA1 (SEQ ID NO: 1203).
Figures 173-109 show D93-AA2 (SEQ ID NO: 1204), D93-AB1 (SEQ ID NO: 1205), D93-AC3 (SEQ ID NO: 1206), D93-AC4 (SEQ ID NO: 1207) and D93-AD1 (SEQ ID NO: 1208) The nucleic acid sequence of
Figure 173-110 shows D93-AE3 (SEQ ID NO: 1209), D93-AE4 (SEQ ID NO: 1210), D93-AF3 (SEQ ID NO: 1211), D93-AG1 (SEQ ID NO: 1212), D93-AH2 (SEQ ID NO: 1213) And the nucleic acid sequence of D93-AH3 (SEQ ID NO: 1214).
Figure 173-111 shows D93-AH4 (SEQ ID NO: 1215), D93-BA10 (SEQ ID NO: 1216), D93-BA12 (SEQ ID NO: 1217), D93-BD11 (SEQ ID NO: 1218), D93-BE11 (SEQ ID NO: 1219) And the nucleic acid sequence of D93-BF12 (SEQ ID NO: 1220).
Figures 173-112 show D94-AA2 (SEQ ID NO: 1221), D94-AA3 (SEQ ID NO: 1222), D94-AB1 (SEQ ID NO: 1223), D94-AB2 (SEQ ID NO: 1224) and D94-AC3 (SEQ ID NO: 1225) The nucleic acid sequence of
Figures 173-113 show D94-AD1 (SEQ ID NO: 1226), D94-AE1 (SEQ ID NO: 1227), D94-AE3 (SEQ ID NO: 1228), D94-AG2 (SEQ ID NO: 1229) and D94-AH1 (SEQ ID NO: 1230) The nucleic acid sequence of
Figure 173-114 shows D94-AH2 (SEQ ID NO: 1231), D94-AH3 (SEQ ID NO: 1232), D95-AC1 (SEQ ID NO: 1233), D95-AD2 (SEQ ID NO: 1234), D96-AA3 (SEQ ID NO: 1235) And the nucleic acid sequence of D96-AA4 (SEQ ID NO: 1236).
Figures 173-115 show D96-AA7 (SEQ ID NO: 1237), D96-AB7 (SEQ ID NO: 2264), D96-AC5 (SEQ ID NO: 1238), D96-AC6 (SEQ ID NO: 1239) and D96-AD6 (SEQ ID NO: 1240) The nucleic acid sequence of
Figure 173-116 shows D96-AE6 (SEQ ID NO: 1241), D96-AG3 (SEQ ID NO: 1242), D96-AH8 (SEQ ID NO: 1243), D97-AA1 (SEQ ID NO: 1244), D97-AB1 (SEQ ID NO: 1245) And the nucleic acid sequence of D97-AB3 (SEQ ID NO: 1246).
Figures 173-117 show D97-AD1 (SEQ ID NO: 1247), D97-AD2 (SEQ ID NO: 1248), D97-AD4 (SEQ ID NO: 1249), D97-AE1 (SEQ ID NO: 1250) and D97-AE2 (SEQ ID NO: 1251) The nucleic acid sequence of
Figures 173-118 show D97-AE4 (SEQ ID NO: 1252), D97-AF3 (SEQ ID NO: 1253), D97-AG2 (SEQ ID NO: 1254), D97-AG3 (SEQ ID NO: 1255) and D97-AH1 (SEQ ID NO: 1256) The nucleic acid sequence of
Figure 173-119 shows D97-AH2 (SEQ ID NO: 1257), D97-AH4 (SEQ ID NO: 1258), D98-AB2 (SEQ ID NO: 1259), D98-AE3 (SEQ ID NO: 1260) and D98-AF1 (SEQ ID NO: 1261). Nucleic acid sequence).
Figure 173-120 shows D98-AH2 (SEQ ID NO: 1262), D99-AC5 (SEQ ID NO: 1263), D99-AC8 (SEQ ID NO: 1264), D99-AD2 (SEQ ID NO: 1265), D99-AD3 (SEQ ID NO: 1266) And the nucleic acid sequence of D99-AD4 (SEQ ID NO: 1267).
Figure 173-121 shows D99-AD6 (SEQ ID NO: 1268), D99-AE1 (SEQ ID NO: 1269), D99-AE5 (SEQ ID NO: 1270), D99-AE6 (SEQ ID NO: 1271), D99-AE7 (SEQ ID NO: 1272) And the nucleic acid sequence of D99-AE8 (SEQ ID NO: 1273).
Figure 173-122 shows D99-AF1 (SEQ ID NO: 1274), D99-AF5 (SEQ ID NO: 1275), D99-AF7 (SEQ ID NO: 1276), D99-AG2 (SEQ ID NO: 1277), D99-AG4 (SEQ ID NO: 1278) , D99-AG7 (SEQ ID NO: 1279) and D99-AH2 (SEQ ID NO: 1280).
Figure 173-123 shows D99-AH10 (SEQ ID NO: 1281), D99-DA3 (SEQ ID NO: 1282), D99-DB5 (SEQ ID NO: 1283), D99-DC2 (SEQ ID NO: 1284) and D99-DC3 (SEQ ID NO: 1285) The nucleic acid sequence of
FIGS. 173-124 are the nucleic acid sequences of D99-DD5 (SEQ ID NO: 1286), D99-DG4 (SEQ ID NO: 1287), D100A-AA4 (SEQ ID NO: 1288) and D101-BG2 (SEQ ID NO: 1289).
Figure 173-125 shows D101A-AC2 (SEQ ID NO: 1290), D101A-AD1 (SEQ ID NO: 1291), D101A-AD2 (SEQ ID NO: 1292), D101A-AE1 (SEQ ID NO: 1293) and D101A-AF1 (SEQ ID NO: 1294) The nucleic acid sequence of
Figures 173-126 show D101C-AA1 (SEQ ID NO: 1295), D101C-AB1 (SEQ ID NO: 1296), D101C-AC1 (SEQ ID NO: 1297), D101C-AD3 (SEQ ID NO: 1298) and D101C-BD12 (SEQ ID NO: 1299) The nucleic acid sequence of
FIGS. 173-127 are the nucleic acid sequences of D101C-BE12 (SEQ ID NO: 1300), D101C-BF12 (SEQ ID NO: 1301), D101C-BG12 (SEQ ID NO: 1302) and D101D-AB6 (SEQ ID NO: 1303).
Figures 173-128 show D101D-AC5 (SEQ ID NO: 1304), D101D-AG5 (SEQ ID NO: 1305), D101D-AG6 (SEQ ID NO: 1306), D101D-AH4 (SEQ ID NO: 1307) and D101D-AH6 (SEQ ID NO: 1308) The nucleic acid sequence of
Figure 173-129 shows D101D-BB11 (SEQ ID NO: 1309), D101D-BD11 (SEQ ID NO: 1310), D107-AA3 (SEQ ID NO: 1311), D107-AB2 (SEQ ID NO: 1312), D107-AC2 (SEQ ID NO: 1313) And the nucleic acid sequence of D107-AC3 (SEQ ID NO: 1314).
Figure 173-130 shows D107-AD2 (SEQ ID NO: 1315), D107-AD3 (SEQ ID NO: 1316), D107-AF1 (SEQ ID NO: 1317), D107-AH1 (SEQ ID NO: 1318), D108-AA6 (SEQ ID NO: 1319) And the nucleic acid sequence of D108-AB4 (SEQ ID NO: 1320).
Figure 173-131 shows D108-AB5 (SEQ ID NO: 1321), D108-AC6 (SEQ ID NO: 1322), D108-AD6 (SEQ ID NO: 1323), D108-AF6 (SEQ ID NO: 1324), D109-AA7 (SEQ ID NO: 1325) And the nucleic acid sequence of D109-AA8 (SEQ ID NO: 1326).
Figure 173-132 shows D109-AA9 (SEQ ID NO: 1327), D109-AB8 (SEQ ID NO: 1328), D109-AC7 (SEQ ID NO: 1329), D109-AC8 (SEQ ID NO: 1330), D109-AC9 (SEQ ID NO: 1331) And the nucleic acid sequence of D109-AE7 (SEQ ID NO: 1332).
Figures 173-133 show D109-AF9 (SEQ ID NO: 1333), D109-AH7 (SEQ ID NO: 1334), D110-AB11 (SEQ ID NO: 1335), D110-AF10 (SEQ ID NO: 1336), D111-AA2 (SEQ ID NO: 1337) And the nucleic acid sequence of D111-AB1 (SEQ ID NO: 1338).
Figures 173-134 show D111-AB3 (SEQ ID NO: 1339), D111-AC3 (SEQ ID NO: 1340), D111-AD1 (SEQ ID NO: 1341), D111-AF1 (SEQ ID NO: 1342) and D111-AG3 (SEQ ID NO: 1343) The nucleic acid sequence of
Figures 173-135 show D111-AH1 (SEQ ID NO: 1344), D111-AH2 (SEQ ID NO: 1345), D112-AD6 (SEQ ID NO: 1346), D112-AE6 (SEQ ID NO: 1347) and D112-AF5 (SEQ ID NO: 1348) The nucleic acid sequence of
Figures 173-136 show D112-AG5 (SEQ ID NO: 1349), D112-AH4 (SEQ ID NO: 1350), D113-AA9 (SEQ ID NO: 1351), D113A-AC3 (SEQ ID NO: 1352) and D113A-AD1 (SEQ ID NO: 1353) The nucleic acid sequence of
Figure 173-137 shows D113A-AD3 (SEQ ID NO: 1354), D113A-AF3 (SEQ ID NO: 1355), D113A-AG2 (SEQ ID NO: 1356), D113A-AH2 (SEQ ID NO: 1357), D113-AB9 (SEQ ID NO: 1358) And the nucleic acid sequence of D113-AC7 (SEQ ID NO: 1359).
Figures 173-138 show D113-AD8 (SEQ ID NO: 1360), D113-AD9 (SEQ ID NO: 1361), D113-AE7 (SEQ ID NO: 1362), D113-AF8 (SEQ ID NO: 1363) and D113-AF9 (SEQ ID NO: 1364). The nucleic acid sequence of
Figures 173-139 show D113-AG7 (SEQ ID NO: 1365), D113-AG9 (SEQ ID NO: 1366), D114-AE10 (SEQ ID NO: 1367), D114-AF11 (SEQ ID NO: 1368) and D115-AA6 (SEQ ID NO: 1369) The nucleic acid sequence of
Figure 173-140 shows D133-AA7 (SEQ ID NO: 1370), D133-AE8 (SEQ ID NO: 1371), D133-AF9 (SEQ ID NO: 1372), D133-AG8 (SEQ ID NO: 1373), D138-AD10 (SEQ ID NO: 1374) And the nucleic acid sequence of D139-AD1 (SEQ ID NO: 1375).
Figure 173-141 shows D140-AA4 (SEQ ID NO: 1376), D140-AD4 (SEQ ID NO: 1377), D140-AF4 (SEQ ID NO: 1378), D141-AA7 (SEQ ID NO: 1379), D141-AB7 (SEQ ID NO: 1380) And the nucleic acid sequence of D142-AB11 (SEQ ID NO: 1381).
Figure 173-142 shows D142-AC10 (SEQ ID NO: 1382), D142-AE10 (SEQ ID NO: 1383), D142-AE11 (SEQ ID NO: 1384), D142-AF10 (SEQ ID NO: 1385), D144-AA1 (SEQ ID NO: 1386) And the nucleic acid sequence of D144A-AA9 (SEQ ID NO: 1387).
Figure 173-143 shows D144A-AB12 (SEQ ID NO: 1388), D144A-AC9 (SEQ ID NO: 1389), D144A-AC10 (SEQ ID NO: 1390), D144A-AD12 (SEQ ID NO: 1391), D144A-AE12 (SEQ ID NO: 1392) And the nucleic acid sequence of D144A-AF11 (SEQ ID NO: 1393).
Figures 173-144 show D144A-AF12 (SEQ ID NO: 1394), D144A-AG11 (SEQ ID NO: 1395), D144A-AH9 (SEQ ID NO: 1396), D144A-AH11 (SEQ ID NO: 1397) and D144-AE4 (SEQ ID NO: 1398) The nucleic acid sequence of
Figure 173-145 shows D144-AH3 (SEQ ID NO: 1399), D145-AA8 (SEQ ID NO: 1400), D145-AC10 (SEQ ID NO: 1401), D145-AD7 (SEQ ID NO: 1402), D145-AD9 (SEQ ID NO: 1403) And the nucleic acid sequence of D145-AD10 (SEQ ID NO: 1404).
Figure 173-146 shows D145-AE7 (SEQ ID NO: 1405), D145-AF7 (SEQ ID NO: 1406), D145-AF8 (SEQ ID NO: 1407), D145-AG8 (SEQ ID NO: 1408), D145-AG9 (SEQ ID NO: 1409) And the nucleic acid sequence of D145-AG10 (SEQ ID NO: 1410).
Figures 173-147 show D145-AH9 (SEQ ID NO: 1411), D146-BB2 (SEQ ID NO: 1412), D146-BC1 (SEQ ID NO: 1413), D146-BD1 (SEQ ID NO: 1414) and D146-BD2 (SEQ ID NO: 1415) The nucleic acid sequence of
Figures 173-148 show the sequences D146-BF2 (SEQ ID NO: 1416), D147-AE2 (SEQ ID NO: 1417), D150-AA2 (SEQ ID NO: 1418), D150-AC2 (SEQ ID NO: 1419) and D150-AD1 (SEQ ID NO: 1420). ) Nucleic acid.
Figure 173-149 shows D151-AA1 (SEQ ID NO: 1421), D152-AA2 (SEQ ID NO: 1422), D152-AB2 (SEQ ID NO: 1423), D152-AC1 (SEQ ID NO: 1424), D152-AD2 (SEQ ID NO: 1425) And the nucleic acid sequence of D152-AG2 (SEQ ID NO: 1426).
Figure 173-150 shows D152-AH2 (SEQ ID NO: 1427), D153-AA7 (SEQ ID NO: 1428), D153-AB2 (SEQ ID NO: 1429), D153-AF2 (SEQ ID NO: 1430), D153-AF7 (SEQ ID NO: 1431) And the nucleic acid sequence of D153-AF9 (SEQ ID NO: 1432).
Figure 173-151 shows the nucleic acid sequences D153-AG6 (SEQ ID NO: 1433), D153-AG7 (SEQ ID NO: 1434), D153-AG9 (SEQ ID NO: 1435), D153-AH6 (SEQ ID NO: 1436) and D153-AH9 (SEQ ID NO: 1) No. 1437).
Figures 173-152 show D154-AC2 (SEQ ID NO: 1438), D154-AE9 (SEQ ID NO: 1439), D155-AB1 (SEQ ID NO: 1440), D155-AC1 (SEQ ID NO: 1441) and D155-AC2 (SEQ ID NO: 1442) The nucleic acid sequence of
Figure 173-153 shows D155-AE1 (SEQ ID NO: 1443), D155-AE2 (SEQ ID NO: 1444), D155-AF1 (SEQ ID NO: 1445), D155-AF2 (SEQ ID NO: 1446) and D155-AH2 (SEQ ID NO: 1447) The nucleic acid sequence of
Figures 173-154 show D156-AB1 (SEQ ID NO: 1448), D156-AC3 (SEQ ID NO: 1449), D156-AC4 (SEQ ID NO: 1450), D156-AD3 (SEQ ID NO: 1451) and D156-AF2 (SEQ ID NO: 1452) The nucleic acid sequence of
Figures 173-155 show D156-AF4 (SEQ ID NO: 1453), D156-AG1 (SEQ ID NO: 1454), D156-AG2 (SEQ ID NO: 1455), D156-AG4 (SEQ ID NO: 1456) and D156-AH1 (SEQ ID NO: 1457) The nucleic acid sequence of
Figures 173-156 show D157-AG4 (SEQ ID NO: 1458), D158-AB8 (SEQ ID NO: 1459), D158-AD5 (SEQ ID NO: 1460), D158-AD6 (SEQ ID NO: 1461) and D158-AD8 (SEQ ID NO: 1462) The nucleic acid sequence of
Figures 173-157 show D158-AE8 (SEQ ID NO: 1463), D159-AD2 (SEQ ID NO: 1464), D160-AB4 (SEQ ID NO: 1465), D160-AC4 (SEQ ID NO: 1466) and D160-AE4 (SEQ ID NO: 1467) The nucleic acid sequence of
Figure 173-158 shows D160-AF4 (SEQ ID NO: 1468), D160-AH4 (SEQ ID NO: 1469), D161-AE5 (SEQ ID NO: 1470), D161-AH5 (SEQ ID NO: 1471), D164-AB1 (SEQ ID NO: 1472) And the nucleic acid sequence of D164-AB3 (SEQ ID NO: 1473).
Figures 173-159 show D164-AC1 (SEQ ID NO: 1474), D164-AC2 (SEQ ID NO: 1475), D164-AC5 (SEQ ID NO: 1476), D164-AE1 (SEQ ID NO: 1477) and D164-AF1 (SEQ ID NO: 1478) The nucleic acid sequence of
Figures 173-160 show D165-AH8 (SEQ ID NO: 1479), D177-BB7 (SEQ ID NO: 1480), D178-AA6 (SEQ ID NO: 1481), D178-AD5 (SEQ ID NO: 1482) and D180-AA9 (SEQ ID NO: 1483). The nucleic acid sequence of
Figures 173-161 show D181-AB6 (SEQ ID NO: 1484), D181-AC6 (SEQ ID NO: 1485), D181-AD7 (SEQ ID NO: 1486), D181-AG7 (SEQ ID NO: 1487) and D181-AH6 (SEQ ID NO: 1488). The nucleic acid sequence of
Figures 173-162 show D182-AA1 (SEQ ID NO: 1489), D182-AA2 (SEQ ID NO: 1490), D182-AA4 (SEQ ID NO: 1491), D182-AB2 (SEQ ID NO: 1492) and D182-AC2 (SEQ ID NO: 1493) The nucleic acid sequence of
Figures 173-163 show D182-AC4 (SEQ ID NO: 1494), D182-AD1 (SEQ ID NO: 1495), D182-AD3 (SEQ ID NO: 1496), D182-AF1 (SEQ ID NO: 1497) and D182-AF4 (SEQ ID NO: 1498) The nucleic acid sequence of
Figure 173-164 shows D182-AG1 (SEQ ID NO: 1499), D183-AC8 (SEQ ID NO: 1500), D185-AB10 (SEQ ID NO: 1501), D185-AC10 (SEQ ID NO: 1502), D185-AF10 (SEQ ID NO: 1503) And the nucleic acid sequence of D185-BA1 (SEQ ID NO: 1504).
Figure 173-165 shows D185-BB3 (SEQ ID NO: 1505), D186-AA3 (SEQ ID NO: 1506), D186-AB4 (SEQ ID NO: 1507), D186-AC2 (SEQ ID NO: 1508), D186-AC3 (SEQ ID NO: 1509) And the nucleic acid sequence of D186-AD2 (SEQ ID NO: 1510).
Figure 173-166 shows D186-AD3 (SEQ ID NO: 1511), D186-AE3 (SEQ ID NO: 1512), D187-AB2 (SEQ ID NO: 1513), D187-AB3 (SEQ ID NO: 1514), D187-AC4 (SEQ ID NO: 1515) , D187-AE4 (SEQ ID NO: 1516) and D187-AF4 (SEQ ID NO: 1517).
Figure 173-167 shows D187-AG1 (SEQ ID NO: 1518), D187-AG2 (SEQ ID NO: 1519), D187-AG3 (SEQ ID NO: 1520), D187-AH2 (SEQ ID NO: 1521), D184-AA1 (SEQ ID NO: 1522) And the nucleic acid sequence of D188-AC7 (SEQ ID NO: 1523).
Figure 173-168 shows D188-AC8 (SEQ ID NO: 1524), D188-AD8 (SEQ ID NO: 1525), D188-AE8 (SEQ ID NO: 1526), D188-AF6 (SEQ ID NO: 1527), D188-AG5 (SEQ ID NO: 1528) And the nucleic acid sequence of D188-AG7 (SEQ ID NO: 1529).
Figure 173-169 shows D188-AH7 (SEQ ID NO: 1530), D189-AA12 (SEQ ID NO: 1531), D189-AB10 (SEQ ID NO: 1532), D189-AE9 (SEQ ID NO: 1533), D189-AE12 (SEQ ID NO: 1534) And the nucleic acid sequence of D189-AF12 (SEQ ID NO: 1535).
Figure 173-170 shows D189-AG10 (SEQ ID NO: 1536), D190-BA6 (SEQ ID NO: 1537), D190-BD6 (SEQ ID NO: 1538), D190-BF6 (SEQ ID NO: 1539), D190-BG6 (SEQ ID NO: 1540) And the nucleic acid sequence of D190-BH6 (SEQ ID NO: 1541).
Figures 173-171 show D191-BC5 (SEQ ID NO: 1542), D191-BD5 (SEQ ID NO: 1543), D191-BF5 (SEQ ID NO: 1544), D191-BG5 (SEQ ID NO: 1545) and D194-AA1 (SEQ ID NO: 1546). The nucleic acid sequence of
Figures 173-172 show D194-AA2 (SEQ ID NO: 1547), D194-AB1 (SEQ ID NO: 1548), D194-AB2 (SEQ ID NO: 1549), D194-AC1 (SEQ ID NO: 1550) and D194-AC2 (SEQ ID NO: 1551) The nucleic acid sequence of
Figures 173-173 show D194-AD1 (SEQ ID NO: 1552), D194-AD2 (SEQ ID NO: 1553), D194-AD3 (SEQ ID NO: 1554), D194-AE1 (SEQ ID NO: 1555) and D194-AE2 (SEQ ID NO: 1556) The nucleic acid sequence of
Figures 173-174 show D194-AE3 (SEQ ID NO: 1557), D194-AF1 (SEQ ID NO: 1558), D194-AF2 (SEQ ID NO: 1559), D194-AG2 (SEQ ID NO: 1560) and D194-AG3 (SEQ ID NO: 1561) The nucleic acid sequence of
Figure 173-175 shows D194-AH1 (SEQ ID NO: 1562), D194-AH2 (SEQ ID NO: 1563), D194-AH3 (SEQ ID NO: 1564), D195-AB6 (SEQ ID NO: 1565), D195-AD5 (SEQ ID NO: 1566) And the nucleic acid sequence of D195-AE4 (SEQ ID NO: 1567).
Figures 173-176 show D195-AE5 (SEQ ID NO: 1568), D195-AG5 (SEQ ID NO: 1569), D195-AH5 (SEQ ID NO: 1570), D196-AD7 (SEQ ID NO: 1571) and D196-AF7 (SEQ ID NO: 1572). The nucleic acid sequence of
Figures 173-177 show D196-AG7 (SEQ ID NO: 1573), D197-AE8 (SEQ ID NO: 1574), D198-AB9 (SEQ ID NO: 1575), D198-AC9 (SEQ ID NO: 1576) and D198-AF9 (SEQ ID NO: 1577). The nucleic acid sequence of
Figures 173-178 show D199-AA10 (SEQ ID NO: 1578), D199-AB10 (SEQ ID NO: 1579), D199-AD10 (SEQ ID NO: 1580), D199-AF10 (SEQ ID NO: 1581) and D199-AG10 (SEQ ID NO: 1582). The nucleic acid sequence of
Figures 173-179 show D200-AB11 (SEQ ID NO: 1583), D200-AC11 (SEQ ID NO: 1584), D200-AD11 (SEQ ID NO: 1585), D200-AE11 (SEQ ID NO: 1586) and D200-AG11 (SEQ ID NO: 1587) The nucleic acid sequence of
Figure 173-180 shows D200-AH11 (SEQ ID NO: 1588), D201-AD12 (SEQ ID NO: 1589), D201-AE12 (SEQ ID NO: 1590), D201-AF12 (SEQ ID NO: 1591), D201-AG12 (SEQ ID NO: 1592) And the nucleic acid sequence of D203-BE11 (SEQ ID NO: 1593).
Figures 173-181 show D203-BF11 (SEQ ID NO: 1594), D204-AA1 (SEQ ID NO: 1595), D204-AA2 (SEQ ID NO: 1596), D204-AA4 (SEQ ID NO: 1597) and D204-AB1 (SEQ ID NO: 1598). The nucleic acid sequence of
Figures 173-182 show D204-AB3 (SEQ ID NO: 1599), D204-AC1 (SEQ ID NO: 1600), D204-AC2 (SEQ ID NO: 1601), D204-AC4 (SEQ ID NO: 1602) and D204-AD1 (SEQ ID NO: 1603) The nucleic acid sequence of
Figures 173-183 show D204-AD2 (SEQ ID NO: 1604), D204-AD3 (SEQ ID NO: 1605), D204-AD4 (SEQ ID NO: 1606), D204-AE3 (SEQ ID NO: 1607) and D204-AE4 (SEQ ID NO: 1608) The nucleic acid sequence of
Figures 173-184 show D204-AF1 (SEQ ID NO: 1609), D204-AF3 (SEQ ID NO: 1610), D204-AF4 (SEQ ID NO: 1611), D204-AG1 (SEQ ID NO: 1612) and D204-AG2 (SEQ ID NO: 1613) The nucleic acid sequence of
Figures 173-185 show D204-AG3 (SEQ ID NO: 1614), D203-BA11 (SEQ ID NO: 1615), D204-AG4 (SEQ ID NO: 1616), D204-AH2 (SEQ ID NO: 1617) and D204-AH1 (SEQ ID NO: 1618) The nucleic acid sequence of
Figures 173-186 show D204-AH3 (SEQ ID NO: 1619), D205-BC9 (SEQ ID NO: 1620), D205-BD9 (SEQ ID NO: 1621), D206-CB3 (SEQ ID NO: 1622), D206-CE3 (SEQ ID NO: 1623) And the nucleic acid sequence of D206-CF3 (SEQ ID NO: 1624).
FIGS. 173-187 are the nucleic acid sequences of D206-CG1 (SEQ ID NO: 1625), D206-CH1 (SEQ ID NO: 1626), D210-BF4 (SEQ ID NO: 1627) and D210-BF6 (SEQ ID NO: 1628).
Figures 173-188 show D210-BH6 (SEQ ID NO: 1629), D211-BA9 (SEQ ID NO: 1630), D211-BB8 (SEQ ID NO: 1631), D211-BB9 (SEQ ID NO: 1632), D211-BC9 (SEQ ID NO: 1633) And the nucleic acid sequence of D211-BD8 (SEQ ID NO: 1634).
FIGS. 173-189 are the nucleic acid sequences of D211-BD9 (SEQ ID NO: 1635), D211-BE7 (SEQ ID NO: 1636), D211-BE8 (SEQ ID NO: 1637) and D211-BF8 (SEQ ID NO: 1638).
Figures 173-190 show D211-BF9 (SEQ ID NO: 1639), D211-BG8 (SEQ ID NO: 1640), D211-BH7 (SEQ ID NO: 1641), D212-BB11 (SEQ ID NO: 1642) and D212-BB12 (SEQ ID NO: 1643) The nucleic acid sequence of
Figure 173-191 shows D212-BD10 (SEQ ID NO: 1644), D212-BD11 (SEQ ID NO: 1645), D212-BE10 (SEQ ID NO: 1646), D212-BE11 (SEQ ID NO: 1647), D212-BF10 (SEQ ID NO: 1648) And the nucleic acid sequence of D212-BF11 (SEQ ID NO: 1649).
Figures 173-192 show D213-BD1 (SEQ ID NO: 1650), D213-BF2 (SEQ ID NO: 1651), D214-AA1 (SEQ ID NO: 1652), D214-AA3 (SEQ ID NO: 1653) and D214-AC1 (SEQ ID NO: 1654) The nucleic acid sequence of
Figures 173-193 show D214-AE1 (SEQ ID NO: 1655), D214-AE3 (SEQ ID NO: 1656), D214-AG1 (SEQ ID NO: 1657), D214-AH2 (SEQ ID NO: 1658) and D214-AH3 (SEQ ID NO: 1659) The nucleic acid sequence of
Figure 173-194 shows D216-AB7 (SEQ ID NO: 1660), D216-AC8 (SEQ ID NO: 1661), D216-AH9 (SEQ ID NO: 1662), D219-BA1 (SEQ ID NO: 1663), D219-BB1 (SEQ ID NO: 1664) And the nucleic acid sequence of D219-BB2 (SEQ ID NO: 1665).
Figure 173-195 shows D219-BC1 (SEQ ID NO: 1666), D219-BD1 (SEQ ID NO: 1667), D219-BD2 (SEQ ID NO: 1668), D219-BE1 (SEQ ID NO: 1669), D219-BE2 (SEQ ID NO: 1670) And the nucleic acid sequence of D219-BF2 (SEQ ID NO: 1671).
Figures 173-196 show D219-BH1 (SEQ ID NO: 1672), D220-BF6 (SEQ ID NO: 1673), D220-BD6 (SEQ ID NO: 1674), D223-BC11 (SEQ ID NO: 1675) and D221-BC7 (SEQ ID NO: 1676) The nucleic acid sequence of
Figure 173-197 shows D227-AE3 (SEQ ID NO: 1677), D223-BB10 (SEQ ID NO: 1678), D221-BF9 (SEQ ID NO: 1679), D229-AD2 (SEQ ID NO: 1680), D229-AE2 (SEQ ID NO: 1681) , D229-AF2 (SEQ ID NO: 1682) and D229-AG1 (SEQ ID NO: 1683).
Figure 173-198 shows D229-AH1 (SEQ ID NO: 1684), D230-AB1 (SEQ ID NO: 1685), D230-AC1 (SEQ ID NO: 1686), D230-AC2 (SEQ ID NO: 1687), D230-AF2 (SEQ ID NO: 1688) And the nucleic acid sequence of D230-AG1 (SEQ ID NO: 1689).
Figures 173-199 show D230-AG2 (SEQ ID NO: 1690), D231-AA1 (SEQ ID NO: 1691), D231-AA2 (SEQ ID NO: 1692), D231-AA3 (SEQ ID NO: 1693), D231-AC1 (SEQ ID NO: 1694) And the nucleic acid sequence of D231-AC2 (SEQ ID NO: 1695).
Figure 173-200 shows D231-AD2 (SEQ ID NO: 1696), D231-AE1 (SEQ ID NO: 1697), D231-AG2 (SEQ ID NO: 1698), D231-AH1 (SEQ ID NO: 1699), D231-AH2 (SEQ ID NO: 1700) And the nucleic acid sequence of D231-AH3 (SEQ ID NO: 1701).
FIGS. 173-201 are the nucleic acid sequences of D232-AD4 (SEQ ID NO: 1702), D232-AE5 (SEQ ID NO: 1703), D232-AE6 (SEQ ID NO: 1704) and D232-AF5 (SEQ ID NO: 1705).
Figure 173-202 shows D233-AA7 (SEQ ID NO: 1706), D233-AA8 (SEQ ID NO: 1707), D233-AB7 (SEQ ID NO: 1708), D233-AC7 (SEQ ID NO: 1709) and D233-AC8 (SEQ ID NO: 1710) The nucleic acid sequence of
Figure 173-203 shows D233-AH9 (SEQ ID NO: 1711), D234-AA11 (SEQ ID NO: 1712), D234-AC11 (SEQ ID NO: 1713), D234-AD11 (SEQ ID NO: 1714), D238-AA2 (SEQ ID NO: 1715) And the nucleic acid sequence of D239-BC3 (SEQ ID NO: 1716).
Figures 173-204 show D239-BD4 (SEQ ID NO: 1717), D239-BD6 (SEQ ID NO: 1718), D239-BE4 (SEQ ID NO: 1719), D240-BB7 (SEQ ID NO: 1720) and D241-BC9 (SEQ ID NO: 1721) The nucleic acid sequence of
Figure 173-205 shows D241-BE9 (SEQ ID NO: 1722), D242-BA11 (SEQ ID NO: 1723), D242-BB11 (SEQ ID NO: 1724), D242-BB12 (SEQ ID NO: 1725), D242-BC12 (SEQ ID NO: 1726) And the nucleic acid sequence of D242-BF12 (SEQ ID NO: 1727).
Figure 173-206 shows D242-BG12 (SEQ ID NO: 1728), D242-BH11 (SEQ ID NO: 1729), D244-AA5 (SEQ ID NO: 1730), D244-AA6 (SEQ ID NO: 1731), D244-AF6 (SEQ ID NO: 1732) And the nucleic acid sequence of D245-AE7 (SEQ ID NO: 1733).
Figures 173-207 show D245-AE8 (SEQ ID NO: 1734), D245-AF7 (SEQ ID NO: 1735), D246-AA12 (SEQ ID NO: 1736), D248-AG4 (SEQ ID NO: 1737) and D249-AG9 (SEQ ID NO: 1738) The nucleic acid sequence of
Figure 173-208 shows D250-AE10 (SEQ ID NO: 1739), D251-AF2 (SEQ ID NO: 1740), D251-AG2 (SEQ ID NO: 1741), D252-AA5 (SEQ ID NO: 1742) and D252-AB5 (SEQ ID NO: 1743) The nucleic acid sequence of
Figure 173-209 shows D252-AC5 (SEQ ID NO: 1744), D252-AD5 (SEQ ID NO: 1745), D252-AF4 (SEQ ID NO: 1746), D252-AF5 (SEQ ID NO: 1747), D252-AG5 (SEQ ID NO: 1748) And the nucleic acid sequence of D254-AE2 (SEQ ID NO: 1749).
Figure 173-210 shows D254-AG2 (SEQ ID NO: 1750), D255-AA5 (SEQ ID NO: 1751), D255-AA6 (SEQ ID NO: 1752), D255-AD5 (SEQ ID NO: 1753) and D255-AD6 (SEQ ID NO: 1754) The nucleic acid sequence of
FIGS. 173-211 are the nucleic acid sequences of D255-AF5 (SEQ ID NO: 1755), D255-AG5 (SEQ ID NO: 1756), D256-AA10 (SEQ ID NO: 1757) and D256-AB10 (SEQ ID NO: 1758).
Figures 173-212 show D256-AC10 (SEQ ID NO: 1759), D256-AE10 (SEQ ID NO: 1760), D256-AF9 (SEQ ID NO: 1761), D256-AF10 (SEQ ID NO: 1762) and D256-AG10 (SEQ ID NO: 1763) The nucleic acid sequence of
Figures 173-213 show D256-AH10 (SEQ ID NO: 1764), D258-AC5 (SEQ ID NO: 1765), D263-AE12 (SEQ ID NO: 1766), D263-AF12 (SEQ ID NO: 1767) and D263-AH12 (SEQ ID NO: 1768) The nucleic acid sequence of
Figures 173-214 show D264-AA1 (SEQ ID NO: 1769), D264-AA2 (SEQ ID NO: 1770), D264-AA3 (SEQ ID NO: 1771), D264-AB2 (SEQ ID NO: 1772) and D264-AC3 (SEQ ID NO: 1773) The nucleic acid sequence of
FIGS. 173-215 are the nucleic acid sequences of D264-AD3 (SEQ ID NO: 1774), D264-AE1 (SEQ ID NO: 1775), D264-AE2 (SEQ ID NO: 1776) and D264-AE3 (SEQ ID NO: 1777).
FIGS. 173-216 are the nucleic acid sequences of D264-AF2 (SEQ ID NO: 1778), D264-AG2 (SEQ ID NO: 1779), D264-AH2 (SEQ ID NO: 1780) and D265-AA4 (SEQ ID NO: 1781).
FIGS. 173-217 are the nucleic acid sequences of D265-AA6 (SEQ ID NO: 1782), D265-AC5 (SEQ ID NO: 1783), D265-AC6 (SEQ ID NO: 1784) and D265-AD4 (SEQ ID NO: 1785).
Figures 173-218 show D265-AD5 (SEQ ID NO: 1786), D266-AB7 (SEQ ID NO: 1787), D266-AB8 (SEQ ID NO: 1788), D266-AC9 (SEQ ID NO: 1789) and D266-AD7 (SEQ ID NO: 1790) The nucleic acid sequence of
Figures 173-219 show D267-AD10 (SEQ ID NO: 1791), D268-AA2 (SEQ ID NO: 1792), D268-AC3 (SEQ ID NO: 1793), D268-AD1 (SEQ ID NO: 1794) and D268-AD2 (SEQ ID NO: 1795) The nucleic acid sequence of
FIGS. 173-220 are the nucleic acid sequences of D268-AD3 (SEQ ID NO: 1796), D268-AE3 (SEQ ID NO: 1797), D268-AG2 (SEQ ID NO: 1798) and D268-AG3 (SEQ ID NO: 1799).
Figure 173-221 shows D269-AA5 (SEQ ID NO: 1800), D269-AD4 (SEQ ID NO: 1801), D269-AE4 (SEQ ID NO: 1802), D269-AF5 (SEQ ID NO: 1803), D269-AF6 (SEQ ID NO: 1804) And the nucleic acid sequence of D270-AA8 (SEQ ID NO: 1805).
Figures 173-222 show D270-AB9 (SEQ ID NO: 1806), D270-AD8 (SEQ ID NO: 1807), D270-AD9 (SEQ ID NO: 1808), D270-AE9 (SEQ ID NO: 1809) and D270-AF8 (SEQ ID NO: 1810) The nucleic acid sequence of
FIGS. 173-223 are the nucleic acid sequences of D271-AG11 (SEQ ID NO: 1811), D271-AH11 (SEQ ID NO: 1812), D276-AD5 (SEQ ID NO: 1813) and D276-AG6 (SEQ ID NO: 1814).
Figures 173-224 show D276-AH4 (SEQ ID NO: 1815), D276-AH6 (SEQ ID NO: 1816), D277-AE8 (SEQ ID NO: 1817), D277-AF9 (SEQ ID NO: 1818) and D277-AH9 (SEQ ID NO: 1819) The nucleic acid sequence of
Figures 173-225 show D278-AF10 (SEQ ID NO: 1820), D279-AA3 (SEQ ID NO: 1821), D279-AB2 (SEQ ID NO: 1822), D279-AC1 (SEQ ID NO: 1823) and D279-AD2 (SEQ ID NO: 1824) The nucleic acid sequence of
Figures 173-226 show D279-AE1 (SEQ ID NO: 1825), D279-AE3 (SEQ ID NO: 1826), D279-AG3 (SEQ ID NO: 1827), D279-BA1 (SEQ ID NO: 1828) and D279-BA2 (SEQ ID NO: 1829) The nucleic acid sequence of
Figures 173-227 show D279-BB3 (SEQ ID NO: 1830), D279-BC2 (SEQ ID NO: 1831), D279-BD2 (SEQ ID NO: 1832), D279-BE2 (SEQ ID NO: 1833) and D279-BF3 (SEQ ID NO: 1834) The nucleic acid sequence of
Figures 173-228 show D279-BG1 (SEQ ID NO: 1835), D279-BH3 (SEQ ID NO: 1836), D280-AB5 (SEQ ID NO: 1837), D280-AB6 (SEQ ID NO: 1838) and D280-AC4 (SEQ ID NO: 1839) The nucleic acid sequence of
Figure 173-229 shows D280-AC6 (SEQ ID NO: 1840), D280-AD4 (SEQ ID NO: 1841), D280-AD5 (SEQ ID NO: 1842), D280-AD6 (SEQ ID NO: 1843) and D280-AE4 (SEQ ID NO: 1844) The nucleic acid sequence of
Figures 173-230 shows D280-AE5 (SEQ ID NO: 1845), D280-AE6 (SEQ ID NO: 1846), D280-AF5 (SEQ ID NO: 1847), D280-AF6 (SEQ ID NO: 1848) and D280-AG4 (SEQ ID NO: 1849) The nucleic acid sequence of
Figures 173-231 show D280-AG5 (SEQ ID NO: 1850), D280-AG6 (SEQ ID NO: 1851), D280-AH4 (SEQ ID NO: 1852), D280-AH5 (SEQ ID NO: 1853) and D280-BC4 (SEQ ID NO: 1854) The nucleic acid sequence of
FIG. 173-232 shows the nucleic acid sequences of D280-BD4 (SEQ ID NO: 1855), D280-BE4 (SEQ ID NO: 1856), D280-BE6 (SEQ ID NO: 1857) and D280-BF4 (SEQ ID NO: 1858).
Figures 173-233 show D280-BG4 (SEQ ID NO: 1859), D280-BH4 (SEQ ID NO: 1860), D280-BH6 (SEQ ID NO: 1861), D281-AA8 (SEQ ID NO: 1862) and D281-AD7 (SEQ ID NO: 1863) The nucleic acid sequence of
Figure 173-234 shows D281-AD8 (SEQ ID NO: 1864), D281-AE7 (SEQ ID NO: 1865), D281-AE8 (SEQ ID NO: 1866), D281-AE9 (SEQ ID NO: 1867), D281-AG7 (SEQ ID NO: 1868) And the nucleic acid sequence of D282-AB10 (SEQ ID NO: 1869).
Figures 173-235 show D282-AB11 (SEQ ID NO: 1870), D282-AD10 (SEQ ID NO: 1871), D282-AH11 (SEQ ID NO: 1872), D282-BA10 (SEQ ID NO: 1873), D282-BB10 (SEQ ID NO: 1874) and D282-BD10 (SEQ ID NO: 1875).
Figures 173-236 show D288-AB3 (SEQ ID NO: 1876), D289-AA4 (SEQ ID NO: 1877), D289-AA6 (SEQ ID NO: 1878), D289-AB4 (SEQ ID NO: 1879) and D289-AB6 (SEQ ID NO: 1880) The nucleic acid sequence of
Figures 173-237 show D289-AD4 (SEQ ID NO: 1881), D289-AE4 (SEQ ID NO: 1882), D289-AE6 (SEQ ID NO: 1883), D289-AF4 (SEQ ID NO: 1884) and D289-AF6 (SEQ ID NO: 1885) The nucleic acid sequence of
Figures 173-238 show D289-AG4 (SEQ ID NO: 1886), D289-AG6 (SEQ ID NO: 1887), D289-AH4 (SEQ ID NO: 1888), D289-AH6 (SEQ ID NO: 1889) and D290-AA7 (SEQ ID NO: 1890). The nucleic acid sequence of
Figures 173-239 show D290-AA8 (SEQ ID NO: 1891), D290-AA9 (SEQ ID NO: 1892), D290-AB7 (SEQ ID NO: 1893), D290-AB9 (SEQ ID NO: 1894) and D290-AC8 (SEQ ID NO: 1895) The nucleic acid sequence of
Figures 173-240 show D290-AC9 (SEQ ID NO: 1896), D290-AD7 (SEQ ID NO: 1897), D290-AD8 (SEQ ID NO: 1898), D290-AD9 (SEQ ID NO: 1899) and D291-AA12 (SEQ ID NO: 1900) The nucleic acid sequence of
Figure 173-241 shows D291-AB10 (SEQ ID NO: 1901), D291-AB11 (SEQ ID NO: 1902), D291-AC12 (SEQ ID NO: 1903), D291-AD11 (SEQ ID NO: 1904) and D291-AD12 (SEQ ID NO: 1905) The nucleic acid sequence of
Figure 173-242 shows D291-AE10 (SEQ ID NO: 1906), D291-AE12 (SEQ ID NO: 1907), D291-AF10 (SEQ ID NO: 1908), D291-AF12 (SEQ ID NO: 1909) and D291-AG11 (SEQ ID NO: 1910) The nucleic acid sequence of
Figure 173-243 shows D291-AG12 (SEQ ID NO: 1911), D291-AH11 (SEQ ID NO: 1912), D292-AA2 (SEQ ID NO: 1913), D292-AA3 (SEQ ID NO: 1914), D292-AB2 (SEQ ID NO: 1915) And the nucleic acid sequence of D292-AC2 (SEQ ID NO: 1916).
Figure 173-244 shows D292-AD1 (SEQ ID NO: 1917), D292-AD2 (SEQ ID NO: 1918), D292-AE1 (SEQ ID NO: 1919), D292-AE2 (SEQ ID NO: 1920), D292-AF1 (SEQ ID NO: 1921) , D292-AF3 (SEQ ID NO: 1922) and D292-AH3 (SEQ ID NO: 1923).
Figures 173-245 show D291-AA10 (SEQ ID NO: 1924), D294-AB7 (SEQ ID NO: 1925), D294-AB8 (SEQ ID NO: 1926), D294-AB9 (SEQ ID NO: 1927) and D294-AC7 (SEQ ID NO: 1928) The nucleic acid sequence of
Figures 173-246 show D294-AC8 (SEQ ID NO: 1929), D294-AE9 (SEQ ID NO: 1930), D294-AG8 (SEQ ID NO: 1931), D294-AH7 (SEQ ID NO: 1932) and D295-AA3 (SEQ ID NO: 1933) The nucleic acid sequence of
Figures 173-247 show D295-AB1 (SEQ ID NO: 1934), D295-AB2 (SEQ ID NO: 1935), D295-AC2 (SEQ ID NO: 1936), D295-AC3 (SEQ ID NO: 1937) and D295-AD1 (SEQ ID NO: 1938) The nucleic acid sequence of
Figures 173-248 show D295-AD2 (SEQ ID NO: 1939), D295-AE3 (SEQ ID NO: 1940), D295-AF1 (SEQ ID NO: 1941), D295-AF2 (SEQ ID NO: 1942) and D295-AG3 (SEQ ID NO: 1943) The nucleic acid sequence of
Figures 173-249 show D295-AH1 (SEQ ID NO: 1944), D296-AA6 (SEQ ID NO: 1945), D296-AE5 (SEQ ID NO: 1946), D296-AF5 (SEQ ID NO: 1947) and D296-AG4 (SEQ ID NO: 1948) The nucleic acid sequence of
Figure 173-250 shows D296-AG5 (SEQ ID NO: 1949), D297-AA7 (SEQ ID NO: 1950), D297-AA8 (SEQ ID NO: 1951), D297-AB7 (SEQ ID NO: 1952) and D297-AE7 (SEQ ID NO: 1953) The nucleic acid sequence of
Figure 173-251 shows D297-AF7 (SEQ ID NO: 1954), D298-AA10 (SEQ ID NO: 1955), D298-AB11 (SEQ ID NO: 1956), D298-AF11 (SEQ ID NO: 1957) and D298-AG12 (SEQ ID NO: 1958) The nucleic acid sequence of
Figures 173-252 show D35-40 (SEQ ID NO: 1959), D35-AC6 (SEQ ID NO: 1960), D35-BB2 (SEQ ID NO: 1961), D55-AB9 (SEQ ID NO: 1962) and D55-BB1 (SEQ ID NO: 1963) The nucleic acid sequence of
Figure 173-253 shows D55-BB2 (SEQ ID NO: 1964), D55-BB3 (SEQ ID NO: 1965), D55-BB7 (SEQ ID NO: 1966), D56-AA1 (SEQ ID NO: 1967), D56-AE4 (SEQ ID NO: 1968) And the nucleic acid sequence of D56-AE9 (SEQ ID NO: 1969).
Figures 173-254 show D56-AD1 (SEQ ID NO: 1970), D56-AG12 (SEQ ID NO: 1971), D56-AH11 (SEQ ID NO: 1972), D57-AA5 (SEQ ID NO: 1973) and D57-AA7 (SEQ ID NO: 1974) The nucleic acid sequence of
Figure 173-255 shows D57-AB10 (SEQ ID NO: 1975), D57-AC2 (SEQ ID NO: 1976), D57-AC3 (SEQ ID NO: 1977), D57-AC6 (SEQ ID NO: 1978), D57-AC9 (SEQ ID NO: 1979) And the nucleic acid sequence of D57-AC11 (SEQ ID NO: 1980).
Figures 173-256 show D57-AD3 (SEQ ID NO: 1981), D57-AE4 (SEQ ID NO: 1982), D57-AE6 (SEQ ID NO: 1983), D57-AE9 (SEQ ID NO: 1984) and D57-AF4 (SEQ ID NO: 1985). The nucleic acid sequence of
Figure 173-257 shows D57-AF11 (SEQ ID NO: 1986), D57-AG3 (SEQ ID NO: 1987), D57-AH11 (SEQ ID NO: 1988), D58-AB2 (SEQ ID NO: 1989), D58-AC8 (SEQ ID NO: 1990) And the nucleic acid sequence of D58-AG9 (SEQ ID NO: 1991).
Figures 173-258 show D58-BA9 (SEQ ID NO: 1992), D58-BB9 (SEQ ID NO: 1993), D58-BC1 (SEQ ID NO: 1994), D58-BC10 (SEQ ID NO: 1995), D58-BC11 (SEQ ID NO: 1996) And the nucleic acid sequence of D58-BD4 (SEQ ID NO: 1997).
Figure 173-259 shows D58-BE8 (SEQ ID NO: 1998), D58-BF4 (SEQ ID NO: 1999), D60-AB4 (SEQ ID NO: 2000), D60-AC4 (SEQ ID NO: 2001), D60-AD11 (SEQ ID NO: 2002) And the nucleic acid sequence of D60-AF9 (SEQ ID NO: 2003).
Figure 173-260 shows D65-AB6 (SEQ ID NO: 2004), D65-AC3 (SEQ ID NO: 2005), D65-AC9 (SEQ ID NO: 2006), D65-AC12 (SEQ ID NO: 2007), D65-AE3 (SEQ ID NO: 2008) And the nucleic acid sequence of D65-AG1 (SEQ ID NO: 2009).
Figure 173-261 shows D65-CE10 (SEQ ID NO: 2010), D65-CE11 (SEQ ID NO: 2011), D65-CF11 (SEQ ID NO: 2012), D65-CH5 (SEQ ID NO: 2013), D66-AA1 (SEQ ID NO: 2014) And the nucleic acid sequence of D66-AA3 (SEQ ID NO: 2015).
Figures 173-262 show D66-AE5 (SEQ ID NO: 2016), D66-AF1 (SEQ ID NO: 2017), D66-AG2 (SEQ ID NO: 2018), D66-AG6 (SEQ ID NO: 2019) and D66-AH3 (SEQ ID NO: 2020) The nucleic acid sequence of
Figure 173-263 shows D66-BA11 (SEQ ID NO: 2021), D66-BD6 (SEQ ID NO: 2022), D66-BD8 (SEQ ID NO: 2023), D67-AA5 (SEQ ID NO: 2024), D67-AD3 (SEQ ID NO: 2025) And the nucleic acid sequence of D67-AE1 (SEQ ID NO: 2026).
Figure 173-264 shows D67-AE4 (SEQ ID NO: 2027), D67-AG4 (SEQ ID NO: 2028), D68-AF3 (SEQ ID NO: 2029), D70A-AE1 (SEQ ID NO: 2030), D70A-AG7 (SEQ ID NO: 2031) And the nucleic acid sequence of D70A-BC6 (SEQ ID NO: 2032).
Figures 173-265 show D70A-BF7 (SEQ ID NO: 2033), D70A-BF8 (SEQ ID NO: 2034), D70A-BH1 (SEQ ID NO: 2035), D70A-BH3 (SEQ ID NO: 2036) and D73A-AA1 (SEQ ID NO: 2037) The nucleic acid sequence of
Figure 173-266 shows D73A-AA3 (SEQ ID NO: 2038), D73A-AA5 (SEQ ID NO: 2039), D73A-AA6 (SEQ ID NO: 2040), D73A-AA8 (SEQ ID NO: 2041), D73A-AA9 (SEQ ID NO: 2042) And the nucleic acid sequence of D73A-AB1 (SEQ ID NO: 2043).
Figures 173-267 show D73A-AB3 (SEQ ID NO: 2044), D73A-AB5 (SEQ ID NO: 2045), D73A-AB10 (SEQ ID NO: 2046), D73A-AC2 (SEQ ID NO: 2047) and D73A-AC5 (SEQ ID NO: 2048) The nucleic acid sequence of
Figure 173-268 shows D73A-AC11 (SEQ ID NO: 2049), D73A-AC12 (SEQ ID NO: 2050), D73A-AD7 (SEQ ID NO: 2051), D73A-AD10 (SEQ ID NO: 2052), D73A-AE6 (SEQ ID NO: 2053) And the nucleic acid sequence of D73A-AE7 (SEQ ID NO: 2054).
Figure 173-269 shows D73A-AE8 (SEQ ID NO: 2055), D73A-AE12 (SEQ ID NO: 2056), D73A-AF5 (SEQ ID NO: 2057), D73A-AF6 (SEQ ID NO: 2058), D73A-AF12 (SEQ ID NO: 2059) And the nucleic acid sequence of D73A-AG4 (SEQ ID NO: 2060).
Figures 173-270 show D73A-AG6 (SEQ ID NO: 2061), D73A-AG10 (SEQ ID NO: 2062), D73A-AH2 (SEQ ID NO: 2063), D73A-AH3 (SEQ ID NO: 2064) and D73A-AH10 (SEQ ID NO: 2065) The nucleic acid sequence of
Figures 173-271 show D93-AD3 (SEQ ID NO: 2066), D97-AD3 (SEQ ID NO: 2067), D97-AE3 (SEQ ID NO: 2068), D97-AF1 (SEQ ID NO: 2069) and D113-AH8 (SEQ ID NO: 2070). The nucleic acid sequence of
Figures 173-272 show D114-AA10 (SEQ ID NO: 2071), D114-AC11 (SEQ ID NO: 2072), D131-AD1 (SEQ ID NO: 2073), D133-AD7 (SEQ ID NO: 2074) and D139-AD2 (SEQ ID NO: 2075) The nucleic acid sequence of
Figure 173-273 shows D139-AF2 (SEQ ID NO: 2076), D144A-AB10 (SEQ ID NO: 2077), D144A-AE9 (SEQ ID NO: 2078), D144A-AG12 (SEQ ID NO: 2079), D145-AC7 (SEQ ID NO: 2080) And the nucleic acid sequence of D145-AH7 (SEQ ID NO: 2081).
Figure 173-274 shows D150-AA3 (SEQ ID NO: 2082), D150-AG3 (SEQ ID NO: 2083), D151-AG3 (SEQ ID NO: 2084), D153-AA8 (SEQ ID NO: 2085), D153-AB7 (SEQ ID NO: 2086) And the nucleic acid sequence of D153-AC9 (SEQ ID NO: 2087).
Figure 173-275 shows D153-AF8 (SEQ ID NO: 2088), D155-AA2 (SEQ ID NO: 2089), D155-AG2 (SEQ ID NO: 2090), D156-AH3 (SEQ ID NO: 2091), D159-AA2 (SEQ ID NO: 2092) And the nucleic acid sequence of D164-AD1 (SEQ ID NO: 2093).
Figure 173-276 shows D181-AG6 (SEQ ID NO: 2094), D182-AB1 (SEQ ID NO: 2095), D182-AF2 (SEQ ID NO: 2096), D185-AE10 (SEQ ID NO: 2097), D186-AH3 (SEQ ID NO: 2098) And the nucleic acid sequence of D188-AC6 (SEQ ID NO: 2099).
Figure 173-277 shows D188-AF5 (SEQ ID NO: 2100), D189-AD12 (SEQ ID NO: 2101), D258-AG6 (SEQ ID NO: 2102), D194-AA3 (SEQ ID NO: 2103), D194-AB3 (SEQ ID NO: 2104) , D198-AD9 (SEQ ID NO: 2105) and D198-AE9 (SEQ ID NO: 2106).
Figures 173-278 show D203-BB11 (SEQ ID NO: 2107), D203-BC11 (SEQ ID NO: 2108), D204-AA3 (SEQ ID NO: 2109), D204-AF2 (SEQ ID NO: 2110), D206-CB1 (SEQ ID NO: 2111) And the nucleic acid sequence of D214-AB1 (SEQ ID NO: 2112).
Figures 173-279 show D214-AF2 (SEQ ID NO: 2113), D216-AA7 (SEQ ID NO: 2114), D216-AD7 (SEQ ID NO: 2115), D219-BA2 (SEQ ID NO: 2116), D219-BF1 (SEQ ID NO: 2117) And the nucleic acid sequence of D229-AB2 (SEQ ID NO: 2118).
Figure 173-280 shows D230-AD2 (SEQ ID NO: 2119), D230-AE2 (SEQ ID NO: 2120), D234-AE12 (SEQ ID NO: 2121), D241-BG10 (SEQ ID NO: 2122), D249-AA9 (SEQ ID NO: 2123) And the nucleic acid sequence of D255-AC5 (SEQ ID NO: 2124).
Figures 173-281 show D270-AE7 (SEQ ID NO: 2125), D270-AE8 (SEQ ID NO: 2126), D276-AH5 (SEQ ID NO: 2127), D279-AA2 (SEQ ID NO: 2128) and D279-AB3 (SEQ ID NO: 2129) The nucleic acid sequence of
FIGS. 173-282 are the nucleic acid sequences of D279-AC2 (SEQ ID NO: 2130), D279-AC3 (SEQ ID NO: 2131), D279-AD3 (SEQ ID NO: 2132) and D279-AE2 (SEQ ID NO: 2133).
Figures 173-283 show D279-AG2 (SEQ ID NO: 2134), D279-AH3 (SEQ ID NO: 2135), D280-BF6 (SEQ ID NO: 2136), D281-AB8 (SEQ ID NO: 2137) and D281-AC7 (SEQ ID NO: 2138) The nucleic acid sequence of
Figures 173-284 show D282-AG10 (SEQ ID NO: 2139), D288-AC2 (SEQ ID NO: 2140), D289-AC6 (SEQ ID NO: 2141), D290-AB8 (SEQ ID NO: 2142) and D291-AD10 (SEQ ID NO: 2143) The nucleic acid sequence of
Figure 173-285 shows D291-AE11 (SEQ ID NO: 2144), D291-AH12 (SEQ ID NO: 2145), D292-AB3 (SEQ ID NO: 2146), D292-AD3 (SEQ ID NO: 2147), D292-AE3 (SEQ ID NO: 2148) And the nucleic acid sequence of D294-AE7 (SEQ ID NO: 2149).
Figure 173-286 shows D419-AH11 (SEQ ID NO: 2150), D295-AE2 (SEQ ID NO: 2151), D295-AG2 (SEQ ID NO: 2152), D295-AH2 (SEQ ID NO: 2153), D295-AH3 (SEQ ID NO: 2154) And the nucleic acid sequence of D296-AD4 (SEQ ID NO: 2155).
Figures 173-287 show D296-AF4 (SEQ ID NO: 2156), D296-AG6 (SEQ ID NO: 2157), D297-AD7 (SEQ ID NO: 2158), D297-AG7 (SEQ ID NO: 2159) and D298-AD11 (SEQ ID NO: 2160) The nucleic acid sequence of
Figures 173-288 show D418-AH9 (SEQ ID NO: 2161), D418-AG10 (SEQ ID NO: 2162), D416-AA6 (SEQ ID NO: 2163), D414-AA2 (SEQ ID NO: 2164) and D269-AD5 (SEQ ID NO: 2165). The nucleic acid sequence of
Figures 173-289 show D268-AE1 (SEQ ID NO: 2166), D258-AF6 (SEQ ID NO: 2167), D256-AG9 (SEQ ID NO: 2168), D255-AH6 (SEQ ID NO: 2169) and D255-AE5 (SEQ ID NO: 2170) The nucleic acid sequence of
Figure 173-290 shows D419-AE11 (SEQ ID NO: 2171), D142-AA10 (SEQ ID NO: 2172), D140-AH4 (SEQ ID NO: 2173), D66-AF7 (SEQ ID NO: 2174) and D66-AA7 (SEQ ID NO: 2175) The nucleic acid sequence of
Figures 173-291 show D65-AF9 (SEQ ID NO: 2176), D65-AB3 (SEQ ID NO: 2177), D65-AB2 (SEQ ID NO: 2178), D144A-AA12 (SEQ ID NO: 2179) and D64-7 (SEQ ID NO: 2180) The nucleic acid sequence of
Figures 173-292 show D109-BF9 (SEQ ID NO: 2181), D109-BG9 (SEQ ID NO: 2182), D118-AA11 (SEQ ID NO: 2183), D142-AB11 (5 ') (SEQ ID NO: 2184) and D142-AE10 ( 5 ′) is the nucleic acid sequence of (SEQ ID NO: 2185).
Figure 173-293 shows D207-AH5 (SEQ ID NO: 2186), D231-AA1 (5 ') (SEQ ID NO: 2187), D232-AD4 (5') (SEQ ID NO: 2188), D232-AE6 (5 ') (sequence) Nos. 2189) and D288-AA3 (SEQ ID NO: 2190).
FIGS. 173-294 are the nucleic acid sequences of D289-AE6 (SEQ ID NO: 2191), D290-AC7 (SEQ ID NO: 2192), and D58-AA2 (SEQ ID NO: 2193).

FIG. 1 shows the nucleic acid sequence of D35-BG11 (SEQ ID NO: 1) and its translation product (SEQ ID NO: 2). FIG. 2 is a schematic diagram of the genomic structure of the tobacco nicotine demethylase gene. FIG. 3 is the genomic tobacco nicotine demethylase nucleic acid sequence (SEQ ID NO: 4) and its translation product (SEQ ID NO: 3). FIG. 3 is the genomic tobacco nicotine demethylase nucleic acid sequence (SEQ ID NO: 4) and its translation product (SEQ ID NO: 3). FIG. 3 is the genomic tobacco nicotine demethylase nucleic acid sequence (SEQ ID NO: 4) and its translation product (SEQ ID NO: 3). FIG. 4 shows the nucleic acid sequence (SEQ ID NO: 5) of the coding region of the tobacco nicotine demethylase gene and its translation product (SEQ ID NO: 6). FIG. 5 is the nucleic acid sequence of an intron (SEQ ID NO: 7) present within the tobacco nicotine demethylase genomic sequence. FIG. 6 is the nucleic acid sequence (SEQ ID NO: 8) of the tobacco demethylase gene promoter. FIG. 7 is the nucleic acid sequence (SEQ ID NO: 9) of the 3 ′ UTR of the tobacco nicotine demethylase gene. FIG. 8 is an electrophoresis image showing tobacco line PCR products with Geno full length (“FL”) primer set. FIG. 9 is an electrophoretic image showing the PCR product of the tobacco line with the primer sets (1), (2), (3) and (4) described in Example 17. The approximate size of the band is 3,500 nucleotides (nt) for FL, 2,600 nt for (1), 1,400 nt for (2), 600 nt for (3) and 1,400 nt for (4) . FIG. 10 shows the nucleic acid sequence of D58-BG7 (SEQ ID NO: 10) and the amino acid sequence of D58-BG7 (SEQ ID NO: 11) (this is a translation of D58-BG7 (SEQ ID NO: 10)). FIG. 11 shows the nucleic acid sequence of D58-AB1 (SEQ ID NO: 12) and the amino acid sequence of D58-AB1 (SEQ ID NO: 13) (this is a translation of D58-AB1 (SEQ ID NO: 12)). FIG. 12 shows the nucleic acid sequence of D186-AH4 (SEQ ID NO: 14) and the amino acid sequence of D186-AH4 (SEQ ID NO: 15) (this is a translation of D186-AH4 (SEQ ID NO: 14)). FIG. 13 shows the nucleic acid sequence of D58-BE4 (SEQ ID NO: 16) and the amino acid sequence of D58-BE4 (SEQ ID NO: 17) (this is a translation of D58-BE4 (SEQ ID NO: 16)). FIG. 14 shows the nucleic acid sequence of D56-AH7 (SEQ ID NO: 18) and the amino acid sequence of D56-AH7 (SEQ ID NO: 19) (which is a translation of D56-AH7 (SEQ ID NO: 18)). FIG. 15 shows the nucleic acid sequence of D13a-5 (SEQ ID NO: 20) and the amino acid sequence of D13a-5 (SEQ ID NO: 21) (this is a translation of D13a-5 (SEQ ID NO: 20)). FIG. 16 shows the nucleic acid sequence of D56-AG10 (SEQ ID NO: 22) and the amino acid sequence of D56-AG10 (SEQ ID NO: 23) (this is a translation of D56-AG10 (SEQ ID NO: 22)). FIG. 17 shows the nucleic acid sequence of D35-33 (SEQ ID NO: 24) and the amino acid sequence of D35-33 (SEQ ID NO: 25) (this is a translation of D35-33 (SEQ ID NO: 24)). FIG. 18 shows the nucleic acid sequence of D34-62 (SEQ ID NO: 26) and the amino acid sequence of D34-62 (SEQ ID NO: 27) (this is a translation of D34-62 (SEQ ID NO: 26)). FIG. 19 shows the nucleic acid sequence of D56-AA7 (SEQ ID NO: 28) and the amino acid sequence of D56-AA7 (SEQ ID NO: 29) (this is a translation of D56-AA7 (SEQ ID NO: 28)). FIG. 20 shows the nucleic acid sequence of D56-AE1 (SEQ ID NO: 30) and the amino acid sequence of D56-AE1 (SEQ ID NO: 31) (this is a translation of D56-AE1 (SEQ ID NO: 30)). FIG. 21 shows the nucleic acid sequence of D35-BB7 (SEQ ID NO: 32) and the amino acid sequence of D35-BB7 (SEQ ID NO: 33) (this is a translation of D35-BB7 (SEQ ID NO: 32)). FIG. 22 shows the nucleic acid sequence of D177-BA7 (SEQ ID NO: 34) and the amino acid sequence of D177-BA7 (SEQ ID NO: 35) (this is a translation of D177-BA7 (SEQ ID NO: 34)). FIG. 23 shows the nucleic acid sequence of D56A-AB6 (SEQ ID NO: 36) and the amino acid sequence of D56A-AB6 (SEQ ID NO: 37) (this is a translation of D56A-AB6 (SEQ ID NO: 36)). FIG. 24 shows the nucleic acid sequence of D144-AE2 (SEQ ID NO: 38) and the amino acid sequence of D144-AE2 (SEQ ID NO: 39) (this is a translation of D144-AE2 (SEQ ID NO: 38)). FIG. 25 shows the nucleic acid sequence of D56-AG11 (SEQ ID NO: 40) and the amino acid sequence of D56-AG11 (SEQ ID NO: 41) (this is a translation of D56-AG11 (SEQ ID NO: 40)). FIG. 26 shows the nucleic acid sequence of D179-AA1 (SEQ ID NO: 42) and the amino acid sequence of D179-AA1 (SEQ ID NO: 43) (this is a translation of D179-AA1 (SEQ ID NO: 42)). FIG. 27 shows the nucleic acid sequence of D56-AC7 (SEQ ID NO: 44) and the amino acid sequence of D56-AC7 (SEQ ID NO: 45) (which is a translation of D56-AC7 (SEQ ID NO: 44)). FIG. 28 shows the nucleic acid sequence of D144-AD1 (SEQ ID NO: 46) and the amino acid sequence of D144-AD1 (SEQ ID NO: 47) (this is a translation of D144-AD1 (SEQ ID NO: 46)). FIG. 29 shows the nucleic acid sequence of D144-AB5 (SEQ ID NO: 48) and the amino acid sequence of D144-AB5 (SEQ ID NO: 49) (this is a translation of D144-AB5 (SEQ ID NO: 48)). FIG. 30 shows the nucleic acid sequence of D181-AB5 (SEQ ID NO: 50) and the amino acid sequence of D181-AB5 (SEQ ID NO: 51) (this is a translation of D181-AB5 (SEQ ID NO: 50)). FIG. 31 shows the nucleic acid sequence of D73-AC9 (SEQ ID NO: 52) and the amino acid sequence of D73-AC9 (SEQ ID NO: 53) (this is a translation of D73-AC9 (SEQ ID NO: 52)). FIG. 32 shows the nucleic acid sequence of D56-AC12 (SEQ ID NO: 54) and the amino acid sequence of D56-AC12 (SEQ ID NO: 55) (this is a translation of D56-AC12 (SEQ ID NO: 54)). FIG. 33 shows the nucleic acid sequence of D58-AB9 (SEQ ID NO: 56) and the amino acid sequence of D58-AB9 (SEQ ID NO: 57) (this is a translation of D58-AB9 (SEQ ID NO: 56)). FIG. 34 shows the nucleic acid sequence of D56-AG9 (SEQ ID NO: 58) and the amino acid sequence of D56-AG9 (SEQ ID NO: 59) (this is a translation of D56-AG9 (SEQ ID NO: 58)). FIG. 35 shows the nucleic acid sequence of D56-AG6 (SEQ ID NO: 60) and the amino acid sequence of D56-AG6 (SEQ ID NO: 61) (this is a translation of D56-AG6 (SEQ ID NO: 60)). FIG. 36 shows the nucleic acid sequence of D35-BG11 (SEQ ID NO: 62) and the amino acid sequence of D35-BG11 (SEQ ID NO: 63) (this is a translation of D35-BG11 (SEQ ID NO: 62)). FIG. 37 shows the nucleic acid sequence of D35-42 (SEQ ID NO: 64) and the amino acid sequence of D35-42 (SEQ ID NO: 65) (which is a translation of D35-42 (SEQ ID NO: 64)). FIG. 38 shows the nucleic acid sequence of D35-BA3 (SEQ ID NO: 66) and the amino acid sequence of D35-BA3 (SEQ ID NO: 67) (this is a translation of D35-BA3 (SEQ ID NO: 66)). FIG. 39 shows the nucleic acid sequence of D34-57 (SEQ ID NO: 68) and the amino acid sequence of D34-57 (SEQ ID NO: 69), which is a translation of D34-57 (SEQ ID NO: 68). FIG. 40 is the nucleic acid sequence of D34-52 (SEQ ID NO: 70) and the amino acid sequence of D34-52 (SEQ ID NO: 71) (this is a translation of D34-52 (SEQ ID NO: 70)). FIG. 41 shows the nucleic acid sequence of D34-25 (SEQ ID NO: 72) and the amino acid sequence of D34-25 (SEQ ID NO: 73) (which is a translation of D34-25 (SEQ ID NO: 72)). FIG. 42 shows the nucleic acid sequence of D56AD10 (SEQ ID NO: 74) and the amino acid sequence of D56AD10 (SEQ ID NO: 75) (which is a translation of D56AD10 (SEQ ID NO: 74)). FIG. 43 shows the nucleic acid sequence of D56-AA11 (SEQ ID NO: 76) and the amino acid sequence of D56-AA11 (SEQ ID NO: 77) (which is a translation of D56-AA11 (SEQ ID NO: 76)). FIG. 44 shows the nucleic acid sequence of D177-BD5 (SEQ ID NO: 78) and the amino acid sequence of D177-BD5 (SEQ ID NO: 79) (this is a translation of D177-BD5 (SEQ ID NO: 78)). FIG. 45 shows the nucleic acid sequence of D56A-AG10 (SEQ ID NO: 80) and the amino acid sequence of D56A-AG10 (SEQ ID NO: 81) (this is a translation of D56A-AG10 (SEQ ID NO: 80)). FIG. 46 shows the nucleic acid sequence of D58-BC5 (SEQ ID NO: 82) and the amino acid sequence of D58-BC5 (SEQ ID NO: 83) (this is a translation of D58-BC5 (SEQ ID NO: 82)). FIG. 47 shows the nucleic acid sequence of D58-AD12 (SEQ ID NO: 84) and the amino acid sequence of D58-AD12 (SEQ ID NO: 85) (this is a translation of D58-AD12 (SEQ ID NO: 84)). FIG. 48 shows the nucleic acid sequence of D56-AC11 (SEQ ID NO: 86) and the amino acid sequence of D56-AC11 (SEQ ID NO: 87) (which is a translation of D56-AC11 (SEQ ID NO: 86)). FIG. 49 shows the nucleic acid sequence of D35-39 (SEQ ID NO: 88) and the amino acid sequence of D35-39 (SEQ ID NO: 89) (which is a translation of D35-39 (SEQ ID NO: 88)). FIG. 50 is the nucleic acid sequence of D58-BH4 (SEQ ID NO: 90) and the amino acid sequence of D58-BH4 (SEQ ID NO: 91), which is a translation of D58-BH4 (SEQ ID NO: 90). FIG. 51 shows the nucleic acid sequence of D177-BD7 (SEQ ID NO: 92) and the amino acid sequence of D177-BD7 (SEQ ID NO: 93) (this is a translation of D177-BD7 (SEQ ID NO: 92)). FIG. 52 shows the nucleic acid sequence of D176-BF2 (SEQ ID NO: 94) and the amino acid sequence of D176-BF2 (SEQ ID NO: 95) (this is a translation of D176-BF2 (SEQ ID NO: 94)). FIG. 53 shows the nucleic acid sequence of D56-AD6 (SEQ ID NO: 96) and the amino acid sequence of D56-AD6 (SEQ ID NO: 97) (this is a translation of D56-AD6 (SEQ ID NO: 96)). FIG. 54 shows the nucleic acid sequence of D73A-AD6 (SEQ ID NO: 98) and the amino acid sequence of D73A-AD6 (SEQ ID NO: 99) (this is a translation of D73A-AD6 (SEQ ID NO: 98)). FIG. 55 shows the nucleic acid sequence of D70A-BA11 (SEQ ID NO: 100) and the amino acid sequence of D70A-BA11 (SEQ ID NO: 101) (this is a translation of D70A-BA11 (SEQ ID NO: 100)). FIG. 56 shows the nucleic acid sequence of D70A-BB5 (SEQ ID NO: 102) and the amino acid sequence of D70A-BB5 (SEQ ID NO: 103) (this is a translation of D70A-BB5 (SEQ ID NO: 102)). FIG. 57 shows the nucleic acid sequence of D70A-AB5 (SEQ ID NO: 104) and the amino acid sequence of D70A-AB5 (SEQ ID NO: 105) (this is a translation of D70A-AB5 (SEQ ID NO: 104)). FIG. 58 shows the nucleic acid sequence of D70A-AA8 (SEQ ID NO: 106) and the amino acid sequence of D70A-AA8 (SEQ ID NO: 107) (which is a translation of D70A-AA8 (SEQ ID NO: 106)). FIG. 59 shows the nucleic acid sequence of D70A-AB8 (SEQ ID NO: 108) and the amino acid sequence of D70A-AB8 (SEQ ID NO: 109) (this is a translation of D70A-AB8 (SEQ ID NO: 108)). FIG. 60 shows the nucleic acid sequence of D70A-BH2 (SEQ ID NO: 110) and the amino acid sequence of D70A-BH2 (SEQ ID NO: 111) (this is a translation of D70A-BH2 (SEQ ID NO: 110)). FIG. 61 shows the nucleic acid sequence of D70A-AA4 (SEQ ID NO: 112) and the amino acid sequence of D70A-AA4 (SEQ ID NO: 113) (which is a translation of D70A-AA4 (SEQ ID NO: 112)). FIG. 62 shows the nucleic acid sequence of D70A-BA1 (SEQ ID NO: 114) and the amino acid sequence of D70A-BA1 (SEQ ID NO: 115) (this is a translation of D70A-BA1 (SEQ ID NO: 114)). FIG. 63 shows the nucleic acid sequence of D70A-BA9 (SEQ ID NO: 116) and the amino acid sequence of D70A-BA9 (SEQ ID NO: 117) (this is a translation of D70A-BA9 (SEQ ID NO: 116)). FIG. 64 shows the nucleic acid sequence of D70A-BD4 (SEQ ID NO: 118) and the amino acid sequence of D70A-BD4 (SEQ ID NO: 119) (this is a translation of D70A-BD4 (SEQ ID NO: 118)). FIG. 65 shows the nucleic acid sequence of D181-AC5 (SEQ ID NO: 120) and the amino acid sequence of D181-AC5 (SEQ ID NO: 121) (this is a translation of D181-AC5 (SEQ ID NO: 120)). FIG. 66 shows the nucleic acid sequence of D144-AH1 (SEQ ID NO: 122) and the amino acid sequence of D144-AH1 (SEQ ID NO: 123) (this is a translation of D144-AH1 (SEQ ID NO: 122)). FIG. 67 shows the nucleic acid sequence of D34-65 (SEQ ID NO: 124) and the amino acid sequence of D34-65 (SEQ ID NO: 125) (which is a translation of D34-65 (SEQ ID NO: 124)). FIG. 68 shows the nucleic acid sequence of D35-BG2 (SEQ ID NO: 126) and the amino acid sequence of D35-BG2 (SEQ ID NO: 127) (this is a translation of D35-BG2 (SEQ ID NO: 126)). FIG. 69 shows the nucleic acid sequence of D73A-AH7 (SEQ ID NO: 128) and the amino acid sequence of D73A-AH7 (SEQ ID NO: 129) (this is a translation of D73A-AH7 (SEQ ID NO: 128)). FIG. 70 shows the nucleic acid sequence of D58-AA1 (SEQ ID NO: 130) and the amino acid sequence of D58-AA1 (SEQ ID NO: 131) (this is a translation of D58-AA1 (SEQ ID NO: 130)). FIG. 71 shows the nucleic acid sequence of D73A-AE10 (SEQ ID NO: 132) and the amino acid sequence of D73A-AE10 (SEQ ID NO: 133) (this is a translation of D73A-AE10 (SEQ ID NO: 132)). FIG. 72 is the nucleic acid sequence of D56A-AC12 (SEQ ID NO: 134) and the amino acid sequence of D56A-AC12 (SEQ ID NO: 135) (this is a translation of D56A-AC12 (SEQ ID NO: 134)). FIG. 73 shows the nucleic acid sequence of D177-BF7 (SEQ ID NO: 136) and the amino acid sequence of D177-BF7 (SEQ ID NO: 137) (this is a translation of D177-BF7 (SEQ ID NO: 136)). FIG. 74 shows the nucleic acid sequence of D73A-AG3 (SEQ ID NO: 138) and the amino acid sequence of D73A-AG3 (SEQ ID NO: 139) (which is a translation of D73A-AG3 (SEQ ID NO: 138)). FIG. 75 shows the nucleic acid sequence of D70A-AA12 (SEQ ID NO: 140) and the amino acid sequence of D70A-AA12 (SEQ ID NO: 141) (this is a translation of D70A-AA12 (SEQ ID NO: 140)). FIG. 76 shows the nucleic acid sequence of D185-BC1 (SEQ ID NO: 142) and the amino acid sequence of D185-BC1 (SEQ ID NO: 143) (this is a translation of D185-BC1 (SEQ ID NO: 142)). FIG. 77 shows the nucleic acid sequence of D185-BG2 (SEQ ID NO: 144) and the amino acid sequence of D185-BG2 (SEQ ID NO: 145) (this is a translation of D185-BG2 (SEQ ID NO: 144)). FIG. 78 shows the nucleic acid sequence of D185-BE1 (SEQ ID NO: 146) and the amino acid sequence of D185-BE1 (SEQ ID NO: 147) (which is a translation of D185-BE1 (SEQ ID NO: 146)). FIG. 79 shows the nucleic acid sequence of D185-BD2 (SEQ ID NO: 148) and the amino acid sequence of D185-BD2 (SEQ ID NO: 149) (this is a translation of D185-BD2 (SEQ ID NO: 148)). FIG. 80 shows the nucleic acid sequence of D176-BG2 (SEQ ID NO: 150) and the amino acid sequence of D176-BG2 (SEQ ID NO: 151) (this is a translation of D176-BG2 (SEQ ID NO: 150)). FIG. 81 shows the nucleic acid sequence of D185-BD3 (SEQ ID NO: 152) and the amino acid sequence of D185-BD3 (SEQ ID NO: 153) (this is a translation of D185-BD3 (SEQ ID NO: 152)). FIG. 82 is the nucleic acid sequence of D176-BC3 (SEQ ID NO: 154) and the amino acid sequence of D176-BC3 (SEQ ID NO: 155), which is a translation of D176-BC3 (SEQ ID NO: 154). FIG. 83 shows the nucleic acid sequence of D176-BB3 (SEQ ID NO: 156) and the amino acid sequence of D176-BB3 (SEQ ID NO: 157) (this is a translation of D176-BB3 (SEQ ID NO: 156)). FIG. 84 shows the nucleic acid sequence of D89-AB1 (SEQ ID NO: 158) and the amino acid sequence of D89-AB1 (SEQ ID NO: 159) (this is a translation of D89-AB1 (SEQ ID NO: 158)). FIG. 85 shows the nucleic acid sequence of D89-AD2 (SEQ ID NO: 160) and the amino acid sequence of D89-AD2 (SEQ ID NO: 161) (this is a translation of D89-AD2 (SEQ ID NO: 160)). FIG. 86 shows the nucleic acid sequence of D90A-BB3 (SEQ ID NO: 162) and the amino acid sequence of D90A-BB3 (SEQ ID NO: 163) (which is a translation of D90A-BB3 (SEQ ID NO: 162)). FIG. 87 shows the nucleic acid sequence of D95-AG1 (SEQ ID NO: 164) and the amino acid sequence of D95-AG1 (SEQ ID NO: 165) (which is a translation of D95-AG1 (SEQ ID NO: 164)). FIG. 88 is the nucleic acid sequence of D96-AB6 (SEQ ID NO: 166) and the amino acid sequence of D96-AB6 (SEQ ID NO: 167) (this is a translation of D96-AB6 (SEQ ID NO: 166)). FIG. 89 is the nucleic acid sequence of D96-AC2 (SEQ ID NO: 168) and the amino acid sequence of D96-AC2 (SEQ ID NO: 169) (this is a translation of D96-AC2 (SEQ ID NO: 168)). FIG. 90 shows the nucleic acid sequence of D98-AA1 (SEQ ID NO: 170) and the amino acid sequence of D98-AA1 (SEQ ID NO: 171) (this is a translation of D98-AA1 (SEQ ID NO: 170)). FIG. 91 shows the nucleic acid sequence of D98-AG1 (SEQ ID NO: 172) and the amino acid sequence of D98-AG1 (SEQ ID NO: 173) (this is a translation of D98-AG1 (SEQ ID NO: 172)). FIG. 92 shows the nucleic acid sequence of D100-BE2 (SEQ ID NO: 174) and the amino acid sequence of D100-BE2 (SEQ ID NO: 175) (this is a translation of D100-BE2 (SEQ ID NO: 174)). FIG. 93 shows the nucleic acid sequence of D100A-AC3 (SEQ ID NO: 176) and the amino acid sequence of D100A-AC3 (SEQ ID NO: 177) (this is a translation of D100A-AC3 (SEQ ID NO: 176)). FIG. 94 shows the nucleic acid sequence of D104A-AE8 (69,1755) (SEQ ID NO: 178) and the amino acid sequence of D104A-AE8 (69,1755) (SEQ ID NO: 179) (this is D104A-AE8 (69,1755) (sequence No. 178). FIG. 95 shows the nucleic acid sequence of D105-AD6 (SEQ ID NO: 180) and the amino acid sequence of D105-AD6 (SEQ ID NO: 181) (this is a translation of D105-AD6 (SEQ ID NO: 180)). FIG. 96 shows the nucleic acid sequence of D109-AH8 (14, 1697) (SEQ ID NO: 182) and the amino acid sequence of D109-AH8 (14, 1697) (SEQ ID NO: 183) (this is D109-AH8 (14, 1697) (sequence No. 182). FIG. 97 shows the nucleic acid sequence of D110-AF12 (166, 1631) (SEQ ID NO: 184) and the amino acid sequence of D110-AF12 (166, 1631) (SEQ ID NO: 185) (this is D110-AF12 (166, 1631) (sequence No. 184). FIG. 98 shows the nucleic acid sequence of D112-AA5 (SEQ ID NO: 186) and the amino acid sequence of D112-AA5 (SEQ ID NO: 187) (this is a translation of D112-AA5 (SEQ ID NO: 186)). FIG. 99 shows the nucleic acid sequence of D120-AH4 (SEQ ID NO: 188) and the amino acid sequence of D120-AH4 (SEQ ID NO: 189) (which is a translation of D120-AH4 (SEQ ID NO: 188)). FIG. 100 shows the nucleic acid sequence of D121-AA8 (SEQ ID NO: 190) and the amino acid sequence of D121-AA8 (SEQ ID NO: 191) (this is a translation of D121-AA8 (SEQ ID NO: 190)). FIG. 101 shows the nucleic acid sequence of D122-AF10 (SEQ ID NO: 192) and the amino acid sequence of D122-AF10 (SEQ ID NO: 193) (this is a translation of D122-AF10 (SEQ ID NO: 192)). FIG. 102 shows the nucleic acid sequence of D128-AB7 (SEQ ID NO: 194) and the amino acid sequence of D128-AB7 (SEQ ID NO: 195) (this is a translation of D128-AB7 (SEQ ID NO: 194)). FIG. 103 shows the nucleic acid sequence of D129-AD10 (SEQ ID NO: 196) and the amino acid sequence of D129-AD10 (SEQ ID NO: 197) (this is a translation of D129-AD10 (SEQ ID NO: 196)). FIG. 104 shows the nucleic acid sequence of D135-AE1 (SEQ ID NO: 198) and the amino acid sequence of D135-AE1 (SEQ ID NO: 199) (this is a translation of D135-AE1 (SEQ ID NO: 198)). FIG. 105 shows the nucleic acid sequence of D141-AD7 (SEQ ID NO: 200) and the amino acid sequence of D141-AD7 (SEQ ID NO: 201) (this is a translation of D141-AD7 (SEQ ID NO: 200)). FIG. 106 shows the nucleic acid sequence of D147-AD3 (SEQ ID NO: 202) and the amino acid sequence of D147-AD3 (SEQ ID NO: 203) (this is a translation of D147-AD3 (SEQ ID NO: 202)). FIG. 107 shows the nucleic acid sequence of D163-AF12 (SEQ ID NO: 204) and the amino acid sequence of D163-AF12 (SEQ ID NO: 205) (this is a translation of D163-AF12 (SEQ ID NO: 204)). FIG. 108 shows the nucleic acid sequence of D163-AG11 (SEQ ID NO: 206) and the amino acid sequence of D163-AG11 (SEQ ID NO: 207) (this is a translation of D163-AG11 (SEQ ID NO: 206)). FIG. 109 shows the nucleic acid sequence of D163-AG12 (SEQ ID NO: 208) and the amino acid sequence of D163-AG12 (SEQ ID NO: 209) (this is a translation of D163-AG12 (SEQ ID NO: 208)). FIG. 110 shows the nucleic acid sequence of D205-BG9 (SEQ ID NO: 210) and the amino acid sequence of D205-BG9 (SEQ ID NO: 211) (this is a translation of D205-BG9 (SEQ ID NO: 210)). FIG. 111 shows the nucleic acid sequence of D207-AA5 (SEQ ID NO: 212) and the amino acid sequence of D207-AA5 (SEQ ID NO: 213) (this is a translation of D207-AA5 (SEQ ID NO: 212)). FIG. 112 shows the nucleic acid sequence of D207-AB4 (SEQ ID NO: 214) and the amino acid sequence of D207-AB4 (SEQ ID NO: 215) (this is a translation of D207-AB4 (SEQ ID NO: 214)). FIG. 113 shows the nucleic acid sequence of D207-AC4 (SEQ ID NO: 216) and the amino acid sequence of D207-AC4 (SEQ ID NO: 217) (this is a translation of D207-AC4 (SEQ ID NO: 216)). FIG. 114 shows the nucleic acid sequence of D209-AA10 (SEQ ID NO: 218) and the amino acid sequence of D209-AA10 (SEQ ID NO: 219), which is a translation of D209-AA10 (SEQ ID NO: 218). FIG. 115 shows the nucleic acid sequence of D209-AA12 (SEQ ID NO: 220) and the amino acid sequence of D209-AA12 (SEQ ID NO: 221) (which is a translation of D209-AA12 (SEQ ID NO: 220)). FIG. 116 shows the nucleic acid sequence of D209-AH10 (SEQ ID NO: 222) and the amino acid sequence of D209-AH10 (SEQ ID NO: 223) (which is a translation of D209-AH10 (SEQ ID NO: 222)). FIG. 117 shows the nucleic acid sequence of D87A-AF3 (SEQ ID NO: 224) and the amino acid sequence of D87A-AF3 (SEQ ID NO: 225) (which is a translation of D87A-AF3 (SEQ ID NO: 224)). FIG. 118 is the nucleic acid sequence of D208-AC8 (SEQ ID NO: 226) and the amino acid sequence of D208-AC8 (SEQ ID NO: 227) (this is a translation of D208-AC8 (SEQ ID NO: 226)). FIG. 119 shows the nucleic acid sequence of D215-AB5 (SEQ ID NO: 228) and the amino acid sequence of D215-AB5 (SEQ ID NO: 229) (this is a translation of D215-AB5 (SEQ ID NO: 228)). FIG. 120 shows the nucleic acid sequence of D103-AH3 (SEQ ID NO: 230) and the amino acid sequence of D103-AH3 (SEQ ID NO: 231) (which is a translation of D103-AH3 (SEQ ID NO: 230)). FIG. 121 shows the nucleic acid sequence of D208-AD9 (SEQ ID NO: 232) and the amino acid sequence of D208-AD9 (SEQ ID NO: 233) (which is a translation of D208-AD9 (SEQ ID NO: 232)). FIG. 122 shows the nucleic acid sequence of D237-AD1 (SEQ ID NO: 234) and the amino acid sequence of D237-AD1 (SEQ ID NO: 235) (this is a translation of D237-AD1 (SEQ ID NO: 234)). FIG. 123 shows the nucleic acid sequence of D125-AF11 (SEQ ID NO: 236) and the amino acid sequence of D125-AF11 (SEQ ID NO: 237) (this is a translation of D125-AF11 (SEQ ID NO: 236)). FIG. 124 shows the nucleic acid sequence of D134-AE11 (SEQ ID NO: 238) and the amino acid sequence of D134-AE11 (SEQ ID NO: 239) (this is a translation of D134-AE11 (SEQ ID NO: 238)). FIG. 125 shows the nucleic acid sequence of D209-AH12 (SEQ ID NO: 240) and the amino acid sequence of D209-AH12 (SEQ ID NO: 241), which is a translation of D209-AH12 (SEQ ID NO: 240). FIG. 126 shows the nucleic acid sequence of D221-BB8 (SEQ ID NO: 242) and the amino acid sequence of D221-BB8 (SEQ ID NO: 243) (which is a translation of D221-BB8 (SEQ ID NO: 242)). FIG. 127 shows the nucleic acid sequence of D222-BH4 (SEQ ID NO: 244) and the amino acid sequence of D222-BH4 (SEQ ID NO: 245) (this is a translation of D222-BH4 (SEQ ID NO: 244)). FIG. 128 shows the nucleic acid sequence of D224-AF10 (SEQ ID NO: 246) and the amino acid sequence of D224-AF10 (SEQ ID NO: 247) (this is a translation of D224-AF10 (SEQ ID NO: 246)). FIG. 129 shows the nucleic acid sequence of D224-BD11 (SEQ ID NO: 248) and the amino acid sequence of D224-BD11 (SEQ ID NO: 249) (this is a translation of D224-BD11 (SEQ ID NO: 248)). FIG. 130 shows the nucleic acid sequence of D228-AD7 (SEQ ID NO: 250) and the amino acid sequence of D228-AD7 (SEQ ID NO: 251) (this is a translation of D228-AD7 (SEQ ID NO: 250)). FIG. 131 shows the nucleic acid sequence of D228-AH8 (SEQ ID NO: 252) and the amino acid sequence of D228-AH8 (SEQ ID NO: 253) (which is a translation of D228-AH8 (SEQ ID NO: 252)). FIG. 132 shows the nucleic acid sequence of D235-AB1 (SEQ ID NO: 254) and the amino acid sequence of D235-AB1 (SEQ ID NO: 255) (this is a translation of D235-AB1 (SEQ ID NO: 254)). FIG. 133 shows the nucleic acid sequence of D243-AA2 (SEQ ID NO: 256) and the amino acid sequence of D243-AA2 (SEQ ID NO: 257) (this is a translation of D243-AA2 (SEQ ID NO: 256)). FIG. 134 shows the nucleic acid sequence of D244-AD4 (SEQ ID NO: 258) and the amino acid sequence of D244-AD4 (SEQ ID NO: 259) (this is a translation of D244-AD4 (SEQ ID NO: 258)). FIG. 135 shows the nucleic acid sequence of D247-AH1 (SEQ ID NO: 260) and the amino acid sequence of D247-AH1 (SEQ ID NO: 261) (which is a translation of D247-AH1 (SEQ ID NO: 260)). FIG. 136 shows the nucleic acid sequence of D248-AA6 (SEQ ID NO: 262) and the amino acid sequence of D248-AA6 (SEQ ID NO: 263) (this is a translation of D248-AA6 (SEQ ID NO: 262)). FIG. 137 shows the nucleic acid sequence of D249-AE8 (SEQ ID NO: 264) and the amino acid sequence of D249-AE8 (SEQ ID NO: 265) (which is a translation of D249-AE8 (SEQ ID NO: 264)). FIG. 138 shows the nucleic acid sequence of D250-AC11 (SEQ ID NO: 266) and the amino acid sequence of D250-AC11 (SEQ ID NO: 267) (this is a translation of D250-AC11 (SEQ ID NO: 266)). FIG. 139 shows the nucleic acid sequence of D259-AB9 (SEQ ID NO: 268) and the amino acid sequence of D259-AB9 (SEQ ID NO: 269) (this is a translation of D259-AB9 (SEQ ID NO: 268)). FIG. 140 shows the nucleic acid sequence of D218A-AC2 (SEQ ID NO: 270) and the amino acid sequence of D218A-AC2 (SEQ ID NO: 271) (which is a translation of D218A-AC2 (SEQ ID NO: 270)). FIG. 141 shows the nucleic acid sequence of D210-BD4 (SEQ ID NO: 272) and the amino acid sequence of D210-BD4 (SEQ ID NO: 273) (this is a translation of D210-BD4 (SEQ ID NO: 272)). FIG. 142 shows the nucleic acid sequence of D233-AG7 (SEQ ID NO: 274) and the amino acid sequence of D233-AG7 (SEQ ID NO: 275) (this is a translation of D233-AG7 (SEQ ID NO: 274)). FIG. 143 shows the nucleic acid sequence of D257-AE4 (SEQ ID NO: 276) and the amino acid sequence of D257-AE4 (SEQ ID NO: 277) (which is a translation of D257-AE4 (SEQ ID NO: 276)). FIG. 144 shows the nucleic acid sequence of D268-AE2 (SEQ ID NO: 278) and the amino acid sequence of D268-AE2 (SEQ ID NO: 279) (which is a translation of D268-AE2 (SEQ ID NO: 278)). FIG. 145 shows the nucleic acid sequence of D283-AC1 (SEQ ID NO: 280) and the amino acid sequence of D283-AC1 (SEQ ID NO: 281) (which is a translation of D283-AC1 (SEQ ID NO: 280)). FIG. 146 shows the nucleic acid sequence of D244-AB6 (SEQ ID NO: 282) and the amino acid sequence of D244-AB6 (SEQ ID NO: 283) (which is a translation of D244-AB6 (SEQ ID NO: 282)). FIG. 147 shows the nucleic acid sequence of D205-BE9 (SEQ ID NO: 284) and the amino acid sequence of D205-BE9 (SEQ ID NO: 285) (this is a translation of D205-BE9 (SEQ ID NO: 284)). FIG. 148 shows the nucleic acid sequence of D136-AF4 (SEQ ID NO: 286) and the amino acid sequence of D136-AF4 (SEQ ID NO: 287) (this is a translation of D136-AF4 (SEQ ID NO: 286)). FIG. 149 shows the nucleic acid sequence of D101-BA2 (SEQ ID NO: 288) and the amino acid sequence of D101-BA2 (SEQ ID NO: 289) (this is a translation of D101-BA2 (SEQ ID NO: 288)). FIG. 150 shows the nucleic acid sequence of D130-AA1 (SEQ ID NO: 290) and the amino acid sequence of D130-AA1 (SEQ ID NO: 291) (this is a translation of D130-AA1 (SEQ ID NO: 290)). FIG. 151 shows the nucleic acid sequence of D136-AD5 (SEQ ID NO: 292) and the amino acid sequence of D136-AD5 (SEQ ID NO: 293) (this is a translation of D136-AD5 (SEQ ID NO: 292)). FIG. 152 shows the nucleic acid sequence of D138-AD12 (SEQ ID NO: 294) and the amino acid sequence of D138-AD12 (SEQ ID NO: 295) (this is a translation of D138-AD12 (SEQ ID NO: 294)). FIG. 153 shows the nucleic acid sequence of D216-AG8 (SEQ ID NO: 296) and the amino acid sequence of D216-AG8 (SEQ ID NO: 297) (which is a translation of D216-AG8 (SEQ ID NO: 296)). FIG. 154 shows the nucleic acid sequence of D243-AB3 (SEQ ID NO: 298) and the amino acid sequence of D243-AB3 (SEQ ID NO: 299) (which is a translation of D243-AB3 (SEQ ID NO: 298)). FIG. 155 shows the nucleic acid sequence of D250-AC11 (SEQ ID NO: 300) and the amino acid sequence of D250-AC11 (SEQ ID NO: 301) (this is a translation of D250-AC11 (SEQ ID NO: 300)). FIG. 156 shows the nucleic acid sequence of D205-AH4 (SEQ ID NO: 302) and the amino acid sequence of D205-AH4 (SEQ ID NO: 303) (this is a translation of D205-AH4 (SEQ ID NO: 302)). FIG. 157 shows the nucleic acid sequence of D267-AF10 (SEQ ID NO: 304) and the amino acid sequence of D267-AF10 (SEQ ID NO: 305) (this is a translation of D267-AF10 (SEQ ID NO: 304)). FIG. 158 shows the nucleic acid sequence of D284-AH5 (SEQ ID NO: 306) and the amino acid sequence of D284-AH5 (SEQ ID NO: 307) (which is a translation of D284-AH5 (SEQ ID NO: 306)). Figure 159A shows the alignment of D58-BG7 (SEQ ID NO: 10), D58-AB1 (SEQ ID NO: 12) and D58-BE4 (SEQ ID NO: 16) and D56-AH7 (SEQ ID NO: 18) and D13a-5 (SEQ ID NO: 20). A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment. Figure 159B shows the alignment of D56-AG10 (SEQ ID NO: 22), D35-33 (SEQ ID NO: 24) and D34-62 (SEQ ID NO: 26) and D56-AA7 (SEQ ID NO: 28), D56-AE1 (SEQ ID NO: 30). And a nucleic acid sequence alignment set consisting of an alignment of D185-BD3 (SEQ ID NO: 152). The percent identity between the sequences in each alignment is shown under each alignment. Figure 159C shows alignment of D56A-AB6 (SEQ ID NO: 36), D35-BB7 (SEQ ID NO: 32), D177-BA7 (SEQ ID NO: 34) and D144-AE2 (SEQ ID NO: 38) and D56-AG11 (SEQ ID NO: 40). And a nucleic acid sequence alignment set consisting of an alignment of D179-AA1 (SEQ ID NO: 42). The percent identity between the sequences in each alignment is shown under each alignment. FIG. 159D shows a set of nucleic acid sequence alignments consisting of an alignment of D56-AC7 (SEQ ID NO: 44) and D144-AD1 (SEQ ID NO: 46) and an alignment of D181-AB5 (SEQ ID NO: 50) and D73-AC9 (SEQ ID NO: 52). It is. The percent identity between the sequences in each alignment is shown under each alignment. Figure 159E shows the nucleic acid sequences D58-AB9 (SEQ ID NO: 56), D56-AG9 (SEQ ID NO: 58), D35-BG11 (SEQ ID NO: 62), D34-25 (SEQ ID NO: 72), D35-BA3 (SEQ ID NO: 66) , D34-52 (SEQ ID NO: 70), D56-AG6 (SEQ ID NO: 60), D35-42 (SEQ ID NO: 64) and D34-57 (SEQ ID NO: 68). The identity (%) between the sequences of the alignment is shown below the alignment. FIG. 159F shows an alignment of D177-BD7 (SEQ ID NO: 92) and D177-BD5 (SEQ ID NO: 78) and D56A-AG10 (SEQ ID NO: 80), D58-AD12 (SEQ ID NO: 84) and D58-BC5 (SEQ ID NO: 82). A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment. FIG.159G shows alignment of D56-AD6 (SEQ ID NO: 96), D56-AC11 (SEQ ID NO: 86), D35-39 (SEQ ID NO: 88) and D58-BH4 (SEQ ID NO: 90) and D73A-AD6 (SEQ ID NO: 98) And a set of nucleic acid sequence alignments consisting of an alignment of D70A-BA11 (SEQ ID NO: 100). The percent identity between the sequences in each alignment is shown under each alignment. FIG.159H shows alignment of D70A-AB5 (SEQ ID NO: 104) and D70A-AA8 (SEQ ID NO: 106) and D70A-AB8 (SEQ ID NO: 108), D70A-BH2 (SEQ ID NO: 110), D70A-AA4 (SEQ ID NO: 112) A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment. FIG.159I shows an alignment of D70A-BA1 (SEQ ID NO: 114) and D70A-BA9 (SEQ ID NO: 116) and D144-AH1 (SEQ ID NO: 122), D34-65 (SEQ ID NO: 124) and D181-AC5 (SEQ ID NO: 120) A set of nucleic acid sequence alignments consisting of the above alignments. The percent identity between the sequences in each alignment is shown under each alignment. FIG. 159J shows the alignment of D58-AA1 (SEQ ID NO: 130), D185-BC1 (SEQ ID NO: 142) and D185-BG2 (SEQ ID NO: 144) and D177-BF7 (SEQ ID NO: 136), D185-BD2 (SEQ ID NO: 148). And a nucleic acid sequence alignment set consisting of an alignment of D185-BE1 (SEQ ID NO: 146). The percent identity between the sequences in each alignment is shown under each alignment. FIG. 159K is an alignment of nucleic acid sequences D70-AA12 (SEQ ID NO: 140) and D176-BF2 (SEQ ID NO: 94). The identity (%) between the sequences of the alignment is shown below the alignment. Figure 160A shows D208-AD9 (SEQ ID NO: 2196), D120-AH4 (SEQ ID NO: 2197), D121-AA8 (SEQ ID NO: 2198), D122-AF10 (SEQ ID NO: 2199), D103-AH3 (SEQ ID NO: 2200), D208 -Sequence alignment of AC8 (SEQ ID NO: 2201) and D235-AB1 (SEQ ID NO: 2202); D244-AD4 (SEQ ID NO: 2203), D244-AB6 (SEQ ID NO: 2204), D285-AA8 (SEQ ID NO: 2205), D285-AB9 A set of amino acid sequence alignments consisting of a sequence alignment of (SEQ ID NO: 2206) and D268-AE2 (SEQ ID NO: 2207); and a sequence alignment of D100A-AC3 (SEQ ID NO: 2208) and D100A-BE2 (SEQ ID NO: 2209). Figure 160B shows the sequence alignment of D205-BG9 (SEQ ID NO: 2210), D205-BE9 (SEQ ID NO: 2211) and D205-AH4 (SEQ ID NO: 2212); D259-AB9 (SEQ ID NO: 2213), D257-AE4 (SEQ ID NO: 2214) ) And D147-AD3 (SEQ ID NO: 2215); D249-AEB (SEQ ID NO: 2216) and D248-AA6 (SEQ ID NO: 2217); D233-AG7 (SEQ ID NO: 2218), D224-BD11 (SEQ ID NO: 2219) and D224-AF10 (SEQ ID NO: 2220); and amino acid sequence alignment consisting of D105-AD6 (SEQ ID NO: 2221), D215-AB5 (SEQ ID NO: 2222) and D135-AE1 (SEQ ID NO: 2223) It is a set of. Figure 160C shows the sequence alignment of D87A-AF3 (SEQ ID NO: 2224) and D210-BD4 (SEQ ID NO: 2225); D89-AB1 (SEQ ID NO: 2226), D89-AD2 (SEQ ID NO: 2227), D163-AG12 (SEQ ID NO: 2228) ), D163-AG11 (SEQ ID NO: 2229) and D163-AF12 (SEQ ID NO: 2230) sequence alignment; D267-AF10 (SEQ ID NO: 2231), D96-AC2 (SEQ ID NO: 2232), D96-AB6 (SEQ ID NO: 2233), D207-AA5 (SEQ ID NO: 2234), D207-AB4 (SEQ ID NO: 2235) and D207-AC4 (SEQ ID NO: 2236); and D98-AG1 (SEQ ID NO: 2237) and D98-AA1 (SEQ ID NO: 2238) A set of amino acid sequence alignments. Figure 160D shows the sequence of D209-AA10 (SEQ ID NO: 2239), D209-AA12 (SEQ ID NO: 2240), D209-AH10 (SEQ ID NO: 2241), D209-AH12 (SEQ ID NO: 2242) and D90a-BB3 (SEQ ID NO: 2243). Alignment; Sequence alignment of D129-AD10 (SEQ ID NO: 2244) and D104A-AE8 (SEQ ID NO: 2245); D228-AH8 (SEQ ID NO: 2246), D228-AD7 (SEQ ID NO: 2247), D250-AC11 (SEQ ID NO: 2248) and A set of amino acid sequence alignments consisting of sequence alignment of D247-AH1 (SEQ ID NO: 2249); and D128-AB7 (SEQ ID NO: 2250), D243-AA2 (SEQ ID NO: 2251) and D125-AF11 (SEQ ID NO: 2252) is there. FIG. 160E is an amino acid sequence alignment of D284-AH5 (SEQ ID NO: 2253) and D110-AF12 (SEQ ID NO: 2254). FIG. 161 is a schematic diagram showing cloning of a cytochrome p450 cDNA fragment by PCR. The primers used for the cloning are as follows: DM (SEQ ID NO: 2255), DM4 (SEQ ID NO: 2256), DM12 (SEQ ID NO: 2257), DM13 (SEQ ID NO: 2258), DM17 (SEQ ID NO: 2259), OLIGO d (T) (SEQ ID NO: 2260), T7 (SEQ ID NO: 2261) and SP6 (SEQ ID NO: 2262). FIG. 162 shows the nucleic acid sequence of D425-AB10 (SEQ ID NO: 367) and the amino acid sequence of D425-AB10 (SEQ ID NO: 368) (this is a translation of D425-AB10 (SEQ ID NO: 367)). FIG. 163 shows the nucleic acid sequence of D425-AB11 (SEQ ID NO: 369) and the amino acid sequence of D425-AB11 (SEQ ID NO: 370) (this is a translation of D425-AB11 (SEQ ID NO: 369)). FIG. 164 shows the nucleic acid sequence of D425-AC9 (SEQ ID NO: 371) and the amino acid sequence of D425-AC9 (SEQ ID NO: 372) (this is a translation of D425-AC9 (SEQ ID NO: 371)). FIG. 165 shows the nucleic acid sequence of D425-AC10 (SEQ ID NO: 373) and the amino acid sequence of D425-AC10 (SEQ ID NO: 374) (this is a translation of D425-AC10 (SEQ ID NO: 373)). FIG. 166 shows the nucleic acid sequence of D425-AC11 (SEQ ID NO: 375) and the amino acid sequence of D425-AC11 (SEQ ID NO: 376), which is a translation of D425-AC11 (SEQ ID NO: 375). FIG. 167 shows the nucleic acid sequence of D425-AG11 (SEQ ID NO: 377) and the amino acid sequence of D425-AG11 (SEQ ID NO: 378) (this is a translation of D425-AG11 (SEQ ID NO: 377)). FIG. 168 shows the nucleic acid sequence of D425-AH7 (SEQ ID NO: 379) and the amino acid sequence of D425-AH7 (SEQ ID NO: 380) (this is a translation of D425-AH7 (SEQ ID NO: 379)). FIG. 169 shows the nucleic acid sequence of D425-AH11 (SEQ ID NO: 381) and the amino acid sequence of D425-AH11 (SEQ ID NO: 382), which is a translation of D425-AH11 (SEQ ID NO: 381). FIG. 170 shows the nucleic acid sequence of D427-AA5 (SEQ ID NO: 383) and the amino acid sequence of D427-AA5 (SEQ ID NO: 384) (this is a translation of D427-AA5 (SEQ ID NO: 383)). FIG. 171 shows the probe set sequences (SEQ ID NO: 385 to SEQ ID NO: 445) of the clones on the GeneChip® microarray. FIG. 171 shows the probe set sequences (SEQ ID NO: 385 to SEQ ID NO: 445) of the clones on the GeneChip® microarray. FIG. 171 shows the probe set sequences (SEQ ID NO: 385 to SEQ ID NO: 445) of the clones on the GeneChip® microarray. Figure 172-1 shows the nucleic acid sequence of D424-AA4 (SEQ ID NO: 446); the nucleic acid sequence of D424-AF5 (SEQ ID NO: 447) and the amino acid sequence of D424-AF5 (SEQ ID NO: 448) (this is D424-AF5 (SEQ ID NO: 448) 447)); a nucleic acid sequence of D425-AA11 (SEQ ID NO: 449) and an amino acid sequence of D425-AA11 (SEQ ID NO: 450) (this is a translator of D425-AA11 (SEQ ID NO: 449)); And a nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of D425-AF11 (SEQ ID NO: 451). FIG. 172-2 shows the amino acid sequence of D425-AF11 (SEQ ID NO: 452) (which is a translation of D425-AF11 (SEQ ID NO: 451)); the nucleic acid sequence of D425-AH10 (SEQ ID NO: 453) and D425-AH10 (SEQ ID NO: 454) amino acid sequence (this is a translation of D425-AH10 (SEQ ID NO: 453)); D426-AA3 (SEQ ID NO: 455) nucleic acid sequence and D426-AA3 (SEQ ID NO: 456) amino acid sequence ( This is a translation of D426-AA3 (SEQ ID NO: 455)); and the nucleic acid sequence of D426-AG1 (SEQ ID NO: 457) and the amino acid sequence of D426-AG1 (SEQ ID NO: 458) (this is D426-AG1 (SEQ ID NO: 458) 457) is a nucleic acid and amino acid sequence group. Figure 172-3 shows the nucleic acid sequence of D427-AA6 (SEQ ID NO: 459) and the amino acid sequence of D427-AA6 (SEQ ID NO: 460) (this is a translation of D427-AA6 (SEQ ID NO: 459)); D427-AB6 (SEQ ID NO: 461) and D427-AB6 (SEQ ID NO: 462) amino acid sequence (which is a translation of D427-AB6 (SEQ ID NO: 461)); D428-AC9 (SEQ ID NO: 463) nucleic acid sequence and A D428-AC9 (SEQ ID NO: 464) amino acid sequence (which is a translation of D428-AC9 (SEQ ID NO: 463)); and a D428-AH10 (SEQ ID NO: 465) nucleic acid and amino acid sequence group. . Figure 172-4 shows the amino acid sequence of D428-AH10 (SEQ ID NO: 466) (which is a translation of D428-AH10 (SEQ ID NO: 465)); the nucleic acid sequence of D429-AA1 (SEQ ID NO: 467) and D429-AA1 (SEQ ID NO: 468) amino acid sequence (this is a translation of D429-AA1 (SEQ ID NO: 467)); D430-AA3 (SEQ ID NO: 469) nucleic acid sequence and D430-AA3 (SEQ ID NO: 470) amino acid sequence ( This is a translation of D430-AA3 (SEQ ID NO: 469)); and the nucleic acid sequence of D431-AE6 (SEQ ID NO: 471) and the amino acid sequence of D431-AE6 (SEQ ID NO: 472) (this is D431-AE6 (SEQ ID NO: 472)) 471) is a nucleic acid and amino acid sequence group. FIG. 172-5 shows the nucleic acid sequence of D113-AE9 (SEQ ID NO: 473) and the amino acid sequence of D113-AE9 (SEQ ID NO: 474) (this is a translation of D113-AE9 (SEQ ID NO: 473)); and D114- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AE12 (SEQ ID NO: 475). Figure 172-6 shows the amino acid sequence of D114-AE12 (SEQ ID NO: 476) (which is a translation of D114-AE12 (SEQ ID NO: 475)); the nucleic acid sequence of D119-AC3 (SEQ ID NO: 477) and D119-AC3 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of (SEQ ID NO: 478) (this is a translation of D119-AC3 (SEQ ID NO: 477)); and the nucleic acid sequence of D132-AA5 (SEQ ID NO: 479). Figure 172-7 shows the amino acid sequence of D132-AA5 (SEQ ID NO: 480) (which is a translation of D132-AA5 (SEQ ID NO: 479)); the nucleic acid sequence of D223-BB10 (SEQ ID NO: 481) and D223-BB10 (SEQ ID NO: 482) amino acid sequence (which is a translation of D223-BB10 (SEQ ID NO: 481)); and D245-AA8 (SEQ ID NO: 483) nucleic acid sequence and D245-AA8 (SEQ ID NO: 484) amino acid sequence (This is a nucleic acid and amino acid sequence group consisting of D245-AA8 (SEQ ID NO: 483)). FIG. 172-8 shows the nucleic acid sequence of D246-AE12 (SEQ ID NO: 485) and the amino acid sequence of D246-AE12 (SEQ ID NO: 486) (which is a translation of D246-AE12 (SEQ ID NO: 485)); and D279- A nucleic acid and amino acid sequence group consisting of a nucleic acid sequence of AD1 (SEQ ID NO: 487) and an amino acid sequence of D279-AD1 (SEQ ID NO: 488) (which is a translation of D279-AD1 (SEQ ID NO: 487)). Figure 172-9 shows the nucleic acid sequence of D282-AA10 (SEQ ID NO: 489) and the amino acid sequence of D282-AA10 (SEQ ID NO: 490), which is a translation of D282-AA10 (SEQ ID NO: 489); and D295- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AA1 (SEQ ID NO: 491). FIG. 172-10 shows the amino acid sequence of D295-AA1 (SEQ ID NO: 492), which is a translation of D295-AA1 (SEQ ID NO: 491); the nucleic acid sequence of D101A-AE2 (SEQ ID NO: 493) and D101A-AE2 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of (SEQ ID NO: 494) (this is a translation of D101A-AE2 (SEQ ID NO: 493)); and the nucleic acid sequence of D108-AA4 (SEQ ID NO: 495). Figure 172-11 shows the amino acid sequence of D108-AA4 (SEQ ID NO: 496) (this is a translation of D108-AA4 (SEQ ID NO: 495)); the nucleic acid sequence of D124-AC5 (5 ') (SEQ ID NO: 497) And the amino acid sequence of D124-AC5 (5 ′) (SEQ ID NO: 498), which is a translation of D124-AC5 (5 ′) (SEQ ID NO: 497); D124-AC5 (3 ′) (SEQ ID NO: 499) And the amino acid sequence of D124-AC5 (3 ′) (SEQ ID NO: 500), which is a translation of D124-AC5 (3 ′) (SEQ ID NO: 499); and D141-AD7 (SEQ ID NO: 501) A nucleic acid and amino acid sequence group consisting of the nucleic acid sequences. FIG. 172-12 shows the amino acid sequence of D141-AD7 (SEQ ID NO: 502), which is a translation of D141-AD7 (SEQ ID NO: 501); and the nucleic acid sequence of D148-AD1 (SEQ ID NO: 503) and D148 A nucleic acid and amino acid sequence group consisting of the amino acid sequence of -AD1 (SEQ ID NO: 504) (this is a translation of D148-AD1 (SEQ ID NO: 503)). FIG. 172-13 shows the nucleic acid sequence of D212-BC11 (SEQ ID NO: 505) and the amino acid sequence of D212-BC11 (SEQ ID NO: 506) (which is a translation of D212-BC11 (SEQ ID NO: 505)); and D217- A nucleic acid and amino acid sequence group consisting of the nucleic acid sequence of AB10 (SEQ ID NO: 507) and the amino acid sequence of D217-AB10 (SEQ ID NO: 508) (which is a translation of D217-AB10 (SEQ ID NO: 507)). Figure 172-14 shows the nucleic acid sequence of D220-BC6 (SEQ ID NO: 509) and the amino acid sequence of D220-BC6 (SEQ ID NO: 510) (this is a translation of D220-BC6 (SEQ ID NO: 509)); D225-AG9 (SEQ ID NO: 511) and D225-AG9 (SEQ ID NO: 512) amino acid sequence (which is a translation of D225-AG9 (SEQ ID NO: 511)); D231-AF1 (SEQ ID NO: 513) nucleic acid sequence and A nucleic acid and amino acid sequence group consisting of the amino acid sequence of D231-AF1 (SEQ ID NO: 514) (this is a translation of D231-AF1 (SEQ ID NO: 513)); and the nucleic acid sequence of D232-AH5 (SEQ ID NO: 515) . Figure 172-15 shows the amino acid sequence of D232-AH5 (SEQ ID NO: 516) (which is a translation of D232-AH5 (SEQ ID NO: 515)); the nucleic acid sequence of D240-BB8 (SEQ ID NO: 517) and D240-BB8 (SEQ ID NO: 518) amino acid sequence (this is a translation of D240-BB8 (SEQ ID NO: 517)); D280-AA6 (SEQ ID NO: 519) nucleic acid sequence and D280-AA6 (SEQ ID NO: 520) amino acid sequence ( This is a translation of D280-AA6 (SEQ ID NO: 519)); and the nucleic acid sequence of D285-AD7 (SEQ ID NO: 521) and the amino acid sequence of D285-AD7 (SEQ ID NO: 522) (this is D285-AD7 (SEQ ID NO: 521)) 521) is a nucleic acid and amino acid sequence group. Figure 172-16 shows the nucleic acid sequence of D285-AH9 (SEQ ID NO: 523) and the amino acid sequence of D285-AH9 (SEQ ID NO: 524) (this is a translation of D285-AH9 (SEQ ID NO: 523)); D99-AB3 (SEQ ID NO: 525) and D99-AB3 (SEQ ID NO: 526) amino acid sequence (which is a translation of D99-AB3 (SEQ ID NO: 525)); and D99-AC2 (SEQ ID NO: 527) nucleic acid sequence And a group of nucleic acid and amino acid sequences consisting of the amino acid sequence of D99-AC2 (SEQ ID NO: 528) (which is a translation of D99-AC2 (SEQ ID NO: 527)). Figure 172-17 shows the nucleic acid sequence of D99-AF11 (SEQ ID NO: 529) and the amino acid sequence of D99-AF11 (SEQ ID NO: 530) (this is a translation of D99-AF11 (SEQ ID NO: 529)); D99-AH4 (SEQ ID NO: 531) and D99-AH4 (SEQ ID NO: 532) amino acid sequence (which is a translation of D99-AH4 (SEQ ID NO: 531)); D99-AH7 (SEQ ID NO: 533) nucleic acid sequence and D99-AH7 (SEQ ID NO: 534) amino acid sequence (this is a translation of D99-AH7 (SEQ ID NO: 533)); D99-DB4 (SEQ ID NO: 535) nucleic acid sequence and D99-DB4 (SEQ ID NO: 536) A nucleic acid and amino acid sequence group consisting of an amino acid sequence (which is a translation of D99-DB4 (SEQ ID NO: 535)). Figure 172-18 shows the nucleic acid sequence of D99-DG4 (SEQ ID NO: 537) and the amino acid sequence of D99-DG4 (SEQ ID NO: 538) (this is a translation of D99-DG4 (SEQ ID NO: 537)); D40-2 (SEQ ID NO: 539) and D40-2 (SEQ ID NO: 540) amino acid sequence (which is a translation of D40-2 (SEQ ID NO: 539)); D301-EE11 (SEQ ID NO: 541) nucleic acid sequence and A nucleic acid and amino acid sequence group consisting of the amino acid sequence of D301-EE11 (SEQ ID NO: 542) (this is a translation of D301-EE11 (SEQ ID NO: 541)); and the nucleic acid sequence of D302-AE10 (SEQ ID NO: 543) . Figure 172-19 shows the amino acid sequence of D302-AE10 (SEQ ID NO: 544) (which is a translation of D302-AE10 (SEQ ID NO: 543)); the nucleic acid sequence of D303-AC6 (SEQ ID NO: 545) and D303-AC6 (SEQ ID NO: 546) amino acid sequence (this is a translation of D303-AC6 (SEQ ID NO: 545)); and D303-AC11 (SEQ ID NO: 547) nucleic acid sequence and D303-AC11 (SEQ ID NO: 548) amino acid sequence This is a nucleic acid and amino acid sequence group consisting of (which is a translation of D303-AC11 (SEQ ID NO: 547)). Figure 173-1 shows 40-17 (SEQ ID NO: 549), 40-20 (SEQ ID NO: 550), 40-23 (SEQ ID NO: 551), 40-26 (SEQ ID NO: 552), D40-27 (SEQ ID NO: 553) , 40-28 (SEQ ID NO: 554) and 40-78 (SEQ ID NO: 555). Figure 173-2 shows 40-83 (SEQ ID NO: 556), D40-86 (SEQ ID NO: 557), D40-97 (SEQ ID NO: 558), 40-100 (SEQ ID NO: 559), 40-107 (SEQ ID NO: 560) , D41-41 (SEQ ID NO: 561) and D41-60 (SEQ ID NO: 562). Figure 173-3 shows D41-65 (SEQ ID NO: 563), D41-67 (SEQ ID NO: 564), D41-69 (SEQ ID NO: 565), D41-99 (SEQ ID NO: 566), D42-AA3 (SEQ ID NO: 567) , D42-AA7 (SEQ ID NO: 568) and D42-AC3 (SEQ ID NO: 569). Figure 173-4 shows D42-AC7 (SEQ ID NO: 570), D42-AC8 (SEQ ID NO: 571), D42-AD3 (SEQ ID NO: 572), D42-AD12 (SEQ ID NO: 573), D42-AF3 (SEQ ID NO: 574) And the nucleic acid sequence of D42-AH3 (SEQ ID NO: 575). Figure 173-5 shows D42-BA2 (SEQ ID NO: 576), D42-BB11 (SEQ ID NO: 577), D42-KC9 (SEQ ID NO: 578), D42-KC10 (SEQ ID NO: 579), D42-LB2 (SEQ ID NO: 580) , D42-LC7 (SEQ ID NO: 581), D42-TG7 (SEQ ID NO: 582) and D42-UG8 (SEQ ID NO: 583). Figure 173-6 shows D42-ZB1 (SEQ ID NO: 584), D300-AA7 (SEQ ID NO: 585), D300-AB3 (SEQ ID NO: 586), D300-AB7 (SEQ ID NO: 587), D300-AB10 (SEQ ID NO: 588) , D300-AB11 (SEQ ID NO: 589) and D300-AC2 (SEQ ID NO: 590). Figure 173-7 shows D300-AC6 (SEQ ID NO: 591), D300-AC7 (SEQ ID NO: 592), D300-AD4 (SEQ ID NO: 593), D300-AD6 (SEQ ID NO: 594), D300-AD10 (SEQ ID NO: 595) , D300-AE3 (SEQ ID NO: 596) and D300-AE9 (SEQ ID NO: 597). Figure 173-8 shows D300-AE11 (SEQ ID NO: 598), D300-AF4 (SEQ ID NO: 599), D300-AF5 (SEQ ID NO: 600), D300-AF7 (SEQ ID NO: 601), D300-AF8 (SEQ ID NO: 602) , D300-AF9 (SEQ ID NO: 603) and D300-AF11 (SEQ ID NO: 604) nucleic acid sequences. Figure 173-9 shows D300-AG1 (SEQ ID NO: 605), D300-AG2 (SEQ ID NO: 606), D300-AG3 (SEQ ID NO: 607), D300-AG10 (SEQ ID NO: 608), D300-AH6 (SEQ ID NO: 609) , D300-AH9 (SEQ ID NO: 610), D300-AH10 (SEQ ID NO: 611) and D300-AH11 (SEQ ID NO: 612). Figure 173-10 shows D300-BA5 (SEQ ID NO: 613), D300-BA6 (SEQ ID NO: 614), D300-BA7 (SEQ ID NO: 615), D300-BA12 (SEQ ID NO: 616), D300-BB6 (SEQ ID NO: 617) , D300-BB7 (SEQ ID NO: 618) and D300-BC1 (SEQ ID NO: 619). Figure 173-11 shows D300-BC4 (SEQ ID NO: 620), D300-BC7 (SEQ ID NO: 621), D300-BC8 (SEQ ID NO: 622), D300-BC9 (SEQ ID NO: 623), D300-BD2 (SEQ ID NO: 624) , D300-BD7 (SEQ ID NO: 625) and D300-BE1 (SEQ ID NO: 626). Figure 173-12 shows D300-BE2 (SEQ ID NO: 627), D300-BE7 (SEQ ID NO: 628), D300-BE8 (SEQ ID NO: 629), D300-BE9 (SEQ ID NO: 630), D300-BE12 (SEQ ID NO: 631) , D300-BF7 (SEQ ID NO: 632), D300-BF11 (SEQ ID NO: 633) and D300-BG1 (SEQ ID NO: 634). Figure 173-13 shows D300-BG2 (SEQ ID NO: 635), D300-BG5 (SEQ ID NO: 636), D300-BH6 (SEQ ID NO: 637), D300-BH9 (SEQ ID NO: 638), D300-CA1 (SEQ ID NO: 639) , D300-CA6 (SEQ ID NO: 640) and D300-CB1 (SEQ ID NO: 641) nucleic acid sequences. Figure 173-14 shows D300-CB12 (SEQ ID NO: 642), D300-CC2 (SEQ ID NO: 643), D300-CD2 (SEQ ID NO: 644), D300-CD3 (SEQ ID NO: 645), D300-CD4 (SEQ ID NO: 646) , D300-CD5 (SEQ ID NO: 647) and D300-CE3 (SEQ ID NO: 648). Figure 173-15 shows D300-CE7 (SEQ ID NO: 649), D300-CF1 (SEQ ID NO: 650), D300-CF2 (SEQ ID NO: 651), D300-CF11 (SEQ ID NO: 652), D300-CG2 (SEQ ID NO: 653) , D300-CG7 (SEQ ID NO: 654), D300-CH1 (SEQ ID NO: 655) and D300-CH3 (SEQ ID NO: 656). Figure 173-16 shows D300-CH5 (SEQ ID NO: 657), D300-CH7 (SEQ ID NO: 658), D300-DA1 (SEQ ID NO: 659), D300-DA4 (SEQ ID NO: 660), D300-DB2 (SEQ ID NO: 661) , D300-DB4 (SEQ ID NO: 662), D300-DB6 (SEQ ID NO: 663) and D300-DB7-c (SEQ ID NO: 664). Figure 173-17 shows D300-DB8 (SEQ ID NO: 665), D300-DC2 (SEQ ID NO: 666), D300-DC4 (SEQ ID NO: 667), D300-DC7 (SEQ ID NO: 668), D300-DC10 (SEQ ID NO: 669) , D300-DC11 (SEQ ID NO: 670) and D300-DD5 (SEQ ID NO: 671). Figure 173-18 shows D300-DD6 (SEQ ID NO: 672), D300-DE2 (SEQ ID NO: 673), D300-DE9 (SEQ ID NO: 674), D300-DE10 (SEQ ID NO: 675), D300-DF5 (SEQ ID NO: 676) , D300-DF6 (SEQ ID NO: 677), D300-DF8 (SEQ ID NO: 678) and D300-DF12 (SEQ ID NO: 679). Figure 173-19 shows D300-DG1 (SEQ ID NO: 680), D300-DG3 (SEQ ID NO: 681), D300-DG9 (SEQ ID NO: 682), D300-DG11 (SEQ ID NO: 683), D300-DH10 (SEQ ID NO: 684) , D301-AB10 (SEQ ID NO: 685) and D301-AC5 (SEQ ID NO: 686). Figure 173-20 shows D301-AC7 (SEQ ID NO: 687), D301-AC9 (SEQ ID NO: 688), D301-AD1 (SEQ ID NO: 689), D301-AD2 (SEQ ID NO: 690), D301-AE4c (SEQ ID NO: 691) , D301-AF12 (SEQ ID NO: 692), D301-AH6 (SEQ ID NO: 693) and D301-AH12 (SEQ ID NO: 694). Figure 173-21 shows D301-BB8 (SEQ ID NO: 695), D301-BB9 (SEQ ID NO: 696), D301-BC1 (SEQ ID NO: 697), D301-BC8 (SEQ ID NO: 698), D301-BD1 (SEQ ID NO: 699) , D301-BD2 (SEQ ID NO: 700) and D301-BF3 (SEQ ID NO: 701). Figure 173-22 shows D301-DA4 (SEQ ID NO: 702), D301-DA8 (SEQ ID NO: 703), D301-DB5 (SEQ ID NO: 704), D301-DC6 (SEQ ID NO: 705), D301-DD4 (SEQ ID NO: 706) , D301-DG1 (SEQ ID NO: 707), D301-DG4c (SEQ ID NO: 708) and D301-EA5 (SEQ ID NO: 709). Figure 173-23 shows D301-EA6 (SEQ ID NO: 710), D301-EA7 (SEQ ID NO: 711), D301-EC9 (SEQ ID NO: 712), D301-EC10 (SEQ ID NO: 713), D301-ED5 (SEQ ID NO: 714) , D301-ED7 (SEQ ID NO: 715) and D301-EE7 (SEQ ID NO: 716). Figure 173-24 shows D301-EF2 (SEQ ID NO: 717), D301-EF5 (SEQ ID NO: 718), D301-EF10 (SEQ ID NO: 719), D301-EG12 (SEQ ID NO: 720), D302-AB8 (SEQ ID NO: 721) , D302-AB9 (SEQ ID NO: 722), D302-AB12 (SEQ ID NO: 723) and D302-AC1 (SEQ ID NO: 724). Figure 173-25 shows D302-AC5 (SEQ ID NO: 725), D302-AC6 (SEQ ID NO: 726), D302-AC7 (SEQ ID NO: 727), D302-AC12 (SEQ ID NO: 728), D302-AD4 (SEQ ID NO: 729) , D302-AD7 (SEQ ID NO: 730) and D302-AE7 (SEQ ID NO: 731) nucleic acid sequences. Figure 173-26 shows D302-AF6 (SEQ ID NO: 732), D302-AF8 (SEQ ID NO: 733), D302-AG2 (SEQ ID NO: 734), D302-AH10 (SEQ ID NO: 735), D302-BA2 (SEQ ID NO: 736) , D302-BA6 (SEQ ID NO: 737), D302-BC3 (SEQ ID NO: 738) and D302-BC6 (SEQ ID NO: 739). Figure 173-27 shows D302-BC9 (SEQ ID NO: 740), D302-BD3 (SEQ ID NO: 741), D302-BD7 (SEQ ID NO: 742), D302-BD9 (SEQ ID NO: 743), D302-BE1 (SEQ ID NO: 744) And the nucleic acid sequence of D302-BE2 (SEQ ID NO: 745). Figure 173-28 shows D302-BE6 (SEQ ID NO: 746), D302-BE7 (SEQ ID NO: 747), D302-BF1 (SEQ ID NO: 748), D302-BF5 (SEQ ID NO: 749), D302-BG1 (SEQ ID NO: 750) , D302-bg3 (SEQ ID NO: 751) and D302-BG7 (SEQ ID NO: 752). Figure 173-29 shows D302-Bg9 (SEQ ID NO: 753), D302-BH1 (SEQ ID NO: 754), D302-bh9 (SEQ ID NO: 755), D302-CA5 (SEQ ID NO: 756), D302-CB7 (SEQ ID NO: 757) , D302-CC1 (SEQ ID NO: 758), D302-CC3 (SEQ ID NO: 759) and D302-CD2 (SEQ ID NO: 760). Figure 173-30 shows D302-CE3 (SEQ ID NO: 761), D302-CE9 (SEQ ID NO: 762), D302-CF2 (SEQ ID NO: 763), D302-CF5 (SEQ ID NO: 764), D302-CF6 (SEQ ID NO: 765) , D302-CH1 (SEQ ID NO: 766) and D302-CH4 (SEQ ID NO: 767). Figure 173-31 shows D302-CH5 (SEQ ID NO: 768), D302-CH6 (SEQ ID NO: 769), D302-DA2 (SEQ ID NO: 770), D302-DA5 (SEQ ID NO: 771), D302-DA9 (SEQ ID NO: 772) , D302-DC4 (SEQ ID NO: 773) and D302-DC7 (SEQ ID NO: 774). Figure 173-32 shows D302-DC8 (SEQ ID NO: 775), D302-DC11 (SEQ ID NO: 776), D302-DD3 (SEQ ID NO: 777), D302-DF5 (SEQ ID NO: 778), D302-DF7 (SEQ ID NO: 779) , D302-DG9 (SEQ ID NO: 780) and D302-DH8 (SEQ ID NO: 781). Figures 173-33 show D303-AA9 (SEQ ID NO: 782), D303-AA10 (SEQ ID NO: 783), D303-AB11 (SEQ ID NO: 784), D303-AC4 (SEQ ID NO: 785), D303-AD3 (SEQ ID NO: 786) , D303-AD11 (SEQ ID NO: 787), D303-AE1 (SEQ ID NO: 788) and D303-ae2 (SEQ ID NO: 789). Figure 173-34 shows D303-ae6 (SEQ ID NO: 790), D303-AF5 (SEQ ID NO: 791), D303-AF12 (SEQ ID NO: 792), D303-AG1 (SEQ ID NO: 793), D303-AG8 (SEQ ID NO: 794) , D303-AG9 (SEQ ID NO: 795), D303-AH5 (SEQ ID NO: 796) and D303-BA1 (SEQ ID NO: 797). Figure 173-35 shows D303-BA5 (SEQ ID NO: 798), D303-BB6 (SEQ ID NO: 799), D303-BC1 (SEQ ID NO: 800), D303-BC2 (SEQ ID NO: 801), D303-BC8 (SEQ ID NO: 802) , D303-BC11 (SEQ ID NO: 803), D303-bd2 (SEQ ID NO: 804) and D303-BD6 (SEQ ID NO: 805). Figure 173-36 shows D303-BD9 (SEQ ID NO: 806), D303-BE3 (SEQ ID NO: 807), D303-BE4 (SEQ ID NO: 808), D303-BE7 (SEQ ID NO: 809), D303-BF1 (SEQ ID NO: 810) , D303-BF6 (SEQ ID NO: 811), D303-BG2 (SEQ ID NO: 812) and D303-BG11 (SEQ ID NO: 813). Figures 173-37 show D303-BH1 (SEQ ID NO: 814), D303-BH6 (SEQ ID NO: 815), D414-AG2 (SEQ ID NO: 816), D35-33 (SEQ ID NO: 817), D35-34 (SEQ ID NO: 818) And the nucleic acid sequence of D35-35 (SEQ ID NO: 819). FIGS. 173-38 are the nucleic acid sequences of D35-41 (SEQ ID NO: 820), D35-43 (SEQ ID NO: 821), D35-51 (SEQ ID NO: 822) and D35-AC4 (SEQ ID NO: 823). Figure 173-39 shows D35-AC12 (SEQ ID NO: 824), D35-AE2 (SEQ ID NO: 825), D35-AE6 (SEQ ID NO: 826), D35-AF1 (SEQ ID NO: 827), D35-AF3 (SEQ ID NO: 828) And the nucleic acid sequence of D35-AG3 (SEQ ID NO: 829). Figure 173-40 shows D35-BA5 (SEQ ID NO: 830), D35-BA12 (SEQ ID NO: 831), D35-BB1 (SEQ ID NO: 832), D35-BB3 (SEQ ID NO: 833) and D35-BB10 (SEQ ID NO: 834) The nucleic acid sequence of Figures 173-41 shows D35-BB12 (SEQ ID NO: 835), D55-AA8 (SEQ ID NO: 836), D55-AB1 (SEQ ID NO: 837), D55-AB5 (SEQ ID NO: 838) and D55-AB6 (SEQ ID NO: 839) The nucleic acid sequence of Figures 173-42 show D55-AB7 (SEQ ID NO: 840), D55-AB10 (SEQ ID NO: 841), D55-AA2 (SEQ ID NO: 842), D55-AC2 (SEQ ID NO: 843) and D55-AC5 (SEQ ID NO: 844) The nucleic acid sequence of Figure 173-43 shows D55-AC7 (SEQ ID NO: 845), D55-BA7 (SEQ ID NO: 846), D55-BA8 (SEQ ID NO: 847), D55-BA10 (SEQ ID NO: 848), D55-BA11 (SEQ ID NO: 849) And the nucleic acid sequence of D55-BB9 (SEQ ID NO: 850). Figure 173-44 shows D55-BB10 (SEQ ID NO: 851), D55-BB12 (SEQ ID NO: 852), D55-BC4 (SEQ ID NO: 853), D55-BC5 (SEQ ID NO: 854), D55-AA6 (SEQ ID NO: 855) And the nucleic acid sequence of D56-AE5 (SEQ ID NO: 856). Figure 173-45 shows D56-AA2 (SEQ ID NO: 857), D56-AA4 (SEQ ID NO: 858), D56-AA8 (SEQ ID NO: 859), D56-AA9 (SEQ ID NO: 860), D56-AB1 (SEQ ID NO: 861) And the nucleic acid sequence of D56-AB7 (SEQ ID NO: 862). Figures 173-46 show D56-AB9 (SEQ ID NO: 863), D56-AC9 (SEQ ID NO: 864), D56-AD3 (SEQ ID NO: 865), D56-AG8 (SEQ ID NO: 866) and D56-AH1 (SEQ ID NO: 867) The nucleic acid sequence of Figures 173-47 show D56-AH10 (SEQ ID NO: 868), D56-AE2 (SEQ ID NO: 869), D57-AB2 (SEQ ID NO: 870), D57-AB5 (SEQ ID NO: 871) and D57-AB6 (SEQ ID NO: 872) The nucleic acid sequence of Figure 173-48 shows D57-AB8 (SEQ ID NO: 873), D57-AB11 (SEQ ID NO: 874), D57-AB12 (SEQ ID NO: 875), D57-AC1 (SEQ ID NO: 876), D57-AC4 (SEQ ID NO: 877) And the nucleic acid sequence of D57-AC12 (SEQ ID NO: 878). Figure 173-49 shows D57-AD5 (SEQ ID NO: 879), D57-AD8 (SEQ ID NO: 880), D57-AD9 (SEQ ID NO: 881), D57-AE1 (SEQ ID NO: 882), D57-AE2 (SEQ ID NO: 883) And the nucleic acid sequence of D57-AE7 (SEQ ID NO: 884). Figure 173-50 shows D57-AF2 (SEQ ID NO: 885), D57-AE12 (SEQ ID NO: 886), D57-AF3 (SEQ ID NO: 887), D57-AF5 (SEQ ID NO: 888) and D57-AF9 (SEQ ID NO: 889) The nucleic acid sequence of FIGS. 173-51 are the nucleic acid sequences of D57-AG5 (SEQ ID NO: 890), D57-AG7 (SEQ ID NO: 891), D57-AG11 (SEQ ID NO: 892) and D57-AH10 (SEQ ID NO: 893). Figures 173-52 show D58-AA3 (SEQ ID NO: 894), D58-AA5 (SEQ ID NO: 895), D58-AB3 (SEQ ID NO: 896), D58-AB5 (SEQ ID NO: 897) and D58-AC1 (SEQ ID NO: 898) The nucleic acid sequence of FIGS. 173-53 are the nucleic acid sequences of D58-AC9 (SEQ ID NO: 899), D58-AC11 (SEQ ID NO: 900), D58-AD2 (SEQ ID NO: 901) and D58-AD5 (SEQ ID NO: 902). Figures 173-54 show D58-AD7 (SEQ ID NO: 903), D58-AD8 (SEQ ID NO: 904), D58-AD10 (SEQ ID NO: 905), D58-AE1 (SEQ ID NO: 906) and D58-AE2 (SEQ ID NO: 907) The nucleic acid sequence of Figures 173-55 show D58-AE5 (SEQ ID NO: 908), D58-AE9 (SEQ ID NO: 909), D58-AE10 (SEQ ID NO: 910), D58-AE11 (SEQ ID NO: 911) and D58-AF3 (SEQ ID NO: 912) The nucleic acid sequence of Figures 173-56 show D58-AF6 (SEQ ID NO: 913), D58-AH4 (SEQ ID NO: 914), D58-AH7 (SEQ ID NO: 915), D58-AH9 (SEQ ID NO: 916) and D58-AH10 (SEQ ID NO: 917) The nucleic acid sequence of Figure 173-57 shows D58-BA5 (SEQ ID NO: 918), D58-BA7 (SEQ ID NO: 919), D58-BB7 (SEQ ID NO: 920), D58-BC5 (SEQ ID NO: 921), D58-BD3 (SEQ ID NO: 922) And the nucleic acid sequence of D58-BD7 (SEQ ID NO: 923). Figures 173-58 show D58-BD8 (SEQ ID NO: 924), D58-BD10 (SEQ ID NO: 925), D58-BE2 (SEQ ID NO: 926), D58-BE11 (SEQ ID NO: 927) and D58-BF1 (SEQ ID NO: 928) The nucleic acid sequence of Figure 173-59 shows D58-BF2 (SEQ ID NO: 929), D58-BG3 (SEQ ID NO: 930), D58-BG5 (SEQ ID NO: 931), D58-BG8 (SEQ ID NO: 932), D58-BG10 (SEQ ID NO: 933) And the nucleic acid sequence of D58-BG12 (SEQ ID NO: 934). Figure 173-60 shows D58-BH10 (SEQ ID NO: 935), D58-BH8 (SEQ ID NO: 936), D58-BH2 (SEQ ID NO: 937), D60-AA1 (SEQ ID NO: 938), D60-AA2 (SEQ ID NO: 939) And the nucleic acid sequence of D60-AA3 (SEQ ID NO: 940). Figure 173-61 shows D60-AA5 (SEQ ID NO: 941), D60-AA6 (SEQ ID NO: 942), D60-AA7 (SEQ ID NO: 943), D60-AA8 (SEQ ID NO: 944), D60-AA10 (SEQ ID NO: 945) And the nucleic acid sequence of D60-AA11 (SEQ ID NO: 946). Figure 173-62 shows D60-AA12 (SEQ ID NO: 947), D60-AB3 (SEQ ID NO: 948), D60-AB5 (SEQ ID NO: 949), D60-AB6 (SEQ ID NO: 950), D60-AB7 (SEQ ID NO: 951) And the nucleic acid sequence of D60-AB8 (SEQ ID NO: 952). Figure 173-63 shows D60-AB11 (SEQ ID NO: 953), D60-AC3 (SEQ ID NO: 954), D60-AC5 (SEQ ID NO: 955), D60-AC7 (SEQ ID NO: 956), D60-AC8 (SEQ ID NO: 957) And the nucleic acid sequence of D60-AC11 (SEQ ID NO: 958). Figure 173-64 shows D60-AC12 (SEQ ID NO: 959), D60-AD3 (SEQ ID NO: 960), D60-AD4 (SEQ ID NO: 961), D60-AD5 (SEQ ID NO: 962), D60-AD7 (SEQ ID NO: 963) And the nucleic acid sequence of D60-AE1 (SEQ ID NO: 964). Figure 173-65 shows D60-AE4 (SEQ ID NO: 965), D60-AE6 (SEQ ID NO: 966), D60-AE8 (SEQ ID NO: 967), D60-AE12 (SEQ ID NO: 968), D60-AF5 (SEQ ID NO: 969) And the nucleic acid sequence of D60-AF8 (SEQ ID NO: 970). Figure 173-66 shows D60-AF11 (SEQ ID NO: 971), D60-AG1 (SEQ ID NO: 972), D60-AG10 (SEQ ID NO: 973), D60-AG12 (SEQ ID NO: 974), D60-AH1 (SEQ ID NO: 975) And the nucleic acid sequence of D60-AH5 (SEQ ID NO: 976). Figures 173-67 show D60-AH6 (SEQ ID NO: 977), D60-AH9 (SEQ ID NO: 978), D60-AH10 (SEQ ID NO: 979), D60-AH11 (SEQ ID NO: 980) and D63-AA12 (SEQ ID NO: 981) The nucleic acid sequence of Figures 173-68 show D64-4 (SEQ ID NO: 982), D64-AB4 (SEQ ID NO: 983), D64-AB10 (SEQ ID NO: 984), D65-AA6 (SEQ ID NO: 985) and D65-AA7 (SEQ ID NO: 986). The nucleic acid sequence of Figure 173-69 shows D65-AA9 (SEQ ID NO: 987), D65-AB4 (SEQ ID NO: 988), D65-AB5 (SEQ ID NO: 989), D65-AB8 (SEQ ID NO: 990), D65-AB9 (SEQ ID NO: 991) And the nucleic acid sequence of D65-AB11 (SEQ ID NO: 992). Figure 173-70 shows D65-AC1 (SEQ ID NO: 993), D65-AC6 (SEQ ID NO: 994), D65-AC10 (SEQ ID NO: 995), D65-AC11 (SEQ ID NO: 996), D65-AD3 (SEQ ID NO: 997) And the nucleic acid sequence of D65-AE1 (SEQ ID NO: 998). Figure 173-71 shows D65-AE2 (SEQ ID NO: 999), D65-AE10 (SEQ ID NO: 1000), D65-AE11 (SEQ ID NO: 1001), D65-AE12 (SEQ ID NO: 1002), D65-AF1 (SEQ ID NO: 1003) And the nucleic acid sequence of D65-AF5 (SEQ ID NO: 1004). Figure 173-72 shows D65-AF7 (SEQ ID NO: 1005), D65-AG11 (SEQ ID NO: 1006), D65-CE1 (SEQ ID NO: 1007), D65-CE2 (SEQ ID NO: 1008), D65-CE7 (SEQ ID NO: 1009) And the nucleic acid sequence of D65-CE9 (SEQ ID NO: 1010). Figure 173-73 shows D65-CE12 (SEQ ID NO: 1011), D65-CF3 (SEQ ID NO: 1012), D65-CF10 (SEQ ID NO: 1013), D65-CF12 (SEQ ID NO: 1014), D65-CG2 (SEQ ID NO: 1015) , D65-CG5 (SEQ ID NO: 1016) and D65-CH3 (SEQ ID NO: 1017). Figure 173-74 shows D65-CH4 (SEQ ID NO: 1018), D65-CH6 (SEQ ID NO: 1019), D65-CH8 (SEQ ID NO: 1020), D65-CH9 (SEQ ID NO: 1021), D65-CH11 (SEQ ID NO: 1022) And the nucleic acid sequence of D65-CH12 (SEQ ID NO: 1023). Figures 173-75 show D66-AA4 (SEQ ID NO: 1024), D66-AA5 (SEQ ID NO: 1025), D66-AA6 (SEQ ID NO: 1026), D66-AA9 (SEQ ID NO: 1027) and D66-AB5 (SEQ ID NO: 1028) The nucleic acid sequence of Figures 173-76 show D66-AB1 (SEQ ID NO: 1029), D66-AB7 (SEQ ID NO: 1030), D66-AC1 (SEQ ID NO: 1031), D66-AC2 (SEQ ID NO: 1032) and D66-AC4 (SEQ ID NO: 1033) The nucleic acid sequence of FIGS. 173-77 are the nucleic acid sequences of D66-AC7 (SEQ ID NO: 1034), D66-AD1 (SEQ ID NO: 1035), D66-AD3 (SEQ ID NO: 1036) and D66-AD6 (SEQ ID NO: 1037). Figure 173-78 shows D66-AD8 (SEQ ID NO: 1038), D66-AE1 (SEQ ID NO: 1039), D66-AE3 (SEQ ID NO: 1040), D66-AE6 (SEQ ID NO: 1041), D66-AE8 (SEQ ID NO: 1042) And the nucleic acid sequence of D66-AF3 (SEQ ID NO: 2263). FIGS. 173-79 are the nucleic acid sequences of D66-AF5 (SEQ ID NO: 1043), D66-AF8 (SEQ ID NO: 1044), D66-AF9 (SEQ ID NO: 1045) and D66-AG1 (SEQ ID NO: 1046). Figure 173-80 shows D66-AG4 (SEQ ID NO: 1047), D66-AG5 (SEQ ID NO: 1048), D66-AH4 (SEQ ID NO: 1049), D66-AH5 (SEQ ID NO: 1050), D66-AH7 (SEQ ID NO: 1051) And the nucleic acid sequence of D66-AH8 (SEQ ID NO: 1052). FIGS. 173-81 are the nucleic acid sequences of D66-AH9 (SEQ ID NO: 1053), D66-BA3 (SEQ ID NO: 1054), D66-BA8 (SEQ ID NO: 1055) and D66-BB1 (SEQ ID NO: 1056). FIGS. 173-82 are the nucleic acid sequences of D66-BB2 (SEQ ID NO: 1057), D66-BB3 (SEQ ID NO: 1058), D66-BB5 (SEQ ID NO: 1059) and D66-BB7 (SEQ ID NO: 1060). Figure 173-83 shows D66-BB9 (SEQ ID NO: 1061), D66-BB10 (SEQ ID NO: 1062), D66-BC6 (SEQ ID NO: 1063), D66-BD1 (SEQ ID NO: 1064) and D66-BD2 (SEQ ID NO: 1065) The nucleic acid sequence of Figures 173-84 show D66-BD9 (SEQ ID NO: 1066), D67-AA2 (SEQ ID NO: 1067), D67-AA3 (SEQ ID NO: 1068), D67-AB1 (SEQ ID NO: 1069) and D67-AC5 (SEQ ID NO: 1070) The nucleic acid sequence of Figures 173-85 show D67-AD1 (SEQ ID NO: 1071), D67-AD4 (SEQ ID NO: 1072), D67-AD6 (SEQ ID NO: 1073), D67-AE2 (SEQ ID NO: 1074) and D67-AE6 (SEQ ID NO: 1075) The nucleic acid sequence of Figures 173-86 show D67-AF6 (SEQ ID NO: 1076), D67-AG1 (SEQ ID NO: 1077), D67-AG3 (SEQ ID NO: 1078), D67-AG5 (SEQ ID NO: 1079) and D67-AG6 (SEQ ID NO: 1080) The nucleic acid sequence of Figures 173-87 show D69-AA5 (SEQ ID NO: 1081), D69-AB1 (SEQ ID NO: 1082), D69-AB8 (SEQ ID NO: 1083), D69-AB9 (SEQ ID NO: 1084) and D69-AC4 (SEQ ID NO: 1085) The nucleic acid sequence of Figure 173-88 shows D70A-AB10 (SEQ ID NO: 1086), D70A-AC2 (SEQ ID NO: 1087), D70A-AC3 (SEQ ID NO: 1088), D70A-AD3 (SEQ ID NO: 1089), D70A-AD5 (SEQ ID NO: 1090) And the nucleic acid sequence of D70A-AD6 (SEQ ID NO: 1091). Figure 173-89 shows D70A-AD11 (SEQ ID NO: 1092), D70A-AE10 (SEQ ID NO: 1093), D70A-AF6 (SEQ ID NO: 1094), D70A-AF8 (SEQ ID NO: 1095), D70A-AF10 (SEQ ID NO: 1096) , D70A-AH5 (SEQ ID NO: 1097) and D70A-AH8 (SEQ ID NO: 1098). Figure 173-90 shows D70A-BA3 (SEQ ID NO: 1099), D70A-BA6 (SEQ ID NO: 1100), D70A-BA10 (SEQ ID NO: 1101), D70A-BB8 (SEQ ID NO: 1102) and D70A-BB11 (SEQ ID NO: 1103) The nucleic acid sequence of Figure 173-91 shows D70A-BC7 (SEQ ID NO: 1104), D70A-BD8 (SEQ ID NO: 1105), D70A-BE1 (SEQ ID NO: 1106), D70A-BE5 (SEQ ID NO: 1107), D70A-BE6 (SEQ ID NO: 1108) And the nucleic acid sequence of D70A-BE8 (SEQ ID NO: 1109). Figure 173-92 shows D70A-BF2 (SEQ ID NO: 1110), D70A-BF3 (SEQ ID NO: 1111), D70A-BF10 (SEQ ID NO: 1112), D70A-BG9 (SEQ ID NO: 1113), D70A-BH8 (SEQ ID NO: 1114) And the nucleic acid sequence of D70-AE2 (SEQ ID NO: 1115). Figure 173-93 shows D70-AE6 (SEQ ID NO: 1116), D70-AF3 (SEQ ID NO: 1117), D70-AG10 (SEQ ID NO: 1118), D70-AH7 (SEQ ID NO: 1119), D70-BE6 (SEQ ID NO: 1120) And the nucleic acid sequence of D70-BE7 (SEQ ID NO: 1121). Figure 173-94 shows D70A-BC12 (SEQ ID NO: 1122), D72-AB2 (SEQ ID NO: 1123), D72-AB6 (SEQ ID NO: 1124), D73A-AA12 (SEQ ID NO: 1125) and D73A-AB4 (SEQ ID NO: 1126) The nucleic acid sequence of Figures 173-95 show D73A-AB9 (SEQ ID NO: 1127), D73A-AB11 (SEQ ID NO: 1128), D73A-AC1 (SEQ ID NO: 1129), D73A-AC3 (SEQ ID NO: 1130) and D73A-AC8 (SEQ ID NO: 1131) The nucleic acid sequence of Figure 173-96 shows D73A-AD3 (SEQ ID NO: 1132), D73A-AE1 (SEQ ID NO: 1133), D73A-AE3 (SEQ ID NO: 1134), D73A-AE4 (SEQ ID NO: 1135), D73A-AE5 (SEQ ID NO: 1136) And the nucleic acid sequence of D73A-AE9 (SEQ ID NO: 1137). Figure 173-97 shows D73A-AE10 (SEQ ID NO: 1138), D73A-AF3 (SEQ ID NO: 1139), D73A-AF4 (SEQ ID NO: 1140), D73A-AF7 (SEQ ID NO: 1141), D73A-AF8 (SEQ ID NO: 1142) And the nucleic acid sequence of D73A-AG11 (SEQ ID NO: 1143). Figure 173-98 shows D73A-AH1 (SEQ ID NO: 1144), D73A-AH5 (SEQ ID NO: 1145), D73A-AH9 (SEQ ID NO: 1146), D73A-AH12 (SEQ ID NO: 1147), D73A-BA9 (SEQ ID NO: 1148) And the nucleic acid sequence of D73A-BB3 (SEQ ID NO: 1149). Figures 173-99 show D73A-BB9 (SEQ ID NO: 1150), D73A-BE1 (SEQ ID NO: 1151), D73A-BF3 (SEQ ID NO: 1152), D73A-BG1 (SEQ ID NO: 1153) and D73A-BG3 (SEQ ID NO: 1154) The nucleic acid sequence of Figure 173-100 shows D73A-BG5 (SEQ ID NO: 1155), D73A-BH1 (SEQ ID NO: 1156), D73-AC1 (SEQ ID NO: 1157), D73-AD8 (SEQ ID NO: 1158) and D73-AD12 (SEQ ID NO: 1159) The nucleic acid sequence of Figure 173-101 shows D80-AB2 (SEQ ID NO: 1160), D81-AA5 (SEQ ID NO: 1161), D81-AB4 (SEQ ID NO: 1162), D81-AB6 (SEQ ID NO: 1163) and D81-AC5 (SEQ ID NO: 1164) The nucleic acid sequence of FIGS. 173-102 are the nucleic acid sequences of D82-AA8 (SEQ ID NO: 1165), D82-AC9 (SEQ ID NO: 1166), D82-AH9 (SEQ ID NO: 1167) and D83-AD10 (SEQ ID NO: 1168). Figure 173-103 shows D83-AG10 (SEQ ID NO: 1169), D84-AD3 (SEQ ID NO: 1170), D84-AE1 (SEQ ID NO: 1171), D84-AF4 (SEQ ID NO: 1172), D84-AG1 (SEQ ID NO: 1173) And the nucleic acid sequence of D87-AA1 (SEQ ID NO: 1174). Figure 173-104 shows D87-AA3 (SEQ ID NO: 1175), D87A-AA1 (SEQ ID NO: 1176), D87A-AB1 (SEQ ID NO: 1177), D87A-AB3 (SEQ ID NO: 1178), D87A-AC1 (SEQ ID NO: 1179) And the nucleic acid sequence of D87A-AC3 (SEQ ID NO: 1180). Figure 173-105 shows D87A-AF2 (SEQ ID NO: 1181), D87A-AG1 (SEQ ID NO: 1182), D87A-AH1 (SEQ ID NO: 1183), D87A-AH3 (SEQ ID NO: 1184), D87-AB2 (SEQ ID NO: 1185) And the nucleic acid sequence of D88-AA10 (SEQ ID NO: 1186). Figures 173-106 show D88-AB3 (SEQ ID NO: 1187), D88-AB6 (SEQ ID NO: 1188), D88-AB7 (SEQ ID NO: 1189), D88-AB8 (SEQ ID NO: 1190), D88-AC5 (SEQ ID NO: 1191) And the nucleic acid sequence of D88-AC9 (SEQ ID NO: 1192). Figures 173-107 show D88-AD8 (SEQ ID NO: 1193), D88-AE5 (SEQ ID NO: 1194), D88-AE6 (SEQ ID NO: 1195), D88-AE8 (SEQ ID NO: 1196) and D91-AA4 (SEQ ID NO: 1197) The nucleic acid sequence of Figure 173-108 shows D92-AD9 (SEQ ID NO: 1198), D92-AE9 (SEQ ID NO: 1199), D92-AF8 (SEQ ID NO: 1200), D92-AG10 (SEQ ID NO: 1201), D92-AH9 (SEQ ID NO: 1202) And the nucleic acid sequence of D93-AA1 (SEQ ID NO: 1203). Figures 173-109 show D93-AA2 (SEQ ID NO: 1204), D93-AB1 (SEQ ID NO: 1205), D93-AC3 (SEQ ID NO: 1206), D93-AC4 (SEQ ID NO: 1207) and D93-AD1 (SEQ ID NO: 1208) The nucleic acid sequence of Figure 173-110 shows D93-AE3 (SEQ ID NO: 1209), D93-AE4 (SEQ ID NO: 1210), D93-AF3 (SEQ ID NO: 1211), D93-AG1 (SEQ ID NO: 1212), D93-AH2 (SEQ ID NO: 1213) And the nucleic acid sequence of D93-AH3 (SEQ ID NO: 1214). Figure 173-111 shows D93-AH4 (SEQ ID NO: 1215), D93-BA10 (SEQ ID NO: 1216), D93-BA12 (SEQ ID NO: 1217), D93-BD11 (SEQ ID NO: 1218), D93-BE11 (SEQ ID NO: 1219) And the nucleic acid sequence of D93-BF12 (SEQ ID NO: 1220). Figures 173-112 show D94-AA2 (SEQ ID NO: 1221), D94-AA3 (SEQ ID NO: 1222), D94-AB1 (SEQ ID NO: 1223), D94-AB2 (SEQ ID NO: 1224) and D94-AC3 (SEQ ID NO: 1225) The nucleic acid sequence of Figures 173-113 show D94-AD1 (SEQ ID NO: 1226), D94-AE1 (SEQ ID NO: 1227), D94-AE3 (SEQ ID NO: 1228), D94-AG2 (SEQ ID NO: 1229) and D94-AH1 (SEQ ID NO: 1230) The nucleic acid sequence of Figure 173-114 shows D94-AH2 (SEQ ID NO: 1231), D94-AH3 (SEQ ID NO: 1232), D95-AC1 (SEQ ID NO: 1233), D95-AD2 (SEQ ID NO: 1234), D96-AA3 (SEQ ID NO: 1235) And the nucleic acid sequence of D96-AA4 (SEQ ID NO: 1236). Figures 173-115 show D96-AA7 (SEQ ID NO: 1237), D96-AB7 (SEQ ID NO: 2264), D96-AC5 (SEQ ID NO: 1238), D96-AC6 (SEQ ID NO: 1239) and D96-AD6 (SEQ ID NO: 1240) The nucleic acid sequence of Figure 173-116 shows D96-AE6 (SEQ ID NO: 1241), D96-AG3 (SEQ ID NO: 1242), D96-AH8 (SEQ ID NO: 1243), D97-AA1 (SEQ ID NO: 1244), D97-AB1 (SEQ ID NO: 1245) And the nucleic acid sequence of D97-AB3 (SEQ ID NO: 1246). Figures 173-117 show D97-AD1 (SEQ ID NO: 1247), D97-AD2 (SEQ ID NO: 1248), D97-AD4 (SEQ ID NO: 1249), D97-AE1 (SEQ ID NO: 1250) and D97-AE2 (SEQ ID NO: 1251) The nucleic acid sequence of Figures 173-118 show D97-AE4 (SEQ ID NO: 1252), D97-AF3 (SEQ ID NO: 1253), D97-AG2 (SEQ ID NO: 1254), D97-AG3 (SEQ ID NO: 1255) and D97-AH1 (SEQ ID NO: 1256) The nucleic acid sequence of Figure 173-119 shows D97-AH2 (SEQ ID NO: 1257), D97-AH4 (SEQ ID NO: 1258), D98-AB2 (SEQ ID NO: 1259), D98-AE3 (SEQ ID NO: 1260) and D98-AF1 (SEQ ID NO: 1261). Nucleic acid sequence). Figure 173-120 shows D98-AH2 (SEQ ID NO: 1262), D99-AC5 (SEQ ID NO: 1263), D99-AC8 (SEQ ID NO: 1264), D99-AD2 (SEQ ID NO: 1265), D99-AD3 (SEQ ID NO: 1266) And the nucleic acid sequence of D99-AD4 (SEQ ID NO: 1267). Figure 173-121 shows D99-AD6 (SEQ ID NO: 1268), D99-AE1 (SEQ ID NO: 1269), D99-AE5 (SEQ ID NO: 1270), D99-AE6 (SEQ ID NO: 1271), D99-AE7 (SEQ ID NO: 1272) And the nucleic acid sequence of D99-AE8 (SEQ ID NO: 1273). Figure 173-122 shows D99-AF1 (SEQ ID NO: 1274), D99-AF5 (SEQ ID NO: 1275), D99-AF7 (SEQ ID NO: 1276), D99-AG2 (SEQ ID NO: 1277), D99-AG4 (SEQ ID NO: 1278) , D99-AG7 (SEQ ID NO: 1279) and D99-AH2 (SEQ ID NO: 1280). Figure 173-123 shows D99-AH10 (SEQ ID NO: 1281), D99-DA3 (SEQ ID NO: 1282), D99-DB5 (SEQ ID NO: 1283), D99-DC2 (SEQ ID NO: 1284) and D99-DC3 (SEQ ID NO: 1285) The nucleic acid sequence of FIGS. 173-124 are the nucleic acid sequences of D99-DD5 (SEQ ID NO: 1286), D99-DG4 (SEQ ID NO: 1287), D100A-AA4 (SEQ ID NO: 1288) and D101-BG2 (SEQ ID NO: 1289). Figure 173-125 shows D101A-AC2 (SEQ ID NO: 1290), D101A-AD1 (SEQ ID NO: 1291), D101A-AD2 (SEQ ID NO: 1292), D101A-AE1 (SEQ ID NO: 1293) and D101A-AF1 (SEQ ID NO: 1294) The nucleic acid sequence of Figures 173-126 show D101C-AA1 (SEQ ID NO: 1295), D101C-AB1 (SEQ ID NO: 1296), D101C-AC1 (SEQ ID NO: 1297), D101C-AD3 (SEQ ID NO: 1298) and D101C-BD12 (SEQ ID NO: 1299) The nucleic acid sequence of FIGS. 173-127 are the nucleic acid sequences of D101C-BE12 (SEQ ID NO: 1300), D101C-BF12 (SEQ ID NO: 1301), D101C-BG12 (SEQ ID NO: 1302) and D101D-AB6 (SEQ ID NO: 1303). Figures 173-128 show D101D-AC5 (SEQ ID NO: 1304), D101D-AG5 (SEQ ID NO: 1305), D101D-AG6 (SEQ ID NO: 1306), D101D-AH4 (SEQ ID NO: 1307) and D101D-AH6 (SEQ ID NO: 1308) The nucleic acid sequence of Figure 173-129 shows D101D-BB11 (SEQ ID NO: 1309), D101D-BD11 (SEQ ID NO: 1310), D107-AA3 (SEQ ID NO: 1311), D107-AB2 (SEQ ID NO: 1312), D107-AC2 (SEQ ID NO: 1313) And the nucleic acid sequence of D107-AC3 (SEQ ID NO: 1314). Figure 173-130 shows D107-AD2 (SEQ ID NO: 1315), D107-AD3 (SEQ ID NO: 1316), D107-AF1 (SEQ ID NO: 1317), D107-AH1 (SEQ ID NO: 1318), D108-AA6 (SEQ ID NO: 1319) And the nucleic acid sequence of D108-AB4 (SEQ ID NO: 1320). Figure 173-131 shows D108-AB5 (SEQ ID NO: 1321), D108-AC6 (SEQ ID NO: 1322), D108-AD6 (SEQ ID NO: 1323), D108-AF6 (SEQ ID NO: 1324), D109-AA7 (SEQ ID NO: 1325) And the nucleic acid sequence of D109-AA8 (SEQ ID NO: 1326). Figure 173-132 shows D109-AA9 (SEQ ID NO: 1327), D109-AB8 (SEQ ID NO: 1328), D109-AC7 (SEQ ID NO: 1329), D109-AC8 (SEQ ID NO: 1330), D109-AC9 (SEQ ID NO: 1331) And the nucleic acid sequence of D109-AE7 (SEQ ID NO: 1332). Figures 173-133 show D109-AF9 (SEQ ID NO: 1333), D109-AH7 (SEQ ID NO: 1334), D110-AB11 (SEQ ID NO: 1335), D110-AF10 (SEQ ID NO: 1336), D111-AA2 (SEQ ID NO: 1337) And the nucleic acid sequence of D111-AB1 (SEQ ID NO: 1338). Figures 173-134 show D111-AB3 (SEQ ID NO: 1339), D111-AC3 (SEQ ID NO: 1340), D111-AD1 (SEQ ID NO: 1341), D111-AF1 (SEQ ID NO: 1342) and D111-AG3 (SEQ ID NO: 1343) The nucleic acid sequence of Figures 173-135 show D111-AH1 (SEQ ID NO: 1344), D111-AH2 (SEQ ID NO: 1345), D112-AD6 (SEQ ID NO: 1346), D112-AE6 (SEQ ID NO: 1347) and D112-AF5 (SEQ ID NO: 1348) The nucleic acid sequence of Figures 173-136 show D112-AG5 (SEQ ID NO: 1349), D112-AH4 (SEQ ID NO: 1350), D113-AA9 (SEQ ID NO: 1351), D113A-AC3 (SEQ ID NO: 1352) and D113A-AD1 (SEQ ID NO: 1353) The nucleic acid sequence of Figure 173-137 shows D113A-AD3 (SEQ ID NO: 1354), D113A-AF3 (SEQ ID NO: 1355), D113A-AG2 (SEQ ID NO: 1356), D113A-AH2 (SEQ ID NO: 1357), D113-AB9 (SEQ ID NO: 1358) And the nucleic acid sequence of D113-AC7 (SEQ ID NO: 1359). Figures 173-138 show D113-AD8 (SEQ ID NO: 1360), D113-AD9 (SEQ ID NO: 1361), D113-AE7 (SEQ ID NO: 1362), D113-AF8 (SEQ ID NO: 1363) and D113-AF9 (SEQ ID NO: 1364). The nucleic acid sequence of Figures 173-139 show D113-AG7 (SEQ ID NO: 1365), D113-AG9 (SEQ ID NO: 1366), D114-AE10 (SEQ ID NO: 1367), D114-AF11 (SEQ ID NO: 1368) and D115-AA6 (SEQ ID NO: 1369) The nucleic acid sequence of Figure 173-140 shows D133-AA7 (SEQ ID NO: 1370), D133-AE8 (SEQ ID NO: 1371), D133-AF9 (SEQ ID NO: 1372), D133-AG8 (SEQ ID NO: 1373), D138-AD10 (SEQ ID NO: 1374) And the nucleic acid sequence of D139-AD1 (SEQ ID NO: 1375). Figure 173-141 shows D140-AA4 (SEQ ID NO: 1376), D140-AD4 (SEQ ID NO: 1377), D140-AF4 (SEQ ID NO: 1378), D141-AA7 (SEQ ID NO: 1379), D141-AB7 (SEQ ID NO: 1380) And the nucleic acid sequence of D142-AB11 (SEQ ID NO: 1381). Figure 173-142 shows D142-AC10 (SEQ ID NO: 1382), D142-AE10 (SEQ ID NO: 1383), D142-AE11 (SEQ ID NO: 1384), D142-AF10 (SEQ ID NO: 1385), D144-AA1 (SEQ ID NO: 1386) And the nucleic acid sequence of D144A-AA9 (SEQ ID NO: 1387). Figure 173-143 shows D144A-AB12 (SEQ ID NO: 1388), D144A-AC9 (SEQ ID NO: 1389), D144A-AC10 (SEQ ID NO: 1390), D144A-AD12 (SEQ ID NO: 1391), D144A-AE12 (SEQ ID NO: 1392) And the nucleic acid sequence of D144A-AF11 (SEQ ID NO: 1393). Figures 173-144 show D144A-AF12 (SEQ ID NO: 1394), D144A-AG11 (SEQ ID NO: 1395), D144A-AH9 (SEQ ID NO: 1396), D144A-AH11 (SEQ ID NO: 1397) and D144-AE4 (SEQ ID NO: 1398) The nucleic acid sequence of Figure 173-145 shows D144-AH3 (SEQ ID NO: 1399), D145-AA8 (SEQ ID NO: 1400), D145-AC10 (SEQ ID NO: 1401), D145-AD7 (SEQ ID NO: 1402), D145-AD9 (SEQ ID NO: 1403) And the nucleic acid sequence of D145-AD10 (SEQ ID NO: 1404). Figure 173-146 shows D145-AE7 (SEQ ID NO: 1405), D145-AF7 (SEQ ID NO: 1406), D145-AF8 (SEQ ID NO: 1407), D145-AG8 (SEQ ID NO: 1408), D145-AG9 (SEQ ID NO: 1409) And the nucleic acid sequence of D145-AG10 (SEQ ID NO: 1410). Figures 173-147 show D145-AH9 (SEQ ID NO: 1411), D146-BB2 (SEQ ID NO: 1412), D146-BC1 (SEQ ID NO: 1413), D146-BD1 (SEQ ID NO: 1414) and D146-BD2 (SEQ ID NO: 1415) The nucleic acid sequence of Figures 173-148 show the sequences D146-BF2 (SEQ ID NO: 1416), D147-AE2 (SEQ ID NO: 1417), D150-AA2 (SEQ ID NO: 1418), D150-AC2 (SEQ ID NO: 1419) and D150-AD1 (SEQ ID NO: 1420). ) Nucleic acid. Figure 173-149 shows D151-AA1 (SEQ ID NO: 1421), D152-AA2 (SEQ ID NO: 1422), D152-AB2 (SEQ ID NO: 1423), D152-AC1 (SEQ ID NO: 1424), D152-AD2 (SEQ ID NO: 1425) And the nucleic acid sequence of D152-AG2 (SEQ ID NO: 1426). Figure 173-150 shows D152-AH2 (SEQ ID NO: 1427), D153-AA7 (SEQ ID NO: 1428), D153-AB2 (SEQ ID NO: 1429), D153-AF2 (SEQ ID NO: 1430), D153-AF7 (SEQ ID NO: 1431) And the nucleic acid sequence of D153-AF9 (SEQ ID NO: 1432). Figures 173-151 show the nucleic acid sequences D153-AG6 (SEQ ID NO: 1433), D153-AG7 (SEQ ID NO: 1434), D153-AG9 (SEQ ID NO: 1435), D153-AH6 (SEQ ID NO: 1436) and D153-AH9 (SEQ ID NO: 1). No. 1437). Figures 173-152 show D154-AC2 (SEQ ID NO: 1438), D154-AE9 (SEQ ID NO: 1439), D155-AB1 (SEQ ID NO: 1440), D155-AC1 (SEQ ID NO: 1441) and D155-AC2 (SEQ ID NO: 1442) The nucleic acid sequence of Figure 173-153 shows D155-AE1 (SEQ ID NO: 1443), D155-AE2 (SEQ ID NO: 1444), D155-AF1 (SEQ ID NO: 1445), D155-AF2 (SEQ ID NO: 1446) and D155-AH2 (SEQ ID NO: 1447) The nucleic acid sequence of Figures 173-154 show D156-AB1 (SEQ ID NO: 1448), D156-AC3 (SEQ ID NO: 1449), D156-AC4 (SEQ ID NO: 1450), D156-AD3 (SEQ ID NO: 1451) and D156-AF2 (SEQ ID NO: 1452) The nucleic acid sequence of Figures 173-155 show D156-AF4 (SEQ ID NO: 1453), D156-AG1 (SEQ ID NO: 1454), D156-AG2 (SEQ ID NO: 1455), D156-AG4 (SEQ ID NO: 1456) and D156-AH1 (SEQ ID NO: 1457) The nucleic acid sequence of Figures 173-156 show D157-AG4 (SEQ ID NO: 1458), D158-AB8 (SEQ ID NO: 1459), D158-AD5 (SEQ ID NO: 1460), D158-AD6 (SEQ ID NO: 1461) and D158-AD8 (SEQ ID NO: 1462) The nucleic acid sequence of Figures 173-157 show D158-AE8 (SEQ ID NO: 1463), D159-AD2 (SEQ ID NO: 1464), D160-AB4 (SEQ ID NO: 1465), D160-AC4 (SEQ ID NO: 1466) and D160-AE4 (SEQ ID NO: 1467) The nucleic acid sequence of Figure 173-158 shows D160-AF4 (SEQ ID NO: 1468), D160-AH4 (SEQ ID NO: 1469), D161-AE5 (SEQ ID NO: 1470), D161-AH5 (SEQ ID NO: 1471), D164-AB1 (SEQ ID NO: 1472) And the nucleic acid sequence of D164-AB3 (SEQ ID NO: 1473). Figures 173-159 show D164-AC1 (SEQ ID NO: 1474), D164-AC2 (SEQ ID NO: 1475), D164-AC5 (SEQ ID NO: 1476), D164-AE1 (SEQ ID NO: 1477) and D164-AF1 (SEQ ID NO: 1478) The nucleic acid sequence of Figures 173-160 show D165-AH8 (SEQ ID NO: 1479), D177-BB7 (SEQ ID NO: 1480), D178-AA6 (SEQ ID NO: 1481), D178-AD5 (SEQ ID NO: 1482) and D180-AA9 (SEQ ID NO: 1483). The nucleic acid sequence of Figures 173-161 show D181-AB6 (SEQ ID NO: 1484), D181-AC6 (SEQ ID NO: 1485), D181-AD7 (SEQ ID NO: 1486), D181-AG7 (SEQ ID NO: 1487) and D181-AH6 (SEQ ID NO: 1488). The nucleic acid sequence of Figures 173-162 show D182-AA1 (SEQ ID NO: 1489), D182-AA2 (SEQ ID NO: 1490), D182-AA4 (SEQ ID NO: 1491), D182-AB2 (SEQ ID NO: 1492) and D182-AC2 (SEQ ID NO: 1493) The nucleic acid sequence of Figures 173-163 show D182-AC4 (SEQ ID NO: 1494), D182-AD1 (SEQ ID NO: 1495), D182-AD3 (SEQ ID NO: 1496), D182-AF1 (SEQ ID NO: 1497) and D182-AF4 (SEQ ID NO: 1498) The nucleic acid sequence of Figure 173-164 shows D182-AG1 (SEQ ID NO: 1499), D183-AC8 (SEQ ID NO: 1500), D185-AB10 (SEQ ID NO: 1501), D185-AC10 (SEQ ID NO: 1502), D185-AF10 (SEQ ID NO: 1503) And the nucleic acid sequence of D185-BA1 (SEQ ID NO: 1504). Figure 173-165 shows D185-BB3 (SEQ ID NO: 1505), D186-AA3 (SEQ ID NO: 1506), D186-AB4 (SEQ ID NO: 1507), D186-AC2 (SEQ ID NO: 1508), D186-AC3 (SEQ ID NO: 1509) And the nucleic acid sequence of D186-AD2 (SEQ ID NO: 1510). Figure 173-166 shows D186-AD3 (SEQ ID NO: 1511), D186-AE3 (SEQ ID NO: 1512), D187-AB2 (SEQ ID NO: 1513), D187-AB3 (SEQ ID NO: 1514), D187-AC4 (SEQ ID NO: 1515) , D187-AE4 (SEQ ID NO: 1516) and D187-AF4 (SEQ ID NO: 1517). Figure 173-167 shows D187-AG1 (SEQ ID NO: 1518), D187-AG2 (SEQ ID NO: 1519), D187-AG3 (SEQ ID NO: 1520), D187-AH2 (SEQ ID NO: 1521), D184-AA1 (SEQ ID NO: 1522) And the nucleic acid sequence of D188-AC7 (SEQ ID NO: 1523). Figure 173-168 shows D188-AC8 (SEQ ID NO: 1524), D188-AD8 (SEQ ID NO: 1525), D188-AE8 (SEQ ID NO: 1526), D188-AF6 (SEQ ID NO: 1527), D188-AG5 (SEQ ID NO: 1528) And the nucleic acid sequence of D188-AG7 (SEQ ID NO: 1529). Figure 173-169 shows D188-AH7 (SEQ ID NO: 1530), D189-AA12 (SEQ ID NO: 1531), D189-AB10 (SEQ ID NO: 1532), D189-AE9 (SEQ ID NO: 1533), D189-AE12 (SEQ ID NO: 1534) And the nucleic acid sequence of D189-AF12 (SEQ ID NO: 1535). Figure 173-170 shows D189-AG10 (SEQ ID NO: 1536), D190-BA6 (SEQ ID NO: 1537), D190-BD6 (SEQ ID NO: 1538), D190-BF6 (SEQ ID NO: 1539), D190-BG6 (SEQ ID NO: 1540) And the nucleic acid sequence of D190-BH6 (SEQ ID NO: 1541). Figures 173-171 show D191-BC5 (SEQ ID NO: 1542), D191-BD5 (SEQ ID NO: 1543), D191-BF5 (SEQ ID NO: 1544), D191-BG5 (SEQ ID NO: 1545) and D194-AA1 (SEQ ID NO: 1546). The nucleic acid sequence of Figures 173-172 show D194-AA2 (SEQ ID NO: 1547), D194-AB1 (SEQ ID NO: 1548), D194-AB2 (SEQ ID NO: 1549), D194-AC1 (SEQ ID NO: 1550) and D194-AC2 (SEQ ID NO: 1551) The nucleic acid sequence of Figures 173-173 show D194-AD1 (SEQ ID NO: 1552), D194-AD2 (SEQ ID NO: 1553), D194-AD3 (SEQ ID NO: 1554), D194-AE1 (SEQ ID NO: 1555) and D194-AE2 (SEQ ID NO: 1556) The nucleic acid sequence of Figures 173-174 show D194-AE3 (SEQ ID NO: 1557), D194-AF1 (SEQ ID NO: 1558), D194-AF2 (SEQ ID NO: 1559), D194-AG2 (SEQ ID NO: 1560) and D194-AG3 (SEQ ID NO: 1561) The nucleic acid sequence of Figure 173-175 shows D194-AH1 (SEQ ID NO: 1562), D194-AH2 (SEQ ID NO: 1563), D194-AH3 (SEQ ID NO: 1564), D195-AB6 (SEQ ID NO: 1565), D195-AD5 (SEQ ID NO: 1566) And the nucleic acid sequence of D195-AE4 (SEQ ID NO: 1567). Figures 173-176 show D195-AE5 (SEQ ID NO: 1568), D195-AG5 (SEQ ID NO: 1569), D195-AH5 (SEQ ID NO: 1570), D196-AD7 (SEQ ID NO: 1571) and D196-AF7 (SEQ ID NO: 1572). The nucleic acid sequence of Figures 173-177 show D196-AG7 (SEQ ID NO: 1573), D197-AE8 (SEQ ID NO: 1574), D198-AB9 (SEQ ID NO: 1575), D198-AC9 (SEQ ID NO: 1576) and D198-AF9 (SEQ ID NO: 1577). The nucleic acid sequence of Figures 173-178 show D199-AA10 (SEQ ID NO: 1578), D199-AB10 (SEQ ID NO: 1579), D199-AD10 (SEQ ID NO: 1580), D199-AF10 (SEQ ID NO: 1581) and D199-AG10 (SEQ ID NO: 1582). The nucleic acid sequence of Figures 173-179 show D200-AB11 (SEQ ID NO: 1583), D200-AC11 (SEQ ID NO: 1584), D200-AD11 (SEQ ID NO: 1585), D200-AE11 (SEQ ID NO: 1586) and D200-AG11 (SEQ ID NO: 1587) The nucleic acid sequence of Figure 173-180 shows D200-AH11 (SEQ ID NO: 1588), D201-AD12 (SEQ ID NO: 1589), D201-AE12 (SEQ ID NO: 1590), D201-AF12 (SEQ ID NO: 1591), D201-AG12 (SEQ ID NO: 1592) And the nucleic acid sequence of D203-BE11 (SEQ ID NO: 1593). Figures 173-181 show D203-BF11 (SEQ ID NO: 1594), D204-AA1 (SEQ ID NO: 1595), D204-AA2 (SEQ ID NO: 1596), D204-AA4 (SEQ ID NO: 1597) and D204-AB1 (SEQ ID NO: 1598). The nucleic acid sequence of Figures 173-182 show D204-AB3 (SEQ ID NO: 1599), D204-AC1 (SEQ ID NO: 1600), D204-AC2 (SEQ ID NO: 1601), D204-AC4 (SEQ ID NO: 1602) and D204-AD1 (SEQ ID NO: 1603) The nucleic acid sequence of Figures 173-183 show D204-AD2 (SEQ ID NO: 1604), D204-AD3 (SEQ ID NO: 1605), D204-AD4 (SEQ ID NO: 1606), D204-AE3 (SEQ ID NO: 1607) and D204-AE4 (SEQ ID NO: 1608) The nucleic acid sequence of Figures 173-184 show D204-AF1 (SEQ ID NO: 1609), D204-AF3 (SEQ ID NO: 1610), D204-AF4 (SEQ ID NO: 1611), D204-AG1 (SEQ ID NO: 1612) and D204-AG2 (SEQ ID NO: 1613) The nucleic acid sequence of Figures 173-185 show D204-AG3 (SEQ ID NO: 1614), D203-BA11 (SEQ ID NO: 1615), D204-AG4 (SEQ ID NO: 1616), D204-AH2 (SEQ ID NO: 1617) and D204-AH1 (SEQ ID NO: 1618) The nucleic acid sequence of Figures 173-186 show D204-AH3 (SEQ ID NO: 1619), D205-BC9 (SEQ ID NO: 1620), D205-BD9 (SEQ ID NO: 1621), D206-CB3 (SEQ ID NO: 1622), D206-CE3 (SEQ ID NO: 1623) And the nucleic acid sequence of D206-CF3 (SEQ ID NO: 1624). FIGS. 173-187 are the nucleic acid sequences of D206-CG1 (SEQ ID NO: 1625), D206-CH1 (SEQ ID NO: 1626), D210-BF4 (SEQ ID NO: 1627) and D210-BF6 (SEQ ID NO: 1628). Figures 173-188 show D210-BH6 (SEQ ID NO: 1629), D211-BA9 (SEQ ID NO: 1630), D211-BB8 (SEQ ID NO: 1631), D211-BB9 (SEQ ID NO: 1632), D211-BC9 (SEQ ID NO: 1633) And the nucleic acid sequence of D211-BD8 (SEQ ID NO: 1634). FIGS. 173-189 are the nucleic acid sequences of D211-BD9 (SEQ ID NO: 1635), D211-BE7 (SEQ ID NO: 1636), D211-BE8 (SEQ ID NO: 1637) and D211-BF8 (SEQ ID NO: 1638). Figures 173-190 show D211-BF9 (SEQ ID NO: 1639), D211-BG8 (SEQ ID NO: 1640), D211-BH7 (SEQ ID NO: 1641), D212-BB11 (SEQ ID NO: 1642) and D212-BB12 (SEQ ID NO: 1643) The nucleic acid sequence of Figure 173-191 shows D212-BD10 (SEQ ID NO: 1644), D212-BD11 (SEQ ID NO: 1645), D212-BE10 (SEQ ID NO: 1646), D212-BE11 (SEQ ID NO: 1647), D212-BF10 (SEQ ID NO: 1648) And the nucleic acid sequence of D212-BF11 (SEQ ID NO: 1649). Figures 173-192 show D213-BD1 (SEQ ID NO: 1650), D213-BF2 (SEQ ID NO: 1651), D214-AA1 (SEQ ID NO: 1652), D214-AA3 (SEQ ID NO: 1653) and D214-AC1 (SEQ ID NO: 1654) The nucleic acid sequence of Figures 173-193 show D214-AE1 (SEQ ID NO: 1655), D214-AE3 (SEQ ID NO: 1656), D214-AG1 (SEQ ID NO: 1657), D214-AH2 (SEQ ID NO: 1658) and D214-AH3 (SEQ ID NO: 1659) The nucleic acid sequence of Figure 173-194 shows D216-AB7 (SEQ ID NO: 1660), D216-AC8 (SEQ ID NO: 1661), D216-AH9 (SEQ ID NO: 1662), D219-BA1 (SEQ ID NO: 1663), D219-BB1 (SEQ ID NO: 1664) And the nucleic acid sequence of D219-BB2 (SEQ ID NO: 1665). Figure 173-195 shows D219-BC1 (SEQ ID NO: 1666), D219-BD1 (SEQ ID NO: 1667), D219-BD2 (SEQ ID NO: 1668), D219-BE1 (SEQ ID NO: 1669), D219-BE2 (SEQ ID NO: 1670) And the nucleic acid sequence of D219-BF2 (SEQ ID NO: 1671). Figures 173-196 show D219-BH1 (SEQ ID NO: 1672), D220-BF6 (SEQ ID NO: 1673), D220-BD6 (SEQ ID NO: 1674), D223-BC11 (SEQ ID NO: 1675) and D221-BC7 (SEQ ID NO: 1676) The nucleic acid sequence of Figure 173-197 shows D227-AE3 (SEQ ID NO: 1677), D223-BB10 (SEQ ID NO: 1678), D221-BF9 (SEQ ID NO: 1679), D229-AD2 (SEQ ID NO: 1680), D229-AE2 (SEQ ID NO: 1681) , D229-AF2 (SEQ ID NO: 1682) and D229-AG1 (SEQ ID NO: 1683). Figure 173-198 shows D229-AH1 (SEQ ID NO: 1684), D230-AB1 (SEQ ID NO: 1685), D230-AC1 (SEQ ID NO: 1686), D230-AC2 (SEQ ID NO: 1687), D230-AF2 (SEQ ID NO: 1688) And the nucleic acid sequence of D230-AG1 (SEQ ID NO: 1689). Figures 173-199 show D230-AG2 (SEQ ID NO: 1690), D231-AA1 (SEQ ID NO: 1691), D231-AA2 (SEQ ID NO: 1692), D231-AA3 (SEQ ID NO: 1693), D231-AC1 (SEQ ID NO: 1694) And the nucleic acid sequence of D231-AC2 (SEQ ID NO: 1695). Figure 173-200 shows D231-AD2 (SEQ ID NO: 1696), D231-AE1 (SEQ ID NO: 1697), D231-AG2 (SEQ ID NO: 1698), D231-AH1 (SEQ ID NO: 1699), D231-AH2 (SEQ ID NO: 1700) And the nucleic acid sequence of D231-AH3 (SEQ ID NO: 1701). FIGS. 173-201 are the nucleic acid sequences of D232-AD4 (SEQ ID NO: 1702), D232-AE5 (SEQ ID NO: 1703), D232-AE6 (SEQ ID NO: 1704) and D232-AF5 (SEQ ID NO: 1705). Figure 173-202 shows D233-AA7 (SEQ ID NO: 1706), D233-AA8 (SEQ ID NO: 1707), D233-AB7 (SEQ ID NO: 1708), D233-AC7 (SEQ ID NO: 1709) and D233-AC8 (SEQ ID NO: 1710) The nucleic acid sequence of Figure 173-203 shows D233-AH9 (SEQ ID NO: 1711), D234-AA11 (SEQ ID NO: 1712), D234-AC11 (SEQ ID NO: 1713), D234-AD11 (SEQ ID NO: 1714), D238-AA2 (SEQ ID NO: 1715) And the nucleic acid sequence of D239-BC3 (SEQ ID NO: 1716). Figures 173-204 show D239-BD4 (SEQ ID NO: 1717), D239-BD6 (SEQ ID NO: 1718), D239-BE4 (SEQ ID NO: 1719), D240-BB7 (SEQ ID NO: 1720) and D241-BC9 (SEQ ID NO: 1721) The nucleic acid sequence of Figure 173-205 shows D241-BE9 (SEQ ID NO: 1722), D242-BA11 (SEQ ID NO: 1723), D242-BB11 (SEQ ID NO: 1724), D242-BB12 (SEQ ID NO: 1725), D242-BC12 (SEQ ID NO: 1726) And the nucleic acid sequence of D242-BF12 (SEQ ID NO: 1727). Figure 173-206 shows D242-BG12 (SEQ ID NO: 1728), D242-BH11 (SEQ ID NO: 1729), D244-AA5 (SEQ ID NO: 1730), D244-AA6 (SEQ ID NO: 1731), D244-AF6 (SEQ ID NO: 1732) And the nucleic acid sequence of D245-AE7 (SEQ ID NO: 1733). Figures 173-207 show D245-AE8 (SEQ ID NO: 1734), D245-AF7 (SEQ ID NO: 1735), D246-AA12 (SEQ ID NO: 1736), D248-AG4 (SEQ ID NO: 1737) and D249-AG9 (SEQ ID NO: 1738) The nucleic acid sequence of Figure 173-208 shows D250-AE10 (SEQ ID NO: 1739), D251-AF2 (SEQ ID NO: 1740), D251-AG2 (SEQ ID NO: 1741), D252-AA5 (SEQ ID NO: 1742) and D252-AB5 (SEQ ID NO: 1743) The nucleic acid sequence of Figure 173-209 shows D252-AC5 (SEQ ID NO: 1744), D252-AD5 (SEQ ID NO: 1745), D252-AF4 (SEQ ID NO: 1746), D252-AF5 (SEQ ID NO: 1747), D252-AG5 (SEQ ID NO: 1748) And the nucleic acid sequence of D254-AE2 (SEQ ID NO: 1749). Figure 173-210 shows D254-AG2 (SEQ ID NO: 1750), D255-AA5 (SEQ ID NO: 1751), D255-AA6 (SEQ ID NO: 1752), D255-AD5 (SEQ ID NO: 1753) and D255-AD6 (SEQ ID NO: 1754) The nucleic acid sequence of FIGS. 173-211 are the nucleic acid sequences of D255-AF5 (SEQ ID NO: 1755), D255-AG5 (SEQ ID NO: 1756), D256-AA10 (SEQ ID NO: 1757) and D256-AB10 (SEQ ID NO: 1758). Figures 173-212 show D256-AC10 (SEQ ID NO: 1759), D256-AE10 (SEQ ID NO: 1760), D256-AF9 (SEQ ID NO: 1761), D256-AF10 (SEQ ID NO: 1762) and D256-AG10 (SEQ ID NO: 1763) The nucleic acid sequence of Figures 173-213 show D256-AH10 (SEQ ID NO: 1764), D258-AC5 (SEQ ID NO: 1765), D263-AE12 (SEQ ID NO: 1766), D263-AF12 (SEQ ID NO: 1767) and D263-AH12 (SEQ ID NO: 1768) The nucleic acid sequence of Figures 173-214 show D264-AA1 (SEQ ID NO: 1769), D264-AA2 (SEQ ID NO: 1770), D264-AA3 (SEQ ID NO: 1771), D264-AB2 (SEQ ID NO: 1772) and D264-AC3 (SEQ ID NO: 1773) The nucleic acid sequence of FIGS. 173-215 are the nucleic acid sequences of D264-AD3 (SEQ ID NO: 1774), D264-AE1 (SEQ ID NO: 1775), D264-AE2 (SEQ ID NO: 1776) and D264-AE3 (SEQ ID NO: 1777). FIGS. 173-216 are the nucleic acid sequences of D264-AF2 (SEQ ID NO: 1778), D264-AG2 (SEQ ID NO: 1779), D264-AH2 (SEQ ID NO: 1780) and D265-AA4 (SEQ ID NO: 1781). FIGS. 173-217 are the nucleic acid sequences of D265-AA6 (SEQ ID NO: 1782), D265-AC5 (SEQ ID NO: 1783), D265-AC6 (SEQ ID NO: 1784) and D265-AD4 (SEQ ID NO: 1785). Figures 173-218 show D265-AD5 (SEQ ID NO: 1786), D266-AB7 (SEQ ID NO: 1787), D266-AB8 (SEQ ID NO: 1788), D266-AC9 (SEQ ID NO: 1789) and D266-AD7 (SEQ ID NO: 1790) The nucleic acid sequence of Figures 173-219 show D267-AD10 (SEQ ID NO: 1791), D268-AA2 (SEQ ID NO: 1792), D268-AC3 (SEQ ID NO: 1793), D268-AD1 (SEQ ID NO: 1794) and D268-AD2 (SEQ ID NO: 1795) The nucleic acid sequence of FIGS. 173-220 are the nucleic acid sequences of D268-AD3 (SEQ ID NO: 1796), D268-AE3 (SEQ ID NO: 1797), D268-AG2 (SEQ ID NO: 1798) and D268-AG3 (SEQ ID NO: 1799). Figure 173-221 shows D269-AA5 (SEQ ID NO: 1800), D269-AD4 (SEQ ID NO: 1801), D269-AE4 (SEQ ID NO: 1802), D269-AF5 (SEQ ID NO: 1803), D269-AF6 (SEQ ID NO: 1804) And the nucleic acid sequence of D270-AA8 (SEQ ID NO: 1805). Figures 173-222 show D270-AB9 (SEQ ID NO: 1806), D270-AD8 (SEQ ID NO: 1807), D270-AD9 (SEQ ID NO: 1808), D270-AE9 (SEQ ID NO: 1809) and D270-AF8 (SEQ ID NO: 1810) The nucleic acid sequence of FIGS. 173-223 are the nucleic acid sequences of D271-AG11 (SEQ ID NO: 1811), D271-AH11 (SEQ ID NO: 1812), D276-AD5 (SEQ ID NO: 1813) and D276-AG6 (SEQ ID NO: 1814). Figures 173-224 show D276-AH4 (SEQ ID NO: 1815), D276-AH6 (SEQ ID NO: 1816), D277-AE8 (SEQ ID NO: 1817), D277-AF9 (SEQ ID NO: 1818) and D277-AH9 (SEQ ID NO: 1819) The nucleic acid sequence of Figures 173-225 show D278-AF10 (SEQ ID NO: 1820), D279-AA3 (SEQ ID NO: 1821), D279-AB2 (SEQ ID NO: 1822), D279-AC1 (SEQ ID NO: 1823) and D279-AD2 (SEQ ID NO: 1824) The nucleic acid sequence of Figures 173-226 show D279-AE1 (SEQ ID NO: 1825), D279-AE3 (SEQ ID NO: 1826), D279-AG3 (SEQ ID NO: 1827), D279-BA1 (SEQ ID NO: 1828) and D279-BA2 (SEQ ID NO: 1829) The nucleic acid sequence of Figures 173-227 show D279-BB3 (SEQ ID NO: 1830), D279-BC2 (SEQ ID NO: 1831), D279-BD2 (SEQ ID NO: 1832), D279-BE2 (SEQ ID NO: 1833) and D279-BF3 (SEQ ID NO: 1834) The nucleic acid sequence of Figures 173-228 show D279-BG1 (SEQ ID NO: 1835), D279-BH3 (SEQ ID NO: 1836), D280-AB5 (SEQ ID NO: 1837), D280-AB6 (SEQ ID NO: 1838) and D280-AC4 (SEQ ID NO: 1839) The nucleic acid sequence of Figure 173-229 shows D280-AC6 (SEQ ID NO: 1840), D280-AD4 (SEQ ID NO: 1841), D280-AD5 (SEQ ID NO: 1842), D280-AD6 (SEQ ID NO: 1843) and D280-AE4 (SEQ ID NO: 1844) The nucleic acid sequence of Figures 173-230 shows D280-AE5 (SEQ ID NO: 1845), D280-AE6 (SEQ ID NO: 1846), D280-AF5 (SEQ ID NO: 1847), D280-AF6 (SEQ ID NO: 1848) and D280-AG4 (SEQ ID NO: 1849) The nucleic acid sequence of Figures 173-231 show D280-AG5 (SEQ ID NO: 1850), D280-AG6 (SEQ ID NO: 1851), D280-AH4 (SEQ ID NO: 1852), D280-AH5 (SEQ ID NO: 1853) and D280-BC4 (SEQ ID NO: 1854) The nucleic acid sequence of FIG. 173-232 shows the nucleic acid sequences of D280-BD4 (SEQ ID NO: 1855), D280-BE4 (SEQ ID NO: 1856), D280-BE6 (SEQ ID NO: 1857) and D280-BF4 (SEQ ID NO: 1858). Figures 173-233 show D280-BG4 (SEQ ID NO: 1859), D280-BH4 (SEQ ID NO: 1860), D280-BH6 (SEQ ID NO: 1861), D281-AA8 (SEQ ID NO: 1862) and D281-AD7 (SEQ ID NO: 1863) The nucleic acid sequence of Figure 173-234 shows D281-AD8 (SEQ ID NO: 1864), D281-AE7 (SEQ ID NO: 1865), D281-AE8 (SEQ ID NO: 1866), D281-AE9 (SEQ ID NO: 1867), D281-AG7 (SEQ ID NO: 1868) And the nucleic acid sequence of D282-AB10 (SEQ ID NO: 1869). Figures 173-235 show D282-AB11 (SEQ ID NO: 1870), D282-AD10 (SEQ ID NO: 1871), D282-AH11 (SEQ ID NO: 1872), D282-BA10 (SEQ ID NO: 1873), D282-BB10 (SEQ ID NO: 1874) and D282-BD10 (SEQ ID NO: 1875). Figures 173-236 show D288-AB3 (SEQ ID NO: 1876), D289-AA4 (SEQ ID NO: 1877), D289-AA6 (SEQ ID NO: 1878), D289-AB4 (SEQ ID NO: 1879) and D289-AB6 (SEQ ID NO: 1880) The nucleic acid sequence of Figures 173-237 show D289-AD4 (SEQ ID NO: 1881), D289-AE4 (SEQ ID NO: 1882), D289-AE6 (SEQ ID NO: 1883), D289-AF4 (SEQ ID NO: 1884) and D289-AF6 (SEQ ID NO: 1885) The nucleic acid sequence of Figures 173-238 show D289-AG4 (SEQ ID NO: 1886), D289-AG6 (SEQ ID NO: 1887), D289-AH4 (SEQ ID NO: 1888), D289-AH6 (SEQ ID NO: 1889) and D290-AA7 (SEQ ID NO: 1890). The nucleic acid sequence of Figures 173-239 show D290-AA8 (SEQ ID NO: 1891), D290-AA9 (SEQ ID NO: 1892), D290-AB7 (SEQ ID NO: 1893), D290-AB9 (SEQ ID NO: 1894) and D290-AC8 (SEQ ID NO: 1895) The nucleic acid sequence of Figures 173-240 show D290-AC9 (SEQ ID NO: 1896), D290-AD7 (SEQ ID NO: 1897), D290-AD8 (SEQ ID NO: 1898), D290-AD9 (SEQ ID NO: 1899) and D291-AA12 (SEQ ID NO: 1900) The nucleic acid sequence of Figure 173-241 shows D291-AB10 (SEQ ID NO: 1901), D291-AB11 (SEQ ID NO: 1902), D291-AC12 (SEQ ID NO: 1903), D291-AD11 (SEQ ID NO: 1904) and D291-AD12 (SEQ ID NO: 1905) The nucleic acid sequence of Figure 173-242 shows D291-AE10 (SEQ ID NO: 1906), D291-AE12 (SEQ ID NO: 1907), D291-AF10 (SEQ ID NO: 1908), D291-AF12 (SEQ ID NO: 1909) and D291-AG11 (SEQ ID NO: 1910) The nucleic acid sequence of Figure 173-243 shows D291-AG12 (SEQ ID NO: 1911), D291-AH11 (SEQ ID NO: 1912), D292-AA2 (SEQ ID NO: 1913), D292-AA3 (SEQ ID NO: 1914), D292-AB2 (SEQ ID NO: 1915) And the nucleic acid sequence of D292-AC2 (SEQ ID NO: 1916). Figure 173-244 shows D292-AD1 (SEQ ID NO: 1917), D292-AD2 (SEQ ID NO: 1918), D292-AE1 (SEQ ID NO: 1919), D292-AE2 (SEQ ID NO: 1920), D292-AF1 (SEQ ID NO: 1921) , D292-AF3 (SEQ ID NO: 1922) and D292-AH3 (SEQ ID NO: 1923). Figures 173-245 show D291-AA10 (SEQ ID NO: 1924), D294-AB7 (SEQ ID NO: 1925), D294-AB8 (SEQ ID NO: 1926), D294-AB9 (SEQ ID NO: 1927) and D294-AC7 (SEQ ID NO: 1928) The nucleic acid sequence of Figures 173-246 show D294-AC8 (SEQ ID NO: 1929), D294-AE9 (SEQ ID NO: 1930), D294-AG8 (SEQ ID NO: 1931), D294-AH7 (SEQ ID NO: 1932) and D295-AA3 (SEQ ID NO: 1933) The nucleic acid sequence of Figures 173-247 show D295-AB1 (SEQ ID NO: 1934), D295-AB2 (SEQ ID NO: 1935), D295-AC2 (SEQ ID NO: 1936), D295-AC3 (SEQ ID NO: 1937) and D295-AD1 (SEQ ID NO: 1938) The nucleic acid sequence of Figures 173-248 show D295-AD2 (SEQ ID NO: 1939), D295-AE3 (SEQ ID NO: 1940), D295-AF1 (SEQ ID NO: 1941), D295-AF2 (SEQ ID NO: 1942) and D295-AG3 (SEQ ID NO: 1943) The nucleic acid sequence of Figures 173-249 show D295-AH1 (SEQ ID NO: 1944), D296-AA6 (SEQ ID NO: 1945), D296-AE5 (SEQ ID NO: 1946), D296-AF5 (SEQ ID NO: 1947) and D296-AG4 (SEQ ID NO: 1948) The nucleic acid sequence of Figure 173-250 shows D296-AG5 (SEQ ID NO: 1949), D297-AA7 (SEQ ID NO: 1950), D297-AA8 (SEQ ID NO: 1951), D297-AB7 (SEQ ID NO: 1952) and D297-AE7 (SEQ ID NO: 1953) The nucleic acid sequence of Figure 173-251 shows D297-AF7 (SEQ ID NO: 1954), D298-AA10 (SEQ ID NO: 1955), D298-AB11 (SEQ ID NO: 1956), D298-AF11 (SEQ ID NO: 1957) and D298-AG12 (SEQ ID NO: 1958) The nucleic acid sequence of Figures 173-252 show D35-40 (SEQ ID NO: 1959), D35-AC6 (SEQ ID NO: 1960), D35-BB2 (SEQ ID NO: 1961), D55-AB9 (SEQ ID NO: 1962) and D55-BB1 (SEQ ID NO: 1963) The nucleic acid sequence of Figure 173-253 shows D55-BB2 (SEQ ID NO: 1964), D55-BB3 (SEQ ID NO: 1965), D55-BB7 (SEQ ID NO: 1966), D56-AA1 (SEQ ID NO: 1967), D56-AE4 (SEQ ID NO: 1968) And the nucleic acid sequence of D56-AE9 (SEQ ID NO: 1969). Figures 173-254 show D56-AD1 (SEQ ID NO: 1970), D56-AG12 (SEQ ID NO: 1971), D56-AH11 (SEQ ID NO: 1972), D57-AA5 (SEQ ID NO: 1973) and D57-AA7 (SEQ ID NO: 1974) The nucleic acid sequence of Figure 173-255 shows D57-AB10 (SEQ ID NO: 1975), D57-AC2 (SEQ ID NO: 1976), D57-AC3 (SEQ ID NO: 1977), D57-AC6 (SEQ ID NO: 1978), D57-AC9 (SEQ ID NO: 1979) And the nucleic acid sequence of D57-AC11 (SEQ ID NO: 1980). Figures 173-256 show D57-AD3 (SEQ ID NO: 1981), D57-AE4 (SEQ ID NO: 1982), D57-AE6 (SEQ ID NO: 1983), D57-AE9 (SEQ ID NO: 1984) and D57-AF4 (SEQ ID NO: 1985). The nucleic acid sequence of Figure 173-257 shows D57-AF11 (SEQ ID NO: 1986), D57-AG3 (SEQ ID NO: 1987), D57-AH11 (SEQ ID NO: 1988), D58-AB2 (SEQ ID NO: 1989), D58-AC8 (SEQ ID NO: 1990) And the nucleic acid sequence of D58-AG9 (SEQ ID NO: 1991). Figures 173-258 show D58-BA9 (SEQ ID NO: 1992), D58-BB9 (SEQ ID NO: 1993), D58-BC1 (SEQ ID NO: 1994), D58-BC10 (SEQ ID NO: 1995), D58-BC11 (SEQ ID NO: 1996) And the nucleic acid sequence of D58-BD4 (SEQ ID NO: 1997). Figure 173-259 shows D58-BE8 (SEQ ID NO: 1998), D58-BF4 (SEQ ID NO: 1999), D60-AB4 (SEQ ID NO: 2000), D60-AC4 (SEQ ID NO: 2001), D60-AD11 (SEQ ID NO: 2002) And the nucleic acid sequence of D60-AF9 (SEQ ID NO: 2003). Figure 173-260 shows D65-AB6 (SEQ ID NO: 2004), D65-AC3 (SEQ ID NO: 2005), D65-AC9 (SEQ ID NO: 2006), D65-AC12 (SEQ ID NO: 2007), D65-AE3 (SEQ ID NO: 2008) And the nucleic acid sequence of D65-AG1 (SEQ ID NO: 2009). Figure 173-261 shows D65-CE10 (SEQ ID NO: 2010), D65-CE11 (SEQ ID NO: 2011), D65-CF11 (SEQ ID NO: 2012), D65-CH5 (SEQ ID NO: 2013), D66-AA1 (SEQ ID NO: 2014) And the nucleic acid sequence of D66-AA3 (SEQ ID NO: 2015). Figures 173-262 show D66-AE5 (SEQ ID NO: 2016), D66-AF1 (SEQ ID NO: 2017), D66-AG2 (SEQ ID NO: 2018), D66-AG6 (SEQ ID NO: 2019) and D66-AH3 (SEQ ID NO: 2020) The nucleic acid sequence of Figure 173-263 shows D66-BA11 (SEQ ID NO: 2021), D66-BD6 (SEQ ID NO: 2022), D66-BD8 (SEQ ID NO: 2023), D67-AA5 (SEQ ID NO: 2024), D67-AD3 (SEQ ID NO: 2025) And the nucleic acid sequence of D67-AE1 (SEQ ID NO: 2026). Figure 173-264 shows D67-AE4 (SEQ ID NO: 2027), D67-AG4 (SEQ ID NO: 2028), D68-AF3 (SEQ ID NO: 2029), D70A-AE1 (SEQ ID NO: 2030), D70A-AG7 (SEQ ID NO: 2031) And the nucleic acid sequence of D70A-BC6 (SEQ ID NO: 2032). Figures 173-265 show D70A-BF7 (SEQ ID NO: 2033), D70A-BF8 (SEQ ID NO: 2034), D70A-BH1 (SEQ ID NO: 2035), D70A-BH3 (SEQ ID NO: 2036) and D73A-AA1 (SEQ ID NO: 2037) The nucleic acid sequence of Figure 173-266 shows D73A-AA3 (SEQ ID NO: 2038), D73A-AA5 (SEQ ID NO: 2039), D73A-AA6 (SEQ ID NO: 2040), D73A-AA8 (SEQ ID NO: 2041), D73A-AA9 (SEQ ID NO: 2042) And the nucleic acid sequence of D73A-AB1 (SEQ ID NO: 2043). Figures 173-267 show D73A-AB3 (SEQ ID NO: 2044), D73A-AB5 (SEQ ID NO: 2045), D73A-AB10 (SEQ ID NO: 2046), D73A-AC2 (SEQ ID NO: 2047) and D73A-AC5 (SEQ ID NO: 2048) The nucleic acid sequence of Figure 173-268 shows D73A-AC11 (SEQ ID NO: 2049), D73A-AC12 (SEQ ID NO: 2050), D73A-AD7 (SEQ ID NO: 2051), D73A-AD10 (SEQ ID NO: 2052), D73A-AE6 (SEQ ID NO: 2053) And the nucleic acid sequence of D73A-AE7 (SEQ ID NO: 2054). Figure 173-269 shows D73A-AE8 (SEQ ID NO: 2055), D73A-AE12 (SEQ ID NO: 2056), D73A-AF5 (SEQ ID NO: 2057), D73A-AF6 (SEQ ID NO: 2058), D73A-AF12 (SEQ ID NO: 2059) And the nucleic acid sequence of D73A-AG4 (SEQ ID NO: 2060). Figures 173-270 show D73A-AG6 (SEQ ID NO: 2061), D73A-AG10 (SEQ ID NO: 2062), D73A-AH2 (SEQ ID NO: 2063), D73A-AH3 (SEQ ID NO: 2064) and D73A-AH10 (SEQ ID NO: 2065) The nucleic acid sequence of Figures 173-271 show D93-AD3 (SEQ ID NO: 2066), D97-AD3 (SEQ ID NO: 2067), D97-AE3 (SEQ ID NO: 2068), D97-AF1 (SEQ ID NO: 2069) and D113-AH8 (SEQ ID NO: 2070). The nucleic acid sequence of Figures 173-272 show D114-AA10 (SEQ ID NO: 2071), D114-AC11 (SEQ ID NO: 2072), D131-AD1 (SEQ ID NO: 2073), D133-AD7 (SEQ ID NO: 2074) and D139-AD2 (SEQ ID NO: 2075) The nucleic acid sequence of Figure 173-273 shows D139-AF2 (SEQ ID NO: 2076), D144A-AB10 (SEQ ID NO: 2077), D144A-AE9 (SEQ ID NO: 2078), D144A-AG12 (SEQ ID NO: 2079), D145-AC7 (SEQ ID NO: 2080) And the nucleic acid sequence of D145-AH7 (SEQ ID NO: 2081). Figure 173-274 shows D150-AA3 (SEQ ID NO: 2082), D150-AG3 (SEQ ID NO: 2083), D151-AG3 (SEQ ID NO: 2084), D153-AA8 (SEQ ID NO: 2085), D153-AB7 (SEQ ID NO: 2086) And the nucleic acid sequence of D153-AC9 (SEQ ID NO: 2087). Figure 173-275 shows D153-AF8 (SEQ ID NO: 2088), D155-AA2 (SEQ ID NO: 2089), D155-AG2 (SEQ ID NO: 2090), D156-AH3 (SEQ ID NO: 2091), D159-AA2 (SEQ ID NO: 2092) And the nucleic acid sequence of D164-AD1 (SEQ ID NO: 2093). Figure 173-276 shows D181-AG6 (SEQ ID NO: 2094), D182-AB1 (SEQ ID NO: 2095), D182-AF2 (SEQ ID NO: 2096), D185-AE10 (SEQ ID NO: 2097), D186-AH3 (SEQ ID NO: 2098) And the nucleic acid sequence of D188-AC6 (SEQ ID NO: 2099). Figure 173-277 shows D188-AF5 (SEQ ID NO: 2100), D189-AD12 (SEQ ID NO: 2101), D258-AG6 (SEQ ID NO: 2102), D194-AA3 (SEQ ID NO: 2103), D194-AB3 (SEQ ID NO: 2104) , D198-AD9 (SEQ ID NO: 2105) and D198-AE9 (SEQ ID NO: 2106). Figures 173-278 show D203-BB11 (SEQ ID NO: 2107), D203-BC11 (SEQ ID NO: 2108), D204-AA3 (SEQ ID NO: 2109), D204-AF2 (SEQ ID NO: 2110), D206-CB1 (SEQ ID NO: 2111) And the nucleic acid sequence of D214-AB1 (SEQ ID NO: 2112). Figures 173-279 show D214-AF2 (SEQ ID NO: 2113), D216-AA7 (SEQ ID NO: 2114), D216-AD7 (SEQ ID NO: 2115), D219-BA2 (SEQ ID NO: 2116), D219-BF1 (SEQ ID NO: 2117) And the nucleic acid sequence of D229-AB2 (SEQ ID NO: 2118). Figure 173-280 shows D230-AD2 (SEQ ID NO: 2119), D230-AE2 (SEQ ID NO: 2120), D234-AE12 (SEQ ID NO: 2121), D241-BG10 (SEQ ID NO: 2122), D249-AA9 (SEQ ID NO: 2123) And the nucleic acid sequence of D255-AC5 (SEQ ID NO: 2124). Figures 173-281 show D270-AE7 (SEQ ID NO: 2125), D270-AE8 (SEQ ID NO: 2126), D276-AH5 (SEQ ID NO: 2127), D279-AA2 (SEQ ID NO: 2128) and D279-AB3 (SEQ ID NO: 2129) The nucleic acid sequence of FIGS. 173-282 are the nucleic acid sequences of D279-AC2 (SEQ ID NO: 2130), D279-AC3 (SEQ ID NO: 2131), D279-AD3 (SEQ ID NO: 2132) and D279-AE2 (SEQ ID NO: 2133). Figures 173-283 show D279-AG2 (SEQ ID NO: 2134), D279-AH3 (SEQ ID NO: 2135), D280-BF6 (SEQ ID NO: 2136), D281-AB8 (SEQ ID NO: 2137) and D281-AC7 (SEQ ID NO: 2138) The nucleic acid sequence of Figures 173-284 show D282-AG10 (SEQ ID NO: 2139), D288-AC2 (SEQ ID NO: 2140), D289-AC6 (SEQ ID NO: 2141), D290-AB8 (SEQ ID NO: 2142) and D291-AD10 (SEQ ID NO: 2143) The nucleic acid sequence of Figure 173-285 shows D291-AE11 (SEQ ID NO: 2144), D291-AH12 (SEQ ID NO: 2145), D292-AB3 (SEQ ID NO: 2146), D292-AD3 (SEQ ID NO: 2147), D292-AE3 (SEQ ID NO: 2148) And the nucleic acid sequence of D294-AE7 (SEQ ID NO: 2149). Figure 173-286 shows D419-AH11 (SEQ ID NO: 2150), D295-AE2 (SEQ ID NO: 2151), D295-AG2 (SEQ ID NO: 2152), D295-AH2 (SEQ ID NO: 2153), D295-AH3 (SEQ ID NO: 2154) And the nucleic acid sequence of D296-AD4 (SEQ ID NO: 2155). Figures 173-287 show D296-AF4 (SEQ ID NO: 2156), D296-AG6 (SEQ ID NO: 2157), D297-AD7 (SEQ ID NO: 2158), D297-AG7 (SEQ ID NO: 2159) and D298-AD11 (SEQ ID NO: 2160) The nucleic acid sequence of Figures 173-288 show D418-AH9 (SEQ ID NO: 2161), D418-AG10 (SEQ ID NO: 2162), D416-AA6 (SEQ ID NO: 2163), D414-AA2 (SEQ ID NO: 2164) and D269-AD5 (SEQ ID NO: 2165). The nucleic acid sequence of Figures 173-289 show D268-AE1 (SEQ ID NO: 2166), D258-AF6 (SEQ ID NO: 2167), D256-AG9 (SEQ ID NO: 2168), D255-AH6 (SEQ ID NO: 2169) and D255-AE5 (SEQ ID NO: 2170) The nucleic acid sequence of Figure 173-290 shows D419-AE11 (SEQ ID NO: 2171), D142-AA10 (SEQ ID NO: 2172), D140-AH4 (SEQ ID NO: 2173), D66-AF7 (SEQ ID NO: 2174) and D66-AA7 (SEQ ID NO: 2175) The nucleic acid sequence of Figures 173-291 show D65-AF9 (SEQ ID NO: 2176), D65-AB3 (SEQ ID NO: 2177), D65-AB2 (SEQ ID NO: 2178), D144A-AA12 (SEQ ID NO: 2179) and D64-7 (SEQ ID NO: 2180) The nucleic acid sequence of Figures 173-292 show D109-BF9 (SEQ ID NO: 2181), D109-BG9 (SEQ ID NO: 2182), D118-AA11 (SEQ ID NO: 2183), D142-AB11 (5 ') (SEQ ID NO: 2184) and D142-AE10 ( 5 ′) is the nucleic acid sequence of (SEQ ID NO: 2185). Figure 173-293 shows D207-AH5 (SEQ ID NO: 2186), D231-AA1 (5 ') (SEQ ID NO: 2187), D232-AD4 (5') (SEQ ID NO: 2188), D232-AE6 (5 ') (sequence) Nos. 2189) and D288-AA3 (SEQ ID NO: 2190). FIGS. 173-294 are the nucleic acid sequences of D289-AE6 (SEQ ID NO: 2191), D290-AC7 (SEQ ID NO: 2192), and D58-AA2 (SEQ ID NO: 2193).

Detailed Description Traditionally, the development of any new desirable plant germplasm has involved multiple stages. Plant breeding begins with the analysis and clarification of current germplasm problems and weaknesses, the establishment of planning goals and the clarification of specific breeding targets. The next step is the selection of germplasm with traits that meet the planned goals. The goal is to combine improved combinations of desirable traits from the parent germplasm in a single variety. Desirable traits include, for example, higher seed yield, disease resistance, insect resistance, drought and heat resistance, and better agricultural quality. However, these processes leading to the final stage of sales and distribution can take 6-12 years after the initial mating. Thus, the development of new varieties is a time consuming process that requires strict future planning, efficient use of resources and minimal policy changes.

  Improving plant varieties by genetic transformation is becoming increasingly important for modern plant breeding. Genes that may be of commercial interest, such as genes that confer certain desirable plant traits of disease resistance, insect resistance or quality improvement, can be incorporated into crop species by various gene transfer techniques. The ability to manipulate gene expression provides a means for obtaining novel properties in transformed plants. In some cases, high or elevated levels of gene expression may be desirable. For example, it is desirable to enhance the production of proteins that themselves maximize disease resistance, yield, flavor or any other commercially desirable plant attributes. Similarly, regulation of endogenous gene expression, for example by gene silencing, can give a more valuable plant or plant product.

  During tobacco ripening or drying (curing), genes identified as ethylene-induced or aging-related genes (eg, SEQ ID NOs: 4, 40, 44, 52, 54, 60, 70, 104, 138, 140 , 158, 162, 188, 212, 226, 234 and 288), activation, upregulation or downregulation of any of the final product quality characteristics (eg disease resistance, insect resistance, quality Can affect those metabolic pathways involved in the formation of a number of secondary metabolites, including terpenoids, polyphenols, alkaloids, and the like. Equally affected by the genes identified herein can include the rate and type of dry matter that accumulates during the aging process or metabolic pathways associated with the distribution of dry matter within the plant during the aging process. Changes in the rate and type of starch accumulation, lignin formation, cellulose precipitation and sugar transport could be shown. Control of the genes identified herein is their metabolic pathways involved in determining aging rate, uniformity of senescence within leaves and between leaves of single plants and induction of senescence by artificial or natural means Can also affect. Senescence-inducing substances or agents that stimulate or activate the genes specified herein include, for example, chemicals such as thin peroxides, insecticides, herbicides, growth regulators, heat treatments, wounds Or increased concentrations of gases such as ozone and carbon dioxide.

Identification of sequences constitutively expressed in tobacco or induced by ethylene or aging In the present invention, RNA was extracted from Nicotiana tissues of converter and nonconverter Nicotiana lines. The extracted RNA was then used for cDNA preparation. The nucleic acid sequences of the present invention were then generated using two methods.

  In the first method, poly A enriched RNA was extracted from plant tissue, and cDNA was prepared by reverse transcription PCR. The single stranded cDNA was then used to obtain a p450-specific PCR population using degenerate primers and oligo d (T) primers. The primer design was based on highly conserved motifs of other plant p450 gene sequences. Specific examples of specific degenerate primers are described in FIG. 1 of patent application publications US 2004/0111759 A1, US 2004/0117869 A1 and US 2004/0103449 A1, which is incorporated herein by reference. ) The sequence of the fragment from the plasmid containing the appropriate size insert was further analyzed. The size of these inserts typically ranges from about 300 to about 800 nucleotides depending on the primer used.

  In the second method, a cDNA library was first constructed. Using the cDNA in the plasmid, a p450-specific PCR population was obtained using degenerate primers and T7 primers. As with the first method, the sequence of the fragment from the plasmid containing the appropriate size insert was further analyzed.

  Nicotiana plant lines known to produce high levels of nornicotine (converters) and plant lines with low levels of nornicotine can be used as starting materials. The leaves can then be removed from the plant and treated with ethylene to activate the p450 enzyme activity as defined herein. Total RNA is extracted using techniques known in the art. Subsequently, a cDNA fragment can be prepared using PCR (RT-PCR) together with an oligo d (T) primer (SEQ ID NO: 2260) as shown in FIG. A cDNA library can then be constructed as described in more detail in the Examples herein.

  The conserved region of p450 type enzyme was used as a template for degenerate primers. Specific examples thereof are shown in FIG. A p450 specific band was amplified by PCR using degenerate primers. A band representing a p450-like enzyme was identified by DNA sequencing. PCR fragments were characterized using BLAST searches, alignments or other means to identify suitable candidates.

PCR primers were generated using sequence information from the identified fragments. These primers combined with the plasmid primers in the cDNA library were used to clone the full length p450 gene. Large scale Southern reverse analysis was performed to examine the differential expression for all fragment clones obtained and in some cases full length clones. In this aspect of the invention, these large scale reverses are used using total labeled cDNA from various tissues as probes that hybridize to the cloned DNA fragments to screen all cloned inserts. Southern assays can be performed. Non-radioactive and radioactive (P ³² ) Northern blot assays were also used to characterize cloned p450 fragments and full-length clones.

  Once a plant cell is obtained that expresses the desired level of p450 enzyme, plant tissue and whole plants can be regenerated from the cell using methods and techniques well known in the art. The regenerated plant can then be regenerated by conventional means and the transgene can be introduced into other strains and cultivars using conventional plant breeding techniques.

  Identified in ethylene-inducible or senescence-related genes such as SEQ ID NOs: 4, 40, 44, 52, 54, 60, 70, 104, 138, 140, 158, 162, 188, 212, 226, 234 and 288 One can encode an enzyme that is an important determinant of tobacco leaf quality parameters important to various tobacco products. Such tobacco products include wet or dry hookah, chewing tobacco, cigarette products, cigar products, cigarillos, pipe tobacco, bidi and similar smoking products. Leaf quality parameters include structural or physical properties indicated by visual attributes such as color, surface regularity, texture or variegation; leaf blade: medium ratio, oiliness, cigarette filling ability, Bulk density, moisture retention and flexibility; flavor, aroma, fermentability, burn rate, burn temperature, chemical or biochemical traits related to absorption and release of artificial flavors; and Production of tobacco components including tar or particulates, alkaloids and other attributes can be included. Enzymatic reactions arising from these ethylene-induced or senescence-related genes can also produce secondary metabolites that affect pathogens or insect interactions that affect tobacco leaf yield and quality. For example, Wagner et al. (Nature Biotechnology, 19: 371-374, 2001) show that repression of the p450 hydroxylase gene significantly increases the accumulation of cembratiene-ol, a secondary metabolite that affects aphid resistance. It was shown to enhance.

Peptide-specific antibodies were produced by deriving the amino acid sequence of the antibody-generated peptide and selecting peptide regions that are unique and antigenic in comparison to other clones. Rabbit antibodies were raised against the synthetic peptide bound to the carrier protein. These antibodies were used for Western blot analysis or other immunological methods on plant tissues. Peptide-specific antibodies were also produced for some full-length clones by deriving the amino acid sequence of the peptides and selecting peptide regions that were unique and potentially antigenic in contrast to other clones. . Rabbit antibodies were raised against the synthetic peptide bound to the carrier protein. Western blot analysis was performed using these antibodies.

Plants with downregulation of gene expression and decreased expression of modified polypeptides of enzyme activity can be transformed into standard gene silencing methods (for review, see Arndt and Rank, Genome 40: 785-797, 1997; Turner and Schuch, Journal of Chemical Technology and Biotechnology 75: 869-882, 2000; and Klink and Wolniak, Journal of Plant Growth Regulation 19 (4): 371-384, 2000). In particular, tobacco nicotine demethylase nucleic acid sequences (eg, SEQ ID NOs: 4, 5, 7, 8 and 9, or fragments thereof, eg, sequences of SEQ ID NOs: 1 and 62) and substantially identical nucleic acid sequences (eg, SEQ ID NO: 188 sequences) can be used to modify tobacco phenotypes or tobacco metabolites, such as nornicotine, in any Nicotiana species. Decreased expression of the tobacco nicotine demethylase gene is e.g. RNA interference (RNAi) (Smith et al., Nature 407: 319-320, 2000; Fire et al., Nature 391: 306-311, 1998; Waterhouse et al., PNAS 95: 13959- 13964, 1998; Stalberg et al., Plant Molecular Biology 23: 671-683, 1993; Brignetti et al., EMBO J. 17: 6739-6746, 1998; Allen et al., Nature Biotechnology 22: 1559-1566, 2004); virus-inducible genes Silencing ("VIGS") (Baulcombe, Current Opinions in Plant Biology, 2: 109-113, 1999; Cogoni and Macino, Genes Dev 10: 638-643, 2000; Ngelbrecht et al., PNAS 91: 10502-10506, 1994) Silencing target genes by introducing plant endogenous genes in sense orientation (Jorgensen et al., Plant Mol Biol 31: 957-973, 1996); antisense gene expression; homologous recombination (Ohl et al., Homologous Recombination and Gene Silencing in Plants. Kluwer, Dordrecht, The Netherlands, 1994); Cre / lox (Qin et al., PNAS 91: 1706-1710, 1994; Koshinsky et al., The Plant Journ al 23: 715-722, 2000; Chou et al., Plant and Animal Genome VII Conference Abstracts. San Diego, CA, 17-21 January, 1999); gene trapping and T-DNA tagging (Burns et al., Genes Dev. 8: 1087) -1105, 1994; Spradling et al., PNAS 92: 10824-10830, 1995; Skarnes et al., Bio / Technology 8, 827-831, 1990; Sundaresan et al., Genes Dev. 9: 1797-1810, 1995); and tobacco polypeptides Can be achieved using any of the other possible gene silencing systems available in the scientific field that cause down-regulation of the expression of or the reduction of its enzymatic activity. As described in further detail herein, any of the nucleic acid sequences described herein can be down-regulated or up-regulated using the techniques described herein and other techniques found in the art. Can be done. A typical method is described in more detail below.

RNA interference
RNA interference (“RNAi”) is a generally applicable method for inducing strong and specific post-translational gene silencing in many organisms such as plants (eg, Bosher et al., Nat. Cell Biol. 2). : E31-36, 2000; and Tavernarakis et al., Nat. Genetics 24: 180-183, 2000). RNAi involves the introduction of RNA with partial or complete double-stranded properties into the intracellular or extracellular environment. Inhibition is specific in that the nucleotide sequence from the portion of the target gene (eg, tobacco nicotine demethylase) is selected to produce inhibitory RNA. The selected portion generally comprises an exon of the target gene, but the selected portion includes untranslated regions (UTRs) and introns (eg, the sequence of SEQ ID NO: 7, or nucleic acid sequences from the desired plant gene, 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294).

  For example, to construct a transformation vector that produces RNA with the ability to form double strands, two nucleic acid sequences (one in sense orientation and the other in antisense orientation) are operably linked, It can be placed under the control of a strong viral promoter, such as a promoter isolated from CaMV 35S or cassava brown streak virus (CBSV). However, it may also be desirable to use an endogenous promoter, such as the nicotine demethylase promoter having the sequence of SEQ ID NO: 8, or a fragment thereof that drives transcription. The length of the tobacco nicotine demethylase nucleic acid sequence included in such a construct is desirably at least 25 nucleotides, but may include sequences comprising tobacco nicotine demethylase up to full length.

  Constructs producing RNA with double-strand-forming ability can be obtained from plants (eg, by Agrobacterium-mediated transformation (Chuang et al., Proc. Natl. Acad. Sci. USA 97: 4985-4990, 2000). Can be introduced into the genome of a tobacco plant) to cause specific and inheritable genetic interference in tobacco nicotine demethylase. The double-stranded RNA can be introduced directly into the cell (ie, into the cell), or outside the cell, eg, in a solution containing the double-stranded RNA, seeds, seedlings or It can be introduced by soaking the plants.

  Depending on the amount of double-stranded RNA material delivered, RNAi can result in partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 99% of the target cells can be obtained. In general, lower amounts of infused material and longer times after administration of dsRNA cause inhibition of a smaller percentage of cells.

  The RNA used in RNAi can include one or more strands of polymerized ribonucleotides, which can include modifications to the phosphate-sugar backbone or nucleoside. The double-stranded structure can be formed by a single self-complementary RNA strand or by two complementary RNA strands, and RNA duplex formation can be initiated inside or outside the cell. The RNA can be introduced in an amount that allows delivery of at least 1 copy / cell. However, higher amounts (eg, at least 5, 10, 100, 500 or 1000 copies / cell) of double-stranded material can result in more efficient suppression. Suppression is sequence specific in that the nucleotide sequence corresponding to the double stranded region of the RNA is targeted for genetic suppression. For suppression, RNA containing the same nucleotide sequence as the target gene portion is preferred. RNA sequences with insertions, deletions and single point mutations when compared to the target sequence may also be effective for suppression. For example, sequence identity can be optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the double-stranded region of the RNA can be functionally defined as a nucleotide sequence that can hybridize to a portion of the target gene transcript.

  Moreover, RNA used for RNAi can be synthesized in vivo or in vitro. For example, endogenous RNA polymerase in the cell can provide transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene or expression construct in vivo, a regulatory region can be used to transcribe the RNA strand.

Triple stranded interference Also by targeting a deoxyribonucleotide sequence complementary to a regulatory region (eg, promoter or enhancer region) of a tobacco gene to form a triple helix structure that prevents transcription of the tobacco gene in the target cell. , Endogenous tobacco nicotine demethylase gene expression, or nucleic acid fragments from the desired plant gene (eg, FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173 -294 (any nucleic acid sequence shown) can be downregulated (generally Helene, Anticancer Drug Des. 6: 569-584, 1991; Helene et al., Ann. NY Acad. Sci. 660: 27- 36, 1992; and Maher, Bioassays 14: 807-815, 1992).

  The nucleic acid molecule used in the formation of a triple helix for transcriptional repression is preferably single-stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides generally promotes triple helix formation by Hoogsteen-type base-pairing rules, which require that a substantial amount of purine or pyrimidine extension be present on one strand of the double helix. Should bring. The nucleotide sequence can be based on pyrimidines. This will give TAT and CGC triplets across the three associated strands of the resulting triple helix. A pyrimidine-rich (pyrimidine-rich) molecule provides base complementarity to a single-stranded purine-rich region of a double helix in a parallel orientation to that strand. Also, purine-rich (eg, containing G residue extensions) nucleic acid molecules can be selected. These molecules form a triple helix with the GC pair-rich DNA double helix, in which the majority of the purine residues are located on one strand of the target double helix and the triple helix in the triple helix. Gives a CGC triplet across the chain.

  Alternatively, the potential sequences that can be targeted for triple helix formation can be increased by generating “switchback” nucleic acid molecules. Switchback molecules are synthesized in an alternating 5′-3 ′, 3′-5 ′ manner. This allows them to base pair with the first strand of the double helix, then base pair with the other, and the presence of a substantial size purine or pyrimidine extension on one strand of the double helix. Eliminate the need for

Ribozymes Ribozymes are RNA molecules that act as enzymes and can be designed to cleave other RNA molecules. Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at specific positions to functionally inactivate the target RNA. In this process, the ribozyme itself is not consumed and can act catalytically to cleave multiple copies of the mRNA target molecule. Thus, ribozymes can also be used as a means to down regulate the expression of tobacco nicotine demethylase. The design and use of target RNA specific ribozymes is described in Haseloff et al. (Nature 334: 585-591, 1988). Preferably, the ribozyme is flanked on both sides of the ribozyme active site by a target sequence (eg, tobacco nicotine demethylase, or a nucleic acid fragment from the desired plant gene, eg, FIG. 1, 3-7, 10-158, 162- 170, 172-1 to 172-19 and any nucleic acid sequence shown in 173-1 to 173-294).

  In addition, ribozyme sequences can be included in the antisense RNA in order to confer RNA cleavage activity to the antisense RNA and enhance the effectiveness of the antisense construct.

Homologous recombination Gene replacement technology is another desirable method for down-regulating expression of a given gene. Gene replacement technology is based on homologous recombination (Schnable et al., Curr. Opinions Plant Biol. 1: 123-129, 1998). Depending on the enzyme of interest (eg tobacco nicotine demethylase or any nucleic acid sequence shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 The nucleic acid sequence of the encoded polypeptide) can be manipulated by mutagenesis (eg, insertion, deletion, duplication or substitution) to reduce enzyme function. The modified sequence can then be introduced into the genome and the existing gene (eg, wild type gene) can be replaced with the sequence by homologous recombination (Puchta et al., Proc. Natl. Acad. Sci. USA 93: 5055-5060, 1996; and Kempin et al., Nature 389: 802-803, 1997). Alternatively, the endogenous tobacco nicotine demethylase gene can be replaced with a gene that does not have demethylase activity (eg, the sequence of SEQ ID NO: 188).

Co-suppression Another desirable method for silencing gene expression is co-suppression (also called sense suppression). This technique involves nucleic acids arranged in a sense orientation (eg, nucleic acid fragments from a desired plant gene, eg, FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173- Any nucleic acid sequence shown in 1-173-294) and has been shown to effectively block transcription of target genes (eg, Napoli et al., Plant Cell, 2: 279-289, 1990 and Jorgensen et al., US Pat. No. 5,034,323).

  In general, sense suppression involves transcription of the introduced sequence. However, the introduced sequence does not contain the coding sequence itself, but the intron or untranslated sequence or other such sequence is substantially identical to the sequence present in the primary transcript of the endogenous gene to be repressed. In some cases, co-suppression can occur. The introduced sequence is generally substantially identical to the endogenous gene targeted for repression. Such identity is typically greater than about 50%, although higher identity (eg, 80% or even 95%) is preferred. Because they cause more effective suppression. The effect of co-suppression can be applied to other proteins within similar gene families that exhibit homology or substantial homology. Segments from genes from one plant can be used directly, for example, to suppress the expression of homologous genes in different plant species.

  In sense suppression, the introduced sequence does not require absolute identity and need not be full length in comparison to the primary transcript or fully processed mRNA. Higher sequence identity in sequences shorter than full length compensates for longer sequences of lower identity. Furthermore, the introduced sequence need not have the same intron or exon pattern, and the identity of the non-coding segment can be equally effective. A sequence of at least 50 base pairs is preferred, with longer introduced sequences being preferred (see, eg, the method described in Jorgensen et al., US Pat. No. 5,034,323).

Antisense suppression In antisense technology, nucleic acid segments from a desired plant gene (eg, FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294). Are cloned and operably linked to the expression control region so that the antisense strand of RNA is synthesized. The construct is then transformed into plants to produce the antisense strand of RNA. In plant cells, antisense RNA has been shown to suppress gene expression.

  The nucleic acid segment to be introduced in antisense suppression is generally substantially the same as at least a portion of the endogenous gene to be suppressed, but need not be the same. The nucleic acid sequence of tobacco nicotine demethylase disclosed herein is such that the inhibitory effect applies to other proteins in the gene family that show homology or substantial homology to the target gene. It can be contained in a vector designed in the above. Segments from genes from one plant can be used directly to suppress the expression of homologous genes, for example in different tobacco varieties.

  The introduced sequence need not be full length relative to the primary transcript or fully processed mRNA. In general, higher homology can be used to compensate for the use of shorter sequences. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective. In general, such antisense sequences will usually be at least 15 base pairs in length, preferably about 15-200 base pairs, more preferably 200-2,000 base pairs or longer. The antisense sequence can be complementary to all or part of the gene to be repressed. As will be appreciated by those skilled in the art, the individual sites to which the antisense sequence binds and the length of the antisense sequence will vary depending on the degree of suppression desired and the uniqueness of the antisense sequence. Transcription constructs that express plant negative regulatory antisense nucleotide sequences include, in the direction of transcription, a promoter, a sequence encoding antisense RNA on the sense strand, and a transcription termination region. Antisense sequences are described, for example, by van der Krol et al. (Gene 72: 45-50, 1988); Rodermel et al. (Cell 55: 673-681, 1988); Mol et al. (FEBS Lett. 268: 427-430, 1990); Weigel And Nilsson (Nature 377: 495-500, 1995); Cheung et al. (Cell 82: 383-393, 1995); and Shewmaker et al. (U.S. Pat.No. 5,107,065) can be constructed and expressed. Is possible.

Dominant negative body A transgenic plant expressing a transgene encoding a dominant negative gene product of a tobacco gene product is used in an artificial environment to show that the transgene down-regulates the tobacco gene product in the transgenic plant. Or it can be assayed in the field. The dominant negative transgene is constructed according to methods known in the art. Typically, a dominant negative gene encodes a mutant negative regulatory polypeptide of a tobacco gene product that, when overexpressed, impairs the activity of the wild type enzyme.

Variants Plants with reduced tobacco gene product expression or reduced enzyme activity can also be generated using standard mutation methods. Such mutagenesis methods include, but are not limited to, treatment of seeds with ethyl methyl sulfate (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon press, pp 317-320, 1965) or UV irradiation, X-ray and fast neutron irradiation (eg Verkerk, Neth. J. Agric. Sci. 19: 197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup rd), 1987), the use of transposons (Fedoroff et al., 1984; US Pat. No. 4,732,856 and US Pat. No. 5,013,658) and the T-DNA insertion method (Hoekema et al., 1983; US Pat. No. 5,149,645). Is included. Types of mutations that can be present in tobacco genes include, for example, point mutations, deletions, insertions, duplications and inversions. Such mutations are desirably present in the coding region of the tobacco gene, although mutations in the promoter region and intron or untranslated region of the tobacco gene may also be desirable.

  For example, T-DNA insertion mutagenesis can be used to create insertion mutations in tobacco genes that down-regulate gene expression. Theoretically, approximately 100,000 independent T-DNA insertions are required to obtain 95% probability of insertion in any given gene (McKinnet, Plant J. 8: 613-622, 1995; and Forsthoefel et al., Aust. J. Plant Physiol. 19: 353-366, 1992). Plant T-DNA tagged lines can be screened using polymerase chain reaction (PCR) analysis. For example, one primer can be designed for one end of T-DNA and the other primer can be designed for the gene of interest, both primers used for the PCR analysis Is possible. If no PCR product is obtained, there is no insertion in the gene of interest. In contrast, when a PCR product is obtained, there is an insertion in the gene of interest.

  The expression of the mutant tobacco gene product can be assessed by standard methods (eg, the methods described herein) and, if desired, can be compared to the expression of the non-mutant enzyme. . Mutant plants with reduced expression of genes encoding tobacco gene products when compared to non-mutant plants are desirable embodiments of the present invention. Plants having mutations within any of the nucleic acid sequences described herein can be used in the breeding schemes described herein.

Overexpression of constitutive or ethylene or aging inducible sequences
In order to enhance desirable traits in tobacco products produced from Nicotiana lines or plants of that line, the nucleic acid sequences of the present invention (e.g., FIGS. 1, 3-7, 10-158, 162-170, 172-1-172). -19 and 173-1 to 173-294 nucleic acid sequences or fragments thereof) can be used. In particular, overexpression of the nucleic acid sequences of the present invention and their translation products can be used to enhance the biosynthesis of desirable flavor and aroma products resulting from secondary metabolites. Further overexpression of nucleic acid sequences encoding tobacco polypeptides can be used to enhance expression of the polypeptide in Nicotiana strains.

  Further desirable traits that can be imparted to Nicotiana strains by overexpressing the nucleic acids of the present invention include bacterial wilt, Granville wilt, blight, potato virus Y, tobacco mosaic virus, tobacco corrosion ( etch) virus, tobacco mottling virus, alfalfa mosaic virus, wildfire, root-knot nematode, southern root-knot nematode, cyst nematode, black root disease, blue mold, race 0 tobacco plague and Includes resistance to race 1 tobacco plague. Other desirable traits that can be enhanced in Nicotiana plants by expressing the nucleic acid sequences of the present invention include increased yield and / or grade, drought, harvestability, retention, improved leaf quality or drought characteristics, length Includes increase or decrease, change in maturation time (eg, early maturity, early to medium, medium, medium to late or late), increase or decrease in stalk size, and increase or decrease in the number of leaves per plant .

Plant promoters Desirable promoters are caulimovirus promoters, such as the cauliflower mosaic virus (CaMV) promoter or the cassava vein mosaic virus (CsVMV) promoter. These promoters provide high levels of expression in most plant tissues, and the activity of these promoters is not dependent on virus-encoded proteins. CaMV is a source of both 35S and 19S promoters. Specific examples of plant expression constructs that use these promoters are known in the art. In most tissues of transgenic plants, the CaMV35S promoter is a strong promoter. The CaMV promoter is also very active in monocotyledonous plants. Furthermore, the activity of this promoter can be further enhanced (ie 2-10 times) by duplication of the CaMV35S promoter.

  Other useful plant promoters include, but are not limited to, the nopaline synthase (NOS) promoter, octopine synthase promoter, sesame mosaic virus (FMV) promoter, rice actin promoter, and ubiquitin promoter system.

  Typical monocotyledonous promoters include, but are not limited to, rice yellow mottle virus, sugarcane badnavirus promoter, rice tungrobacilliform virus promoter, corn streak virus element , And wheat dwarf virus promoters.

  For some applications, it may be desirable to produce tobacco gene products, such as dominant negative mutant gene products, in appropriate tissues, in appropriate amounts, or at appropriate growth points. For this purpose, a group of gene promoters that have been shown to be regulated in response to inducible signals such as environment, hormones and / or developmental stimuli, each of which is its own distinct form embodied in its regulatory sequence. Has a characteristic). These include, but are not limited to, heat-regulated gene expression, light-regulated gene expression (eg, pea rbcS-3A; maize rbcS promoter; chlorophyll a / b binding protein gene found in pea; or Arabssu Promoter), hormone-regulated gene expression (eg, abscisic acid (ABA) responsive element from wheat Em gene; barley and Arabidopsis ABA-inducible HVA1 and HVA22 and rd29A promoters) and wound-inducible gene expression (eg, wunl ), Gene-specific gene expression (eg tuber-specific storage protein gene; 23 kDa zein gene from corn as described; or kidney bean β-phaseolin gene), or pathogen-inducible promoter (eg PR -1, prp-1 or β-1,3 gluca Over lactamase promoter, fungal inducible wirla promoter and nematode inducible promoter of wheat, tobacco and parsley, respectively TobRB7-5A and Hmg-1) are included.

Plant Expression Vectors Typically, plant expression vectors include (1) a cloned plant gene under transcriptional control of 5 ′ and 3 ′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors optionally have a promoter regulatory region (eg, one that confers inducible or constitutive, pathogen or wound inducible, environmental or developmental regulatory, or cell or tissue specific expression), transcription initiation. It may also contain a start site, ribosome binding site, RNA processing signal, transcription termination site and / or polyadenylation signal.

  Plant expression vectors may also optionally contain RNA processing signals such as introns, which have been shown to be important for efficient RNA synthesis and accumulation. The location of the RNA splice sequence can dramatically affect the level of transgene expression in plants. In view of this fact, it is possible to place an intron upstream or downstream of the tobacco nicotine demethylase coding sequence in the transgene to modify the level of gene expression.

  In addition to the 5 ′ regulatory control sequences described above, the expression vector may also include regulatory control regions that are generally present in the 3 ′ region of plant genes. For example, a 3 ′ terminator region can be included in the expression vector to increase the stability of the mRNA. One such terminator region may be derived from the potato PI-II terminator region. Also other commonly used terminators are derived from octopine or nopaline synthase signals.

  Plant expression vectors typically also contain a dominant selectable marker gene to identify transformed cells. Useful selection genes for plant systems include transposon Tn5 aminoglycoside phosphotransferase gene (AphII), genes encoding antibiotic resistance genes such as hygromycin, kanamycin, bleomycin, neomycin, G418, streptomycin or spectinomycin A gene encoding resistance is included. Genes required for photosynthesis can also be used as selection markers in photosynthesis deficient strains. Finally, a gene encoding herbicide resistance can be used as a selectable marker. Useful herbicide resistance genes include the bar gene that encodes the enzyme phosphinotricin acetyltransferase and confers resistance to the broad spectrum herbicide Basta® (Bayer Cropscience Deutschland GmbH, Langenfeld, Germany). Other selectable markers include genes that confer resistance to other such herbicides such as glyphosate, and imidazolinones, sulfonylureas, triazolopyrimidine herbicides, chlorosulfuron, bromoxynyl, dalapon, and the like. In addition, genes encoding dihydrofolate reductase can be used with molecules such as methotrexate.

  Efficient use of a selectable marker is facilitated by determining the sensitivity of the plant cell to an individual selection factor and measuring the concentration of this factor that effectively kills most if not all of the transformed cells. Some useful concentrations of antibiotics for tobacco transformation include, for example, 20-100 μg / ml (kanamycin), 20-50 μg / ml (hygromycin) or 5-10 μg / ml (bleomycin). A useful method for selection of transformants for herbicide resistance is described, for example, by Vasil (Cell Culture and Somatic Cell Genetics of Plants, Vol I, II, III Laboratory Procedures and Their Applications, Academic Press, New York, 1984). Have been described.

  In addition to a selectable marker, it may be desirable to use a reporter gene. In some cases, the reporter gene is used in the absence of a selectable marker. Reporter genes are genes that are typically not present or expressed in the recipient organism or tissue. Reporter genes typically encode proteins that result in some phenotypic change or enzymatic property. Specific examples of such genes are described in Weising et al. (Ann. Rev. Genetics 22: 421, 1988), incorporated herein by reference. Preferred reporter genes include but are not limited to the glucuronidase (GUS) gene and the GFP gene.

  Following construction of a plant expression vector, several standard methods are available for introducing the vector into a plant host to create a transgenic plant. These methods include (1) Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes) (eg, Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, London, Academic Press, 1987; and Lichtenstein, CP and Draper, J., In: DNA Cloning, Vol II, DM Glover, Oxford, IRI Press, 1985; U.S. Pat. And 5,464,763; and European Patent Nos. 0131624, 0159418, 0120516, 0176112, 0116718, 0290799, 0292435, 0320500 and 0627752, and European Patent Application Nos. 0267159 and 00604622), (2) particle delivery systems (eg, US Pat. Nos. 4,945,050 and 5,141,131), (3) microinjection method, (4) polyethylene glycol (PEG) method, (5) liposome-mediated DNA uptake, (6) electroporation (See, for example, WO 87/06614 and US Pat. Nos. 5,384,253, 5,472,869, 5,641,664, 5,679,558, 5,712,135, 6,002,070 and 6,074,877), (7) vortex method, or (8) the so-called whiskers method ( See, for example, Coffee et al., US Pat. Nos. 5,302,523 and 5,464,765). Types of plant tissue that can be transformed with expression vectors include embryonic tissue, callus tissue types I and II, hypocotyl, meristem, and the like.

  Following introduction into plant tissue, the expression of structural genes can be assayed by any means known in the art, as a transcribed mRNA, as a synthesized protein, or as a secondary alkaloid in tobacco Expression can be measured as the amount of gene silencing produced as measured by metabolite monitoring by chemical analysis of (as described herein; US Pat. No. 5,583,021 incorporated herein by reference) (See also issue). Techniques for in vitro culture of plant tissue and in many cases regeneration to whole plants are known (see, eg, US Pat. Nos. 5,595,733 and 5,766,900). Methods for transferring introduced expression complexes to commercially useful cultivars are known to those skilled in the art.

  Once plant cells expressing the desired level of the desired gene product are obtained, plant tissues and whole plants can be regenerated therefrom using methods and techniques well known in the art. The regenerated plant can then be regenerated by conventional means and the introduced gene can be transferred to other strains and cultivars using conventional plant breeding techniques.

  A transgenic tobacco plant may contain nucleic acids of any part of the genomic gene in different orientations for down-regulation or overexpression (eg, antisense orientation or sense orientation, respectively). In order to enhance the expression of the gene product in Nicotiana strains, overexpression of nucleic acid sequences encoding the entire amino acid sequence of a full-length tobacco gene or a functional part is desirable.

Measurement of transcription or translation level of tobacco genes Gene expression can be performed, for example, by standard Northern blot analysis using tobacco genes or gene fragments as hybridization probes (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York , NY, (2001), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, (1989)). RNA expression levels are measured by quantitative rtPCR (Kawasaki et al., In PCR Technology: Principles and Applications of DNA Amplification (HA Erlich) Stockton Press (1989); Wang et al. In PCR Protocols: A Guide to Methods and Applications (MA Innis Et al.) Academic Press (1990); and Freeman et al., Biotechniques 26: 112-122 and 124-125, 1999) can also be assisted by reverse transcription PCR (rtPCR). Further well-known techniques for measuring tobacco gene expression include in situ hybridization and fluorescence in situ hybridization (eg, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY). , (2001)). The standard techniques described above can also be useful for comparing expression levels between plants, for example, between plants having mutations in the tobacco gene and control plants.

  If desired, use the same general approach and standard protein analysis techniques such as Bradford assay, spectrophotometric assay, and immunological detection techniques such as Western blotting or immunoprecipitation with antibodies specific for the desired polypeptide. Used for expression of tobacco genes (eg nucleic acid sequences shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or fragments thereof) It can be measured at the level of protein production (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (2001), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, (1989)).

  The activity of any polypeptide described herein can be assayed using standard methods in the art. For example, the activity of p450 is typically assayed using a fluorescence-based assay (see, eg, Donato et al., Drug Metab Dispos. 32: 699-706, 2004). In particular, the activity of nicotine demethylase can be assayed as described herein using a yeast microsome assay.

Identification of Tobacco Gene Product Modulators Isolation of cDNA also facilitates identification of molecules that increase or decrease gene product expression. In one approach, various candidate molecules are placed in the medium of cells expressing tobacco mRNA (eg, prokaryotic cells, eg, E. coli or eukaryotic cells, eg, yeast, mammalian cells, insect cells or plant cells). Add by concentration. The expression of the gene product is then measured in the presence and absence of the candidate molecule using, for example, standard methods such as those described herein.

  Candidate modulators can be purified (or substantially purified) molecules, or can be a component of a compound mixture. In mixed compound assays, candidate compound pools (eg, those obtained by standard purification techniques, such as HPLC), until a single compound or minimal compound mixture is shown to modify tobacco nicotine demethylase gene expression. The expression of the gene product is tested against progressively smaller subgroups. In one embodiment of the invention, molecules that promote reduced expression of gene products may be particularly desirable. Modulators that have been found to be effective at the level of gene product expression or activity may prove useful in in planta.

  For agriculture, molecules, compounds or substances identified using the methods disclosed herein can be used as chemicals applied as sprays or dusts on plant foliage. The molecule, compound or substance can be applied to a plant with another molecule that provides some benefit to the plant.

Applications Regulation of endogenous genes corresponding to any of the sequences described herein, such as gene silencing, can give a more valuable plant or plant product. In particular, sequences identified herein as being induced by ethylene or associated with aging (eg, SEQ ID NOs: 4, 40, 44, 52, 54, 60, 70, 104, 138, 140 , 158, 162, 188, 212, 226, 234 and 288 or the nucleic acids shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 Can be used to influence the metabolic pathways involved in the formation of numerous secondary metabolites, including terpenoids, polyphenols, alkaloids, etc. that affect the quality characteristics of the final product. Is possible. Similarly, the genes identified herein can be used to regulate the rate and type of dry matter that accumulates during the aging process or the metabolic pathways associated with the distribution of dry matter within the plant during the aging process. The regulation of the genes identified herein can be achieved by stimulating or activating the genes identified herein by stimulating or activating the rate of senescence, uniformity of senescence within leaves and between leaves of a single plant. It can also be used to influence the metabolic pathways involved in determining the induction of aging by substances or activities that control the quality of products or manufactured goods that contain other plant components.

  The promoter regions of the genes described herein can be used to drive the expression of any desired gene product that improves crop quality or enhances a particular trait. Induction expressed during specific periods of the plant life cycle in constructs for introduction into plants to express unique genes involved in the biosynthesis of flavor and aroma products resulting from secondary metabolites Sex promoters can be used. Tobacco gene promoters can also be used to increase or modify the expression of structural carbohydrates or proteins that affect end-use properties. Furthermore, tobacco gene promoters could be combined with heterologous genes including genes involved in the biosynthesis of nutritional products, pharmaceutical substances or industrial substances. Regulation of promoter sequences can be used to down-regulate endogenous tobacco genes, including genes involved in alkaloid biosynthesis and / or other pathways. Desirably, tobacco gene promoter regions or other transcriptional regulatory regions are used to modify chemical properties such as nornicotine content and nitrosamine levels in plants. Also, a factor that is associated with or regulates expression of a tobacco gene product (eg, p450) is identified using a promoter motif that can be readily identified within the promoter sequence using standard methods in the art. It is possible.

  Further, any of the sequences of the present invention (for example, the nucleic acid sequences shown in FIGS. 1, 3 to 7, 10 to 158, 162 to 170, 172-1 to 172-19 and 173-1 to 173-294, or fragments thereof) It can be used in methods of reducing gene expression of a gene product (eg, p450) or modifying its enzymatic activity using standard methods described herein. Such techniques include, but are not limited to, RNA interference, triple strand interference, ribozyme, homologous recombination, virus-induced gene silencing, antisense and co-suppression techniques, expression of dominant negative gene products, standards Production of mutant genes using conventional mutagenesis techniques. For example, decreased p450 expression to modify fatty acids associated with phytopathogen interactions and disease resistance, or to modify the plant profile of selected fatty acids to modify the flavor or aroma of the plant or plant component Alternatively, modification of p450 enzyme activity can be used.

  In addition, to screen for related genes in other tobacco Nicotiana species, or to determine whether a plant has a mutation in the corresponding endogenous gene, to isolate related genes, promoters or regulatory regions As a marker, any part of a tobacco gene such as a promoter, coding sequence, intron or 3′UTR or a fragment thereof can be used by standard methods. Also, a portion of the tobacco gene can be used to monitor gene flow through breeding operations to track the transfer or loss of a particular gene.

  For example, Nicotiana tabacum, like some of the other Nicotiana species, is an allotetraploid and can be used to identify homologous genes or related genes in the parental genome that are different from the genome in which the original gene resides. I will. Markers for related genes could also be used to screen existing tobacco germplasm, segregated or synthetic populations generated by crossing, populations generated by mutagenesis or various tissue culture methods. Accordingly, the nucleic acid sequences described herein (e.g., the nucleic acid sequences shown in Figures 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or their Fragments) are plant components (eg, lignin, cellulose, etc.) that are disease resistant, insect resistant, flavor and fragrant, herbicide resistant, increase leaf yield or affect leaves or are associated with structural traits or fiber content Can be used to identify or affect genes involved in quality factors affecting or undesired components.

Tobacco products with reduced product nitrosamine content are produced using any tobacco plant material described herein by standard methods known in the art. In one embodiment, the tobacco product is manufactured using tobacco plant material obtained from dried tobacco plants. Dried tobacco plants can contain reduced nicotine demethylase activity. Alternatively, the dried tobacco plant may have been bred to contain reduced nicotine demethylase activity. For example, a dry tobacco plant can be a tobacco plant obtained from a cross that includes a tobacco plant identified as having mutated expression of nicotine demethylase. Desirably, the tobacco product is less than about 5 mg / g, less than about 4.5 mg / g, less than about 4.0 mg / g, less than about 3.5 mg / g, less than about 3.0 mg / g, less than about 2.5 mg / g, about Less than 2.0 mg / g, less than about 1.5 mg / g, less than about 1.0 mg / g, less than about 750 μg / g, less than about 500 μg / g, less than about 250 μg / g, less than about 100 μg / g, about 75 <μg / g, <50 μg / g, <25 μg / g, <10 μg / g, <7.0 μg / g, <5.0 μg / g, <4.0 μg / g, <2.0 μg <g / g, <1.0 μg / g, <0.5 μg / g, <0.4 μg / g, <0.2 μg / g, <0.1 μg / g, <0.05 μg / g, or <0.01 μg / g containing a reduced amount of nornicotine or NNN less than g, or where the percentage of secondary alkaloids relative to the total alkaloid content contained therein is less than 90%, less than 70%, less than 50%, 30 <%, <10%, <5%, <4%, <3%, <2%, <1.5%, <1%, <0.75%, <0.5% Less than 0.25%, or less than 0.1%. The expression "reduced amount" (reduced amount) is found in wild-type tobacco plants or plant components or tobacco products from similarly processed varieties that have not been transgenic for nornicotine or NNN reduction Mean lower amounts of nornicotine or NNN or both in tobacco plants or plant components or tobacco products. In one embodiment, a similarly processed wild-type tobacco plant that has been similarly processed as a control to determine whether a decrease in nornicotine or NNN or both was obtained by the methods described herein. Is used. In another embodiment, reduced nitrosamine content using standard methods, for example, by monitoring the presence or absence of a gene or gene product (eg, nicotine demethylase) or a specific intragenic mutation. Evaluate plants with In yet another embodiment, the nitrosamine content of the plant obtained from the breeding program is compared to the nitrosamine content of the recipient line and / or donor line used to breed the plant with reduced nitrosamine levels. . Other suitable controls known in the art are also used as needed. Nornicotine and NNN or both levels are measured according to methods well known in the tobacco field.

  The following examples are illustrative of methods for practicing the present invention, and are to be understood as merely exemplary of the invention as defined in the appended claims. It should not be construed as limiting the scope of the invention.

Plant tissue development and ethylene treatment
Plant growth Plants were sown in pots and grown in a greenhouse for 4 weeks. The 4 week old seedlings were transplanted into individual pots and grown for 2 months in the greenhouse. During growth, the plants were watered twice daily with water containing 150 ppm NPK fertilizer. The enlarged green leaves were cut from the plant and treated with ethylene as described below.

Cell line 78379
Tobacco line 78379 is a Burley tobacco line published by the University of Kentucky, which was used as a source of plant material. 100 plants were cultivated as standard in the field of tobacco growth, transplanted and identified with different numbers (1-100). Fertilization and field management were performed as recommended.

  Three quarters of those 100 plants converted 20-100% of nicotine into nornicotine. A quarter of those 100 plants converted less than 5% of nicotine into nornicotine. Plant number 87 showed the lowest conversion rate (2%), while plant number 21 showed 100% conversion. Plants with a conversion rate of less than 3% were classified as non-converters. To examine genetic and phenotypic differences, plant number 87 and plant number 21 self-pollinated seeds and crossed (21 × 87 and 87 × 21) seeds were obtained. Plants from 21 that had bred were converted, and 99% of those that bred from 87 were unconverted. The remaining 1% of plants from 87 showed low conversion (5-15%). All plants from reciprocal crosses were transformants.

Cell line 4407
Nicotiana line 4407 is a Burley line, which was used as a source of plant material. Uniform and representative plants (100) were selected and identified by numbers. Of those 100 plants, 97 were non-converters and three were converters. Plant number 56 showed the lowest conversion (1.2%), while plant number 58 showed the highest level of conversion (96%). These two plants were used to obtain self-pollinated seeds as well as cross seeds.

  The self-propagating plants from 58 were isolated with a converter: nonconverter ratio of 3: 1. Plants 58-33 and 58-25 were identified as homozygous transformants and homozygous non-transformed transformants, respectively. Stable conversion of 58-33 was confirmed by subsequent analysis.

Cell line PBLB01
PBLB01 is a Burley line developed by ProfiGen, Inc., which was used as a plant material source. A converter plant was selected from the original species of PBLB01.

Ethylene Treatment Method Green leaves were cut from 2-3 month greenhouse plants and sprayed with 0.3% ethylene solution (Prep brand Ethephon (Rhone-Poulenc)). Each spray leaf was hung in a drying shelf equipped with a humidifier and covered with plastic. During the treatment, the sample leaves were periodically sprayed with ethylene solution. Approximately 24-48 hours after ethylene treatment, leaves were collected for RNA extraction. Another small sample was taken for metabolic component analysis to determine the concentration of leaf metabolites and more detailed components of interest (eg, various alkaloids).

As an example, alkaloid analysis could be performed as follows. Samples (0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH and 5 ml extraction solution containing quinoline and t-butyl ether as internal standards. Samples were analyzed on an HP 6890 GC equipped with an FID detector. A temperature of 250 ° C. was used for the detector and injector. An HP column (30 m-0.32 nm-1 mm) consisting of fused silica crosslinked with 5% phenol and 95% methylsilicon was used at a temperature gradient of 110-185 ° C. at 10 ° C./min. The column was operated with helium as the carrier gas at 100 ° C., 1.7 cm ³ min ⁻¹ flow rate, 40: 1 split ratio, 2: 1 injection volume.

RNA isolation
For RNA extraction, middle leaves from 2 month old greenhouse plants were treated with ethylene as described above. The 0 and 24-48 hour samples were used for RNA extraction. In some cases, leaf samples during the aging process were taken from plants 10 days after removal of the flower head. These samples were also used for extraction. Total RNA was isolated using the Rneasy Plant Mini Kit® (Qiagen, Inc., Valencia, California) according to the manufacturer's protocol.

  Using a mortar and pestle treated with DEPC, the tissue sample was ground under liquid nitrogen to obtain a fine powder. Approximately 100 micrograms of ground tissue was transferred to a 1.5 ml sterile Eppendorf tube. The sample tube was placed in liquid nitrogen until all samples were collected. Subsequently, 450 μl of buffer RLT provided with the kit was added to each tube (with the addition of mercaptoethanol). The sample was vortexed vigorously and incubated at 56 ° C. for 3 minutes. The lysate was then applied to a QIAshredder® centrifuge column placed in a 2 ml collection tube and centrifuged at maximum speed for 2 minutes. The effluent was collected and 0.5 volume of ethanol was added to the clarified lysate. The sample was mixed well and transferred to an Rneasy® mini centrifuge column placed in a 2 ml collection tube. The sample was centrifuged at 10,000 rpm for 1 minute. Next, 700 μl of buffer RW1 was pipetted onto an Rneasy® column and centrifuged at 10,000 rpm for 1 minute. Buffer RPE was pipetted onto an Rneasy® column in a new collection tube and centrifuged at 10,000 rpm for 1 minute. Buffer RPE was again added to the Rneasy® column and centrifuged at maximum speed for 2 minutes to dry the membrane. To remove ethanol carryover, the membrane was placed in a separate collection tube and centrifuged for an additional 1 minute at maximum speed. The Rneasy® column was transferred into a new 1.5 ml collection tube and 40 μl of RNase-free water was pipetted directly onto the Rneasy® membrane. The final elution tube was centrifuged at 10,000 rpm for 1 minute. Qualitative and quantitative analysis of total RNA was performed by denaturing formaldehyde gel and spectrophotometer.

  Poly (A) RNA was isolated using an Origotex® poly A + RNA purification kit (Qiagen Inc.) according to the manufacturer's protocol. Approximately 200 μg of total RNA in a maximum volume of 250 μl was used. A volume of 250 μl buffer OBB and 15 μl Oligotex® suspension was added to the 250 μl total RNA. The contents were mixed well by pipetting and incubated on a heating block at 70 ° C. for 3 minutes. The sample was then left at room temperature for 20 minutes. Oligotex®: mRNA complex was pelleted by centrifugation at maximum speed for 2 minutes. All the suspension except 50 ml was removed from the microcentrifuge tube. The sample was further processed with OBB buffer. The Oligotex®: mRNA pellet was resuspended in 400 μl buffer OW2 by vortexing. This mixture was transferred onto a small centrifuge column placed in a new tube and centrifuged at maximum speed for 1 minute. The centrifuge column was transferred to a new tube and an additional 400 μl of buffer OW2 was added to the column. The tube was then centrifuged for 1 minute at maximum speed. The centrifuge column was transferred to the final 1.5 ml microcentrifuge tube. The sample was eluted with 60 μl of warm (70 ° C.) buffer OEB. Poly A product was analyzed by denaturing formaldehyde gel and spectrophotometer.

Reverse transcription PCR
First strand cDNA was obtained using SuperScript reverse transcriptase according to the manufacturer's protocol (Invitrogen, Carlsbad, California). The poly A + enriched RNA / oligo dT primer mix consists of less than 5 μg total RNA, 1 μl 10 mM dNTP mix, 1 μl oligo d (T) _12-18 (0.5 μg / μl) and up to 10 μl DEPC-treated water. there were. Each sample was incubated at 65 ° C. for 5 minutes and then placed on ice for at least 1 minute. The reaction mixture was prepared by adding each of the following components in turn: 2 μl 10 × RT buffer, 4 μl 25 mM MgCl ₂ , 2 μl 0.1 M DTT and 1 μl RNase OUT Recombinant RNase Inhibitor. 9 μl of reaction mixture additive was pipetted into each RNA / primer mixture and mixed gently. It was incubated at 42 ° C. for 2 minutes and 1 μl Super Script II RT was added to each tube. The tube was incubated at 42 ° C. for 50 minutes. The reaction was terminated at 70 ° C. for 15 minutes and cooled on ice. The sample was collected by centrifugation and 1 μl RNase H was added to each tube and incubated at 37 ° C. for 20 minutes. A second PCR was performed using 200 pmol forward primer and 100 pmol reverse primer (18 nt oligo d (T) followed by one random base).

  The reaction conditions were 94 ° C. for 2 minutes, then 94 ° C. for 1 minute, 45 ° C.-60 ° C. for 2 minutes, 72 ° C. for 3 minutes, 40 cycles of PCR, and 72 ° C. for another 10 minutes of extension. Ten μl of the amplified sample was analyzed by electrophoresis using a 1% agarose gel. The correct size fragment was purified from an agarose gel.

Preparation of PCR fragment population The PCR fragment from Example 3 was ligated into pGEM-T Easy Vector (Promega, Madison, Wisconsin) according to the manufacturer's instructions. Ligation products were transformed into JM109 competent cells and plated on LB media plates for blue / white selection. Colonies were selected and grown overnight at 37 ° C. in 96 well plates containing 1.2 ml LB medium. Frozen stocks of all selected colonies were made. Plasmid DNA was purified from the plates using a Beckman's Biomeck 2000 miniprep robotics with Wizard SV Miniprep kit (Promega). Plasmid DNA was eluted with 100 μl water and stored in a 96 well plate. The plasmid was digested with EcoR1 and analyzed using a 1% agarose gel to confirm the amount of DNA and the size of the insert. Plasmids containing 400-600 bp inserts were sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, California). The sequence was aligned with the GenBank database by BLAST search (see, eg, FIGS. 159A-159K). p450 related fragments were identified and further analyzed. Alternatively, the p450 fragment was isolated from a subtraction library. These fragments were also analyzed as described above.

Construction of cDNA Library A cDNA library was constructed by preparing total RNA from leaves treated with ethylene as follows. First, total RNA was extracted from ethylene-treated leaves of tobacco line 58-33 using a modified acid phenol and chloroform extraction protocol. Use 1 gram of tissue, grind it, then 5 ml of extraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA) with 5 ml of phenol (pH 5.5) and 5 ml of chloroform. The protocol was modified to vortex in (0.5% SDS). The extracted sample was centrifuged and the supernatant was saved. This extraction process was repeated 2-3 times until the supernatant was clear. About 5 ml of chloroform was added to remove traces of phenol. RNA was precipitated from the combined supernatant fractions by adding 3 volumes of ethanol and 1/10 volume of 3M NaOAc (pH 5.2) and storing at −20 ° C. for 1 hour. After transfer to a Corex glass container, the RNA fraction was centrifuged at 9,000 rpm at 4 ° C. for 45 minutes. The pellet was washed with 70% ethanol and centrifuged at 9,000 RPM at 4 ° C. for 5 minutes. After drying the pellet, the pelleted RNA was dissolved in 0.5 ml RNase-free water. Total RNA was qualitatively and quantitatively analyzed by denaturing formaldehyde gel and spectrophotometer, respectively.

  Using the resulting total RNA, poly A + RNA was isolated using an oligo (dT) cellulose protocol (Invitrogen) and a microcentrifuge column (Invitrogen) according to the following protocol. About 20 mg of total RNA was subjected to purification twice to obtain high quality poly A + RNA. In order to ensure high quality of mRNA, poly A + RNA products were analyzed by performing a denatured formaldehyde gel of known full-length gene followed by RT-PCR.

Next, using poly A + RNA as a template, a cDNA library was obtained using a cDNA synthesis kit, a ZAP-cDNA synthesis kit and a ZAP-cDNA Gigapack III gold cloning kit (Stratagene, La Jolla, California). The method was performed according to the manufacturer's protocol as indicated. A cDNA library was constructed using approximately 8 μg of poly A + RNA. Analysis of the primary library showed approximately 2.5 × 10 ⁶ to 1 × 10 ⁷ pfu. The library quality background test was completed by a complementation assay using IPTG and X-gal. In this case, recombinant plaques were expressed more than 100 times the background reaction.

  A more quantitative analysis of the library by random PCR showed that the average size of the insert cDNA was approximately 1.2 kb. The method used a two-step PCR method. In the first step, a reverse primer was designed based on the preliminary sequence information obtained from the p450 fragment. The designed reverse primer and T3 (forward) primer were used to amplify the corresponding gene from the cDNA library. The PCR reaction was subjected to agarose electrophoresis and the corresponding high molecular weight band was excised, purified, cloned and sequenced. In the second step, the reverse primer (designed from the 3'UTR of p450) and a new primer designed from the 5'UTR of p450 and the start coding region as a forward primer were used in subsequent PCR to complete A long p450 clone was obtained.

  The p450 fragment was obtained by PCR amplification from the constructed cDNA library described in Example 3, except for the reverse primer. A T7 primer located on the plasmid downstream of the cDNA insert was used as a reverse primer. PCR fragments were isolated, cloned and sequenced as described in Example 4.

  The full-length p450 gene was isolated from the constructed cDNA library by this PCR method. A full length gene was obtained using a gene specific reverse primer (designed from the downstream sequence of the p450 fragment) and a forward primer (T3 on the library plasmid). PCR fragments were isolated, cloned and sequenced. A second PCR step was performed as needed. In this second step, a new primer designed from the 5′UTR of the cloned p450 along with a reverse primer designed from the 3′UTR of the p450 clone is used in subsequent PCR reactions to generate the full-length p450 clone. Obtained. The clone was then sequenced.

Cloned Fragment Characterization-Reverse Southern Blot Analysis A non-radioactive large scale reverse Southern blot assay was performed on all p450 clones identified in the previous example to detect differential expression. It was observed that the level of expression between different p450 clusters was very different. Furthermore, real-time detection was performed on clones showing high expression.

Non-radioactive Southern blotting was performed as follows.
1) Total RNA from transformed (58-33) and untransformed (58-25) and untransformed leaves treated with ethylene using the Qiagen Rnaeasy kit as described in Example 2 Extracted.

  2) A probe was prepared by labeling the single-stranded cDNA derived from the poly A + -enriched RNA obtained in the above step with a biotin tail. This labeled single-stranded cDNA was prepared by RT-PCR of converted and non-converted total RNA (Invitrogen) as described in Example 3 except that biotinylated oligo dT was used as a primer (Promega). These were used as probes for hybridizing to cloned DNA.

  3) Plasmid DNA was digested with the restriction enzyme EcoR1 and run on an agarose gel. The gel was simultaneously dried and transferred to two nylon membranes (Biodyne B). One membrane was hybridized with the converter probe and the other was hybridized with the non-converter probe. Prior to hybridization, the membrane was subjected to UV crosslinking (autocrosslinking setting, 254 nm, Stratagene, Stratalinker).

  Alternatively, the insert was PCR amplified from each plasmid using primers located on both arms of the p-GEM plasmid, T3 and SP6. PCR products were analyzed by running on a 96-well Ready-to-run agarose gel. The identified insert was doted onto two nylon membranes. One membrane was hybridized with the converter probe and the other was hybridized with the non-converter probe.

  4) The membrane was washed according to the manufacturer's instructions (Enzo MaxSence kit, Enzo Diagnostics, Inc, Farmingdale, NY) except that the washing stringency was changed. The membrane is prehybridized with hybridization buffer (2 × SSC buffered formamide containing surfactant and hybridization enhancer) for 30 minutes at 42 ° C. and hybridized overnight at 42 ° C. with 10 μl denaturing probe. It was. The membrane was then washed in 1 × hybridization wash buffer for 10 minutes at room temperature (1 time) and 15 minutes at 68 ° C. (4 times). The membrane was ready for the detection method.

  5) The washed membranes were detected by alkaline phosphatase labeling followed by NBT / BCIP colorimetric detection as described in the manufacturer's detection instructions (Enzo Diagnostics, Inc.). The membrane is blocked with 1 × blocking solution for 1 hour at room temperature, washed with 1 × detection reagent for 10 minutes (3 times), washed with 1 × predevelopment reaction buffer for 5 minutes (2 times), then the The blot was developed for 30-45 minutes in developer solution until dots appeared. All reagents were obtained from the manufacturer (Enzo Diagnostics, Inc). Large scale reverse Southern assays were also performed using the KPL Southern hybridization and detection kit according to the manufacturer's instructions (KPL, Gaithersburg, Maryland).

Clone characterization-Northern blot analysis Instead of Southern blot analysis, several membranes were hybridized and detected as described in the Examples of Northern blot assays. Northern hybridization was used to detect differentially expressed mRNA in Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450 (Megaprime DNA Labeling Systems, Amersham Biosciences). The following components were mixed: 25 ng denatured DNA template; 4 ul of each unlabeled dTTP, dGTP and dCTP; 5 μl reaction buffer; P ³² labeled dATP and 2 ul Klenow I; and H ₂ O (the reaction in 50 μl) adjust). The mixture was incubated at 37 ° C. for 1-4 hours and stopped with 2 μl 0.5 M EDTA. The probe was denatured by incubation at 95 ° C. for 5 minutes before use.

RNA samples were prepared from fresh leaves treated and untreated with ethylene from several pairs of tobacco lines. In some cases, poly A + enriched RNA was used. Approximately 15 μg total RNA or 1.8 μg mRNA (RNA and mRNA extraction method as described in Example 5) was made up to volume with DEPC H ₂ O (5-10 μl). The same volume of loading buffer (1 × MOPS; 18.5% formaldehyde; 50% formamide; 4% Ficoll400; bromophenol blue) and 0.5 μl EtBr (0.5 μg / μl) were added. The sample was then denatured in preparation for the separation of the RNA by electrophoresis.

  Samples were formaldehyde gel (1% agarose, 1 × MOPS, 0.6M formaldehyde) containing 1 × MOP buffer (0.4 M morpholinopropane sulfate; 0.1 M Na-3 × H2O; 10 mM EDTA; adjusted to pH 7.2 with NaOH) ) Subjected to the above electrophoresis. RNA was transferred to Hybond-N + membrane (Nylon, Amersham Pharmacia Biotech) for 24 hours by capillary method in 10 × SSC buffer (1.5 M NaCl; 0.15 M Na citrate). Prior to hybridization, the membrane containing the RNA sample was subjected to UV crosslinking (autocrosslinking setting, 254 nm, Stratagene, Stratalinker).

  Pre-hybridize the membrane with 5-10 ml prehybridization buffer (5x SSC; 50% formaluminide; 5x Denhardt's solution; 1% SDS; 100 μg / ml heat-denatured shear heterologous DNA) at 42 ° C for 1-4 hours Made soy. The old prehybridization buffer was discarded and new prehybridization buffer and probe were added. The hybridization was performed overnight at 42 ° C. The membrane was washed with 2 × SSC for 15 minutes at room temperature and then with 2 × SSC.

As shown in Table 1 below, Northern and reverse Southern blots were useful in determining which genes were induced by ethylene treatment in comparison to uninduced plants. Interestingly, not all fragments were similarly affected in the transformants and non-transformants. Some of the cytochrome p450 fragments were partially sequenced to determine their structural relevance. This information was then used to isolate and characterize the full length gene clone of interest.

  Northern analysis was performed using full-length clones on tobacco tissue from converter and non-converter Burley lines derived by ethylene treatment. This analysis was used to identify full-length clones that showed increased expression in ethylene-induced converter lines compared to ethylene-induced non-converter Burley lines. By doing so, the functional relevance of the full-length clone can be determined by comparing the biochemical differences in leaf components between converter and non-converter lines.

As shown in Table 2 below, 6 clones were significantly higher represented by ++ and +++ in the transformed ethylene treated tissue compared to the expression of the non-transformed treated tissue represented by + Expression was shown. All of these clones showed little or no expression in converter and non-converter lines not treated with ethylene.

Immunodetection of polypeptides encoded by cloned genes.
Peptide regions corresponding to 20-22 amino acids in length from 3 p450 clones (1) have lower or no homology to other clones, and (2) good Selected for having hydrophilicity and antigenicity. The amino acid sequences of peptide regions selected from each p450 clone are listed below. The synthetic peptide was conjugated with KHL (keyhole limpet hemocyanin) and then injected into rabbits. Antisera were collected 2 and 4 weeks after the fourth injection (Alpha Diagnostic Intl. Inc. San Antonio, TX).

D234-AD1 DIDGSKSKLVKAHRKIDEILG (SEQ ID NO: 2266)
D90a-BB3 RDAFREKETFDENDVEELNY (SEQ ID NO: 163)
D89-AB1 FKNNGDEDRHFSQKLGDLADKY (SEQ ID NO: 2267)
Antisera were tested for cross-activity against target proteins from tobacco plant tissue by Western blot analysis. Crude protein extracts were obtained from ethylene-treated (0-40 hours) middle leaves of converter and non-converter lines. The protein concentration of the extract was measured using RC DC Protein Assay Kit (BIO-RAD) according to the manufacturer's protocol.

  2 μg of protein was loaded on each lane and the protein was separated on a 10% to 20% gradient gel using the Laemmli SDS-PAGE system. The protein was transferred from the gel to PROTRAN Nitrocellulose Transfer Membranes (Schleicher & Schuell) using a Trans-Blot Semi-Dry cell (BIO-RAD). The target p450 protein was detected and visualized using the ECL Advance Western Blotting Detection Kit (Amersham Biosciences). Primary antibodies against the synthetic KLH conjugate were prepared in rabbits. A secondary antibody against rabbit IgG conjugated to peroxidase was purchased from Sigma. Both primary and secondary antibodies were used at a dilution of 1: 1000. The antibody showed strong reactivity against a single band on the Western blot. This indicates that the antiserum was monospecific for the target peptide of interest. The antiserum was also cross-reactive with the KLH conjugated synthetic peptide.

Nucleic acid identity, structural relevance of isolated nucleic acid fragments and GeneChip® hybridization
More than 100 cloned p450 fragments were sequenced with Northern blot analysis to determine their structural relevance. The approach used a forward primer based on one of two common p450 motifs located near the carboxyl terminus of the p450 gene. The forward primer corresponded to cytochrome p450 motif FXPERF (SEQ ID NO: 2268) or GRRXCP (A / G) (SEQ ID NO: 2269). As reverse primers, standard primers from the plasmid SP6 or T7 located on both arms of the poly A tail or pGEM plasmid were used. The protocol used is described below.

  Spectrophotometry was used to estimate the concentration of starting double stranded DNA according to the manufacturer's protocol (Beckman Coulter). The template was diluted with water to an appropriate concentration, denatured by heating at 95 ° C. for 2 minutes, and then placed on ice. The sequencing reaction was prepared on ice using 0.5-10 μl denatured DNA template, 2 μl 1.6 pmol forward primer, 8 μl DTCS Quick Start Master Mix, bringing the total volume to 20 μl with water. The thermal cycle program consisted of 30 cycles of the following cycles: 96 ° C. for 20 seconds, 50 ° C. for 20 seconds and 60 ° C. for 4 minutes followed by 4 ° C. maintenance.

  The sequencing reaction was stopped by adding 5 μl of stop buffer (equal volumes of 3M NaOAc and 100 mM EDTA and 1 μl of 20 mg / ml glycogen). The sample was precipitated with 60 μl cold 95% ethanol and centrifuged at 6000 × g for 6 minutes. Ethanol was discarded. The pellet was washed twice with 200 μl cold 70% ethanol. After the pellet dried, 40 μl of SLS solution was added and the pellet was resuspended. The mineral oil layer was overlaid and the sample was placed on a CEQ 8000 Automated Sequencer for further analysis.

  To confirm the nucleic acid sequence, use a forward primer for the FXPERF (SEQ ID NO: 2268) or GRRXCP (A / G) (SEQ ID NO: 2269) region of the p450 gene, or a reverse primer for the plasmid or poly A tail. The sequence was resequenced in both directions. All sequencing was performed at least twice in both directions.

  The nucleic acid sequences of cytochrome p450 fragments were compared to each other with respect to the coding region corresponding to the first nucleic acid after the region encoding the GRRXCP (A / G) (SEQ ID NO: 2269) motif to the stop codon. This region was selected as an indicator of genetic diversity between p450 proteins. As with other plant species, a number of genetically distinct p450 genes over 70 genes were observed. Upon comparison of nucleic acid sequences, it has been found that the genes can be placed in different sequences based on sequence identity. The best unique groupings of p450 members have been found to be sequences having 75% or more nucleic acid identity sequences (eg, published in US 2004/0162420 patent application incorporated herein by reference). (See Table 1). A decrease in identity (%) gave a significantly larger group. A preferred grouping is a sequence having 81% or more nucleic acid identity, a more preferred grouping is a sequence having 91% or more nucleic acid identity, and the most preferred grouping is 99% or more. Of sequences having the same nucleic acid identity. Most of those groups contained at least 2 members, and often 3 or more members. Others were not found repeatedly. This suggests that the approach adopted was able to isolate both low and high expression mRNA in the tissues used.

  Based on 75% or more nucleic acid identity, it was found that two cytochrome p450 groups contain nucleic acid sequence identity to a former tobacco cytochrome gene that is genetically different from that group. Group 23 showed nucleic acid identity to GenBank sequences GI: 1171579 (SEQ ID NO: 2270) (CAA64635) and GI: 14423327 (SEQ ID NO: 2271) (or AAK62346) in the parameters used in Table 3A. GI: 1171579 (SEQ ID NO: 2270) has nucleic acid identity ranging from 96.9% to 99.5% for members of group 23, while GI: 14423327 (SEQ ID NO: 2271) is 95.4% to It showed identity in the range of 96.9%. Group 31 members had 76.7% -97.8% nucleic acid identity to the sequence reported in the GenBank of GI: 14423319 (SEQ ID NO: 2272) (AAK62342). None of the other p450 identity groups in Table 3A contained the parameter identity used in Table 3A for the previously reported Nicotiana p450 gene.

  In order to preferentially identify and isolate additional members of each group from Nicotiana plants, it would be possible to derive consensus sequences with appropriate nucleic acid degenerate probes for the group.

Table 3A : Nicotiana p450 nucleic acid sequence identity group
Group fragment
1 D58-BG7 (SEQ ID NO: 10), D58-AB1 (SEQ ID NO: 12); D58-BE4 (SEQ ID NO: 16)
2 D56-AH7 (SEQ ID NO: 18); D13a-5 (SEQ ID NO: 20)
3 D56-AG10 (SEQ ID NO: 22); D35-33 (SEQ ID NO: 24); D34-62 (SEQ ID NO: 26)
4 D56-AA7 (SEQ ID NO: 28); D56-AE1 (SEQ ID NO: 30); 185-BD3 (SEQ ID NO: 152)
5 D35-BB7 (SEQ ID NO: 32); D177-BA7 (SEQ ID NO: 34); D56A-AB6 (SEQ ID NO: 36); D144-AE2 (SEQ ID NO: 38)
6 D56-AG11 (SEQ ID NO: 40); D179-AA1 (SEQ ID NO: 42)
7 D56-AC7 (SEQ ID NO: 44); D144-AD1 (SEQ ID NO: 46)
8 D144-AB5 (SEQ ID NO: 48)
9 D181-AB5 (SEQ ID NO: 50); D73-AC9 (SEQ ID NO: 52)
10 D56-AC12 (SEQ ID NO: 54)
11 D58-AB9 (SEQ ID NO: 56); D56-AG9 (SEQ ID NO: 58); D56-AG6 (SEQ ID NO: 60); D35-BG11 (SEQ ID NO: 62); D35-42 (SEQ ID NO: 64); D35-BA3 ( SEQ ID NO: 66); D34-57 (SEQ ID NO: 68); D34-52 (SEQ ID NO: 70); D34-25 (SEQ ID NO: 72)
12 D56-AD10 (SEQ ID NO: 74)
13 56-AA11 (SEQ ID NO: 76)
14 D177-BD5 (SEQ ID NO: 78); D177-BD7 (SEQ ID NO: 92)
15 D56A-AG10 (SEQ ID NO: 80); D58-BC5 (SEQ ID NO: 82); D58-AD12 (SEQ ID NO: 84)
16 D56-AC11 (SEQ ID NO: 86); D35-39 (SEQ ID NO: 88); D58-BH4 (SEQ ID NO: 90); D56-AD6 (SEQ ID NO: 96)
17 D73A-AD6 (SEQ ID NO: 98); D70A-BA11 (SEQ ID NO: 100)
18 D70A-AB5 (SEQ ID NO: 104); D70A-AA8 (SEQ ID NO: 106)
19 D70A-AB8 (SEQ ID NO: 108); D70A-BH2 (SEQ ID NO: 110); D70A-AA4 (SEQ ID NO: 112)
20 D70A-BA1 (SEQ ID NO: 114); D70A-BA9 (SEQ ID NO: 116)
21 D70A-BD4 (SEQ ID NO: 118)
22 D181-AC5 (SEQ ID NO: 120); D144-AH1 (SEQ ID NO: 122); D34-65 (SEQ ID NO: 124)
23 D35-BG2 (SEQ ID NO: 126)
24 D73A-AH7 (SEQ ID NO: 128)
25 D58-AA1 (SEQ ID NO: 130); D185-BC1 (SEQ ID NO: 142); D185-BG2 (SEQ ID NO: 144)
26 D73-AE10 (SEQ ID NO: 132)
27 D56-AC12 (SEQ ID NO: 134)
28 D177-BF7 (SEQ ID NO: 136); D185-BE1 (SEQ ID NO: 146); D185-BD2 (SEQ ID NO: 148)
29 D73A-AG3 (SEQ ID NO: 138)
30 D70A-AA12 (SEQ ID NO: 140); D176-BF2 (SEQ ID NO: 94)
31 D176-BC3 (SEQ ID NO: 154)
32 D176-BB3 (SEQ ID NO: 156)
33 D186-AH4 (SEQ ID NO: 14)

GeneChip® microarray hybridization (Affymetrix Inc .; Santa Clara, CA) to identify genes with a differential expression pattern between non-converters and transformants that are close to isogenic lines after ethylene activation It was used. The chip size is 18 microns, the array format is 100-2187, and accommodates 528 probe sets (11,628 probes). Seven pairs of hybridizations were used to independently verify the microarray results. These are one pair of 4407-33 / 4407-25 untreated burley tobacco samples (converter / non-converter), four pairs of ethylene treated 4407-33 / 4407-25 samples, ethylene treated One pair of dark tobacco NL Madole / 181, another pair of lines close to homogeneity in terms of nicotine conversion, and one pair of naturally aged leaves of 4407 = 33/25 (Table 3B).

  All 14 sets of hybridization were successful, as shown by an expression report generated using a detector by Genome Explorations, Inc. (Memphis, TN). The main report included analysis of noise, scale factor, background, total probe set, number and percentage of probe sets present and absent, and signal intensity of housekeeping controls. The software GCOS was then used with other software to analyze and display the data. Signal comparisons between treatment pairs were made, and overall data for all individual probes of all hybridizations was summarized and expression data was also analyzed. Results based on signal intensity indicated that only two genes (D121-AA8 and D120-AH4) and one fragment (D35-BG11, which is a partial fragment of D121-AA8), were found in non-converter lines in ethylene-treated converter lines. When compared with, it was shown to receive reproducible induction. The signal of the gene in a converter line, for example Burley tobacco variety 4407-33, was measured as a ratio to the signal of the gene in the related non-converter isogenic line 4407-25. In the absence of ethylene treatment, the ratio of converter signal to non-converter signal for all genes was close to 1.00. To eliminate the effect of background differences, normalized signal ratios were also calculated. The normalized signal ratio is obtained by dividing the treatment pair ratio by the corresponding non-pair ratio. Upon ethylene treatment and analysis, the two genes (D121-AA8 and D120-AH4) were confirmed to be induced in the converter line in contrast to the non-converter line, as measured by four different analyses. These two genes shared 99.8% relative homology and their relative hybridization signals in the transformant varieties were about 2-22 times higher than the signals in their non-transformant counterparts. Based on the normalized ratio, two actin-like internal control clones were not induced in the converter lines. In addition, the fragment (D35-BG11), whose entire coding region is contained within the D121-AA8 and D120-AH4 genes, is highly expressed in the same sample of paired isogenic and non-converter lines. Was guided to. Furthermore, the D121-AA8 and D120-AH4 genes were strongly induced (8-28 fold) in the transformant lines of the isogenic tobacco pair NL Madole and 181. This therefore indicates that ethylene induction of these genes in the converter lineage was an in planta response. The same gene was identified in a comparison made from 4407-33 / 25 natural aging sample hybridization. RT-PCR assays of these materials using primers specific for D121-AA8 demonstrated microarray results for this gene.

  Based on these results, the D121-AA8 gene (its cDNA sequence is the sequence of sequence code 5; FIG. 4) was identified as the tobacco nicotine demethylase gene of interest. Considering the nomenclature of p450, D121-AA8 was confirmed to be most similar to p450 within the CYP82E family (The Arabidopsis Genome Initiative (AGI) and The Arabidopsis Information Resource (TAIR); Frank, Plant Physiol. 110: 1035-1046, 1996; Whitbred et al., Plant Physiol. 124: 47-58, 2000); Schopfer and Ebel, Mol. Gen. Genet. 258: 315-322, 1998; and Takemoto et al., Plant Cell Physiol. 40 : 1232-1242, 1999).

Biochemical analysis of enzyme activity Biochemical analysis (eg, as described in previously filed applications incorporated herein by reference) is a sequence in which the sequence of SEQ ID NO: 5 is tobacco nicotine demethylase (SEQ ID NO: 3 Confirms that it encodes Figures 3 and 4).

  In particular, the function of the candidate clone D121-AA8 was confirmed as a coding gene for nicotine demethylase by assaying the enzymatic activity of p450 heterologously expressed in yeast cells as follows.

1． Construction of Yeast Expression Vector The putative protein coding sequences D120-AH4, D121-AA8, 208-AC-8 and D208-AD9 of the cDNA encoding tobacco nicotine demethylase (D121-AA8) were cloned into the yeast expression vector pYeDP60. Appropriate BamHI and MfeI sites (underlined below) were introduced by PCR primers containing these sequences upstream of the translation start codon (ATG) or downstream of the stop codon (TAA). The MfeI on the vector on the amplified PCR product is compatible with the EcoRI site. The primers used to amplify D121-AA8 cDNA were 5'-TAGCTACGC GGATCC ATGCTTTCTCCCATAGAAGCC-3 '(SEQ ID NO: 2194) and 5'-CTGGATCA CAATTG TTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTCAGGTGCCAGGC-3' (SEQ ID NO: 2297). A segment of the sequence encoding the 9 additional amino acids at the C-terminus of the protein containing 6 histidines was incorporated into the reverse primer to facilitate expression of 6-His tagged p450 when induced. . After enzymatic digestion, the PCR product was ligated into the pYeDP60 vector in sense orientation relative to the GAL10-CYC1 promoter. Proper construction of the yeast expression vector was confirmed by restriction enzyme analysis and DNA sequencing. In addition, the expression of p450 protein was visualized on SDS-PAGE gel electrophoresis for the surfactant phase of yeast microsomes. Based on the gene sequence, the expected size of the p450 protein is 59 kD and the results were confirmed by gel analysis.

2． Yeast transformation
A WAT11 yeast system modified to express Arabidopsis NADPH-cytochrome p450 reductase ATR1 was transformed with the pYeDP60-p450 cDNA plasmid. 50 μl of WAT11 yeast cell suspension was mixed with ˜1 μg of plasmid DNA in a cuvette with a 0.2 cm electrode gap. One pulse of 2.0 kV was applied by an Eppendorf electroporator (Model 2510). Cells were spread on SGI plates (5 g / L bactocasamino acid, 6.7 g / L yeast nitrogen base without amino acids, 20 g / L glucose, 40 mg / L DL-tryptophan, 20 g / L agar). Transformants were confirmed by PCR analysis performed directly on randomly selected colonies.

3． P450 expression in transformed yeast cells Using a single yeast colony, add 30ml SGI medium (5g / L bactocasamino acid, 6.7 g / L yeast nitrogen base without amino acids, 20 g / L glucose, 40 mg / L DL-tryptophan) and grown at 30 ° C. for about 24 hours. An aliquot of this culture is diluted 1:50 in 1000 mL of YPGE medium (10 g / L yeast extract, 20 g / L bactopeptone, 5 g / L glucose, 30 ml / L ethanol), and a Diastix urine analysis reagent strip ( Bayer, Elkhart, IN) were grown until glucose was completely consumed as indicated by colorimetric changes. Induction of cloned P450 was initiated by adding DL-galactose to a final concentration of 2%. The cultures were grown for an additional 20 hours before being used for in vivo activity assays or microsome preparation.

  WAT11 yeast cells expressing pYeDP60-CYP71D20 (p450 catalyzing the hydroxylation of 5-epi-Aristroken and 1-deoxycapsidiol in Nicotiana tabacum) were used as controls for p450 expression and enzyme activity assays.

  In order to evaluate in detail the effectiveness of p450 yeast expression, reduced CO difference spectroscopy was performed. The reduced CO spectrum showed a peak in 450 nm protein from all four p450 transformed yeast lines. Similar peaks were not observed in control microsomes derived from control untransformed yeast cells or blank vector control yeast cells. These results indicated that the p450 protein was effectively expressed in the yeast system containing pYeDP60-CYP 450. The concentration of expressed p450 protein in yeast microsomes was 45-68 nmole / mg total protein.

Four. In Vitro Enzyme Assay Nicotine demethylase activity in transformed yeast cells was assayed by feeding DL-nicotine (pyrrolidine-2- ¹⁴ C) to yeast cultures. ¹⁴ C-labeled nicotine (54 mCi / mmol) was added to 75 μl of galactose-induced culture at a final concentration of 55 μM. The assay culture was incubated for 6 hours with shaking in a 14 ml polypropylene tube and extracted with 900 μl of methanol. After centrifugation, 20 μl of the methanol extract was separated by rp-HPLC, and the nornicotine fraction was quantified by LSC.

  Control cultures of WAT11 (pYeDP60-CYP71D20) did not convert nicotine to nornicotine. This indicates that the WAT11 yeast strain does not contain endogenous enzyme activity that catalyzes the process of bioconversion from nicotine to nornicotine. In contrast, yeast expressing the tobacco nicotine demethylase gene produced detectable amounts of nornicotine. This indicates the nicotine demethylase activity of the translation product of SEQ ID NO: 4 or SEQ ID NO: 5.

Five. Preparation of yeast microsomes After 20 minutes induction with galactose, yeast cells were collected by centrifugation and TES-M buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6 M sorbitol, 10 mM 2-mercaptoethanol) And washed twice. Extract the pellet into extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6 M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serum albumin, protease inhibitor cocktail (Roche) (1 tablet / 50 ml) ). The cells were then disrupted with glass beads (diameter 0.5 mm, Sigma) and the cell extract was centrifuged at 20,000 × g for 20 minutes to remove cell debris. The supernatant was subjected to ultracentrifugation at 100,000 × g for 60 minutes. The resulting pellet contained the microsomal fraction. The microsomal fraction was suspended in TEG-M buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2-mercaptoethanol) at a protein concentration of 1 mg / mL. Microsome preparations were stored in a liquid nitrogen refrigerator until use.

6． Enzyme Activity Assay in Yeast Microsome Preparation A nicotine demethylase activity assay in a yeast microsome preparation was performed. In particular, DL-nicotine (pyrrolidine-2- ¹⁴ C) was obtained from Moravek Biochemicals and had a specific activity of 54 mCi / mmol. Chlorpromazine (CPZ) and oxidized cytochrome c (cyt.C) (both are P450 inhibitors) were purchased from Sigma. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a typical electron donor to cytochrome P450 via NADPH: cytochrome P450 reductase. NADPH was excluded from the control incubation. The usual enzyme assay included microsomal protein (approximately 1 mg / ml), 6 mM NADPH and 55 μM ¹⁴ C labeled nicotine. The concentrations of CPZ and Cyt.C were 1 mM and 100 μM, respectively, when they were used. The reaction was carried out for 1 hour at 25 ° C. and stopped by the addition of 300 μl methanol to 25 μl reaction mixture each time. After centrifugation, 20 μl of the methanol extract was separated on a reverse phase high performance liquid chromatography (HPLC) system (Agilent) using an Inertsil ODS-3 3 μ (150 × 4.6 mm) chromatography column from Varian. The isocratic mobile phase was a 60:40 (v / v) ratio mixture of methanol and 50 mM potassium phosphate buffer (pH 6.25) with a flow rate of 1 ml / min. Nornicotine peaks determined by comparison with authentic unlabeled nornicotine were collected and subjected to a 2900 tri-carb Liquid Scintillation Counter (LSC) (Perkin Elmer) for quantification. The activity of nicotine demethylase is calculated based on the production of ¹⁴ C-labeled nornicotine over a 1 hour incubation.

P450-like activity was observed in microsomal preparations from control yeast cells expressing CYP71D20 and in three test p450 yeast cultures transformed with the genes D120-AH4, D208-AC8 and D208-AD9. However, the controls and their three test p450s showed no nornicotine conversion formation. This suggests that they did not contain endogenous or inducible enzymes that could catalyze nicotine demethylation. In contrast, results from HPLC and LSC analysis resulted from nicotine demethylation when using microsomal samples obtained from yeast cells expressing the tobacco nicotine demethylase gene (D121-AA8) A detectable amount of nornicotine was shown. These results indicate that nicotine demethylase activity results from the D121-AA8 gene product. Nicotine demethylase activity requires NADPH and has been shown to be suppressed by p450 specific inhibitors, consistent with tobacco nicotine demethylase being p450. The enzyme activity for tobacco nicotine demethylase (D121-AA8) was approximately 10.8 pKat / mg protein as calculated by radioactivity intensity and protein concentration. The results of a series of typical enzyme assays obtained for the yeast cells are shown in the following table (Table 4).

  When NADPH was removed from assays using microsomes from D121-AA8 yeast cells, inhibition of nicotine demethylase activity occurred. Therefore, nornicotine did not form (Table 4). If two known P450 inhibitors chlorpromazine (CPZ, 1 mM) and oxidized cytochrome c (cytC, 100 μM) were added separately into the enzyme assay mixture and incubated for 1 hour followed by methanol stop solution, Nicotine demethylase activity was significantly reduced (Table 4). Taken together, these experiments showed that D121-AA8 encodes a cytochrome p450 protein that catalyzes the conversion of nicotine to nornicotine when expressed in yeast.

Related Amino Acid Sequence Identity of Isolated Nucleic Acid Fragments The amino acid sequence of the nucleic acid sequence obtained for the cytochrome p450 fragment from Example 8 was deduced. The deduced region corresponded to the amino acid immediately after the GXRXCP (A / G) (SEQ ID NO: 2273) sequence motif to the carboxyl terminus or stop codon. When comparing the sequence identity of the fragments, a unique grouping was found for sequences with 70% or more amino acid identity. A preferred grouping is a sequence having 80% or more amino acid identity, a more preferred grouping is a sequence having 90% or more amino acid identity, and the most preferred grouping is 99% or more. The sequence having the amino acid identity of Since some of their unique nucleic acid sequences were found to have complete amino acid identity to other fragments, the only member with the same amino acid was reported.

  The amino acid identity for group 19 in Table 5 corresponded to three different groups based on their nucleic acid sequence. The amino acid sequences of group members and their identity are shown in Figure 159H. Amino acid differences are shown.

At least one member of each amino acid identity group was selected for gene cloning and functional studies using plants. Also, group members that were differentially affected by ethylene treatment or other biological differences as assessed by Northern and Southern analysis were selected for gene cloning and functional studies. Peptide specific antibodies can be prepared based on sequence identity and specific sequences to aid gene cloning, expression studies and whole plant evaluation.

Related amino acid sequence identity of full-length clones The nucleic acid sequence of the full-length Nicotiana gene cloned in Example 5 was deduced with respect to their full amino acid sequence. UXXRXXZ (SEQ ID NO: 2274), PXRFXF (SEQ ID NO: 2275) or GXRXC (SEQ ID NO: 2276) at the carboxyl terminus (where U is E or K, X is any amino acid, Z is P, T, S) The cytochrome p450 gene was identified by the presence of three conserved p450 domain motifs corresponding to (or M). All p450 genes were characterized for amino acid identity using the BLAST program that compares full-length sequences against each other and against known tobacco genes. The program used NCBI special BLAST tool (Align two sequences (b12seq), http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html) . The two sequences were aligned with BLASTN without filter for nucleic acid sequences and BLASTP for amino acid sequences. Based on their amino acid identity (%), each sequence was grouped into identity groups. In this case, the grouping contained members that shared at least 85% identity to another member. A preferred grouping is a sequence having 90% or more amino acid identity, a more preferred grouping is a sequence having 95% or more amino acid identity, and the most preferred grouping is 99% or more. The sequence having the amino acid identity of Using these criteria, 25 unique groups were identified. They are shown in Table 6. The amino acid sequence of the full-length nicotine demethylase gene was deduced to have the sequence set forth in SEQ ID NO: 5 (FIG. 4).

In the parameters used in Table 6 for amino acid identity, the three groups were found to contain 85% or more identity to known tobacco genes. Group 5 members had up to 96% amino acid identity with respect to the full-length sequence relative to GeneBank sequence GI: 14423327 (SEQ ID NO: 2271) (or AAK62346). Group 23 has up to 93% amino acid identity to GI: 14423328 (SEQ ID NO: 2277) (or AAK62347), Group 24 is 92 to GI: 14423318 (SEQ ID NO: 2278) (or AAK62343 ; SEQ ID NO: 2300 ) % Identity.

  Full-length genes were further grouped based on the highly conserved amino acid homology between the GXRXC p450 domain near the carboxyl terminus (SEQ ID NO: 2276) and the UXXRXXZ p450 domain (SEQ ID NO: 2274). As shown in FIGS. 160A-160E, individual clones were aligned based on sequence homology between conserved domains and placed in various identity groups. In some cases, the nucleic acid sequence of the clone was unique, but the amino acid sequence for the region was identical. A preferred grouping is a sequence having 90% or more amino acid identity, a more preferred grouping is a sequence having 95% or more amino acid identity, and the most preferred grouping is 99% or more. The sequence having the amino acid identity of This final grouping was similar to that based on identity with respect to the entire amino acid sequence of the clones except group 17 (Table 6), which was classified into two different groups.

In the parameters used for amino acid identity in Table 7, the three groups were found to contain 90% or more identity to known tobacco genes. Group 5 members had up to 93.4% amino acid identity with respect to the full-length sequence against GeneBank sequence GI: 14423326 (SEQ ID NO: 2279) (or AAK62346). Group 23 has up to 91.8% amino acid identity to GI: 14423328 (SEQ ID NO: 2277) (or AAK62347), Group 24 has 92% identity to GI: 14423318 (SEQ ID NO: 2278) (or AAK62342) Had.

Nicotiana cytochrome P450 clone lacking one or more of tobacco P450 specific domains
Four clones had high nucleic acid homology ranging from 90% to 99% to the other tobacco cytochrome genes reported in Table 6. Those four clones included D136-AD5 (SEQ ID NO: 292), D138-AD12 (SEQ ID NO: 294), D243-AB3 (SEQ ID NO: 298) and D250-AC11 (SEQ ID NO: 300). However, due to nucleotide frameshifts, these genes did not contain one or more of the three C-terminal cytochrome p450 domains and were excluded from the identity groups shown in Table 6 or Table 7.

  The amino acid identity of one clone D95-AG1 did not contain the third domain GXRXC (SEQ ID NO: 2276) that was used to group the p450 tobacco genes in Table 6 or Table 7. The nucleic acid sequence of this clone has low homology to other tobacco cytochrome genes, and therefore this clone represents a novel group of Nicotiana cytochrome p450 genes.

Use of Nicotiana Cytochrome P450 Fragments and Clones in Altering Control of Tobacco Quality The use of tobacco p450 nucleic acid fragments or whole genes can be used in plants with altered tobacco phenotypes or tobacco components, and more importantly, metabolite alterations. Useful in identification and selection. By various transformation systems that incorporate nucleic acid fragments or full-length genes selected from those described herein in an orientation such as for down-regulation (eg, antisense orientation) or overexpression (eg, sense orientation), It is possible to produce transgenic tobacco plants. For overexpression to the full-length gene, any nucleic acid sequence encoding the entire or functional part or amino acid sequence of the full-length gene according to the present invention is desirable. Such nucleic acid sequences are desirably effective to enhance the expression of certain enzymes and produce phenotypic effects in Nicotiana. Homozygous Nicotiana strains are obtained by a series of backcrosses, such as analyzing concentrations of endogenous p450 RNA, transcripts, p450-expressed peptides and plant metabolites using techniques generally available to those skilled in the art, etc. (But not limited to) are evaluated for phenotypic changes. The changes shown in tobacco plants provide information regarding the functional role of the selected gene of interest or are useful as a preferred Nicotiana plant species.

Cloning of Genomic Tobacco Nicotine Demethylase from Transformer Burley Tobacco
Genomic DNA was extracted from the transformant burley tobacco plant line 4407-33 (Nicotiana tabacum variety 4407 line) using the Qiagen Plant Easy kit as described in the previous examples (see also the manufacturer's instructions) .

  Primers were designed based on the 5 ′ promoter and 3 ′ UTR region cloned in the previous example. The forward primer is 5'-GGC TCT AGA TAA ATC TCT TAA GTT ACT AGG TTC TAA-3 '(SEQ ID NO: 2280) and 5'-TCT CTA AAG TCC CCT TCC-3' (SEQ ID NO: 2288), the reverse primer is 5′-GGC TCT AGA AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3 ′ (SEQ ID NO: 2281) and 5′-CCA GCA TTC CTC AAT TTC-3 ′ (SEQ ID NO: 2289). PCR was applied to 4407-33 genomic DNA using 100 μl of reaction mixture. Pfx high fidelity enzyme was used for PCR amplification. PCR products were visualized on a 1% agarose gel after electrophoresis. A single band with a molecular weight of about 3.5 kb was observed and was excised from the gel. The resulting band was purified using a gel purification kit (Qiagen; based on manufacturer's procedure). The purified DNA was digested with the enzyme XbaI (NEB; used according to manufacturer's instructions). The pBluescript plasmid was digested with XbaI using the same method. The fragment was gel purified and ligated into the pBluescript plasmid. The ligation mixture was transformed into competent cells GMlO9 and plated on LB plates containing 100 mg / l ampicillin with blue / white selection. White colonies were picked and grown in 10 ml LB liquid medium containing ampicillin. DNA was extracted by miniprep. Plasmid DNA containing the insert was sequenced using a CEQ2000 sequencer (Beckman, Fullerton, California) according to the manufacturer's instructions. T3 and T7 primers and 8 other internal primers were used for sequencing. The sequences were assembled and analyzed to obtain the genomic sequence (SEQ ID NO: 4; FIG. 3). Based on the genomic sequence, it was confirmed that the nicotine demethylase gene in both transformed and non-transformed tobacco lines does not contain a transposable element.

  Comparison of the sequence of SEQ ID NO: 5 with SEQ ID NO: 4 allowed the determination of a single intron (identified as the sequence of SEQ ID NO: 7; FIG. 5) of the coding part code of the gene. As shown in FIG. 2, the genomic structure of tobacco nicotine demethylase contains two exons flanking a single intron. The first exon extends to nucleotides 2010-2949 of SEQ ID NO: 4, which encodes amino acids 1-313 of SEQ ID NO: 3, and the second exon extends to nucleotides 3947-4562 of SEQ ID NO: 4, which are SEQ ID NO: 4 It encodes 3 amino acids 314-517. Thus, the intron extends to nucleotides 2950-3946 of SEQ ID NO: 4. The intron sequence is shown in FIG. It is the sequence of SEQ ID NO: 7. The translation product of the genomic DNA sequence is shown in FIG. The tobacco nicotine demethylase amino acid sequence contains an endoplasmic reticulum membrane anchor motif.

Cloning of 5 'flanking sequences (SEQ ID NO: 8) and 3' UTR (SEQ ID NO: 9) from transgenic tobacco
A. Isolation of total DNA from converter tobacco leaf tissue Genomic DNA was isolated from leaves of converter tobacco 4407-33. DNA isolation was performed using the DNeasy Plant Mini Kit from Qiagen, Inc. (Valencia, Ca) according to the manufacturer's protocol. The manufacturer's manual Dneasy 'Plant Mini and DNeasy Plant Maxi Handbook, Qiagen January 2004 is incorporated herein by reference. The method of DNA preparation included the following steps. Tobacco leaf tissue (about 20 mg dry weight) was ground to a fine powder under liquid nitrogen for 1 minute. The tissue powder was transferred into a 1.5 ml tube. Buffer AP1 (400 μl) and 4 μl RNase stock solution (100 mg / ml) were added to a maximum of 100 mg ground leaf tissue and vortexed vigorously. The mixture was incubated at 65 ° C. for 10 minutes and mixed 2-3 times during the incubation by inverting the tube. Buffer AP2 (130 μl) was then added to the lysate. The mixture was mixed and incubated on ice for 5 minutes. The lysate was applied to a QIAshredder Mini spin column and centrifuged for 2 minutes (14,000 rpm). The effluent fraction was transferred to a new tube without disturbing the cell debris pellet. Buffer AP3 / E (1.5 volumes) was then added to the clarified lysate and mixed by pipetting. The mixture from the previous step (650 μl) containing the possible precipitate was applied to the DNeasy Mini spin column. The mixture was centrifuged at> 6000 × g (> 8000 rpm) for 1 minute and the effluent was discarded. This was repeated for the remaining samples and the effluent and collection tube discarded. The DNeasy Mini spin column was placed in a new 2 ml collection tube. Buffer AW (500 μl) was then added to the DNeasy column and centrifuged for 1 minute (> 8000 rpm). The spill was discarded. The collection tube was reused in the next step. Buffer AW (500 μl) was then added to the DNeasy column and centrifuged for 2 minutes (> 14,000 rpm) to dry the membrane. The DNeasy column was transferred to a 1.5 ml tube. Buffer AE (100 μl) was then pipetted onto the DNeasy membrane. The mixture was incubated at room temperature (15-25 ° C.) for 5 minutes and then eluted by centrifugation for 1 minute (> 8000 rpm).
The quality and quantity of the DNA was assessed by running samples on an agarose gel.

B. Cloning of the 5 ′ flanking sequence of the structural gene To clone 750 nucleotides of the 5 ′ flanking sequence of the structural gene from SEQ ID NO: 5, a modified inverse PCR method was used. First, an appropriate restriction enzyme was selected based on the restriction site in the known sequence fragment and the restriction site distance downstream of the 5 ′ flanking sequence. Two primers were designed based on this known fragment. The forward primer was placed downstream of the reverse primer. A reverse primer was placed in the 3 ′ portion of the known fragment.

The cloning operation included the following steps.
Purified genomic DNA (5 μg) was digested with 20-40 units of the appropriate restriction enzymes (EcoRI and SpeI) in a 50 μl restriction mixture. To confirm whether the DNA was completely digested, agarose gel electrophoresis was performed with 1/10 volume of the reaction mixture. After complete digestion, direct ligation was performed by overnight ligation at 4 ° C. 200 μl of reaction mixture containing 10 μl of digested DNA and 0.2 μl of T4 DNA ligase (NEB) was ligated overnight at 4 ° C. After an artificial small circular genome was obtained, PCR was performed on the ligation reaction. PCR was performed using two primers from two different orientations of known fragments and 10 μl of ligation reaction in a 50 μl reaction mixture. A gradient PCR program using an annealing temperature of 45-56 ° C was applied.

  To examine the PCR reaction, agarose gel electrophoresis was performed. The desired band was excised from the gel and the band was purified using QIAGEN's QIAquick gel purification kit. Purified PCR fragments were ligated into pGEM-T Easy Vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV Miniprep kit (Promega, Madison, Wis.) According to the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). About 758 nt of the 5 ′ flanking sequence (nucleotides 1241-2009 of SEQ ID NO: 4) was cloned by the above method.

C. Cloning of the longer 5 'flanking sequence of the structural gene (SEQ ID NO: 8; Figure 6) To clone additional 5' flanking sequences of the structural gene D121-AA8, the BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto , CA) was used according to the manufacturer's user manual. The manufacturer's manual BD GenomeWalker August, 2004 is incorporated herein by reference. Tobacco genomic DNA was tested for size and purity by running samples on a 0.5% agarose gel. A total of four blunt end reactions (DRA I, STU I, ECOR V, PVU II) were prepared for the construction of the tobacco 33 library genome walking. After purification of the digested DNA, the digested genomic DNA was ligated to a genomic walker adapter. The primary PCR reaction was applied to these four digested DNAs by using the gene specific primer from D121-AA8 (CTCTATTGATACTAGCTGGTTTTGGAC; SEQ ID NO: 2282) and the adapter primer AP1. The primary PCR product was used directly as a template for nested PCR. The adapter nested primer supplied with the kit and the nested primer (GGAGGGAGAGTATAACTTACGGATTC; SEQ ID NO: 2283) from the known clone D121-AA8 (SEQ ID NO: 5) were used in the PCR reaction. PCR products were examined by gel electrophoresis. The desired band was excised from the gel and the band was purified using QIAGEN's QIAquick gel purification kit. Purified PCR fragments were ligated into pGEM-T Easy Vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV Miniprep kit (Promega, Madison, Wis.) According to the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). An additional approximately 853 nt of the 5 ′ flanking sequence containing nucleotides 399-1240 of SEQ ID NO: 4 was cloned by the above method.

  Perform a second round of genome walking in the same way except using the following primers GWR1A (5'-AGTAACCGATTGCTCACGTTATCCTC -3 ') (SEQ ID NO: 2284) and GWR2A (5'-CTCTATTCAACCCCACACGTAACTG -3') (SEQ ID NO: 2285). went. An additional approximately 398 nt of the flanking sequence containing nucleotides 1-398 of SEQ ID NO: 4 was cloned by this method.

  A search for regulatory elements indicates that in addition to the “TATA”, “CAAT” and “GAGA” boxes, several MYB-like recognition sites and organ-specific elements are present in the tobacco nicotine demethylase promoter region. It was. Putative elicitor response elements and nitrogen regulatory elements identified using standard methods are also present in the promoter region.

D. Cloning the 3 'flanking sequence of the structural gene To clone additional 3' flanking sequences of the structural gene D121-AA8, use the BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto, CA) according to the manufacturer's user manual. used. The cloning procedure is the same as described in section C of this example, except for gene specific primers. The first primer was designed from the vicinity of the end of the D121-AA8 structural gene (5′-CTA AAC TCT GGT CTG ATC CTG ATA CTT-3 ′) (SEQ ID NO: 2286). The nested primer was designed further downstream from primer 1 of the D121-AA8 structural gene (CTA TAC GTA AGG TAA ATC CTG TGG AAC) (SEQ ID NO: 2287). The final PCR product was examined by gel electrophoresis. The desired band was cut from the gel. The PCR fragment was purified using QIAGEN's QIAquick gel purification kit. Purified PCR fragments were ligated into pGEM-T Easy Vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV Miniprep kit (Promega, Madison, Wis.) According to the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). Further, about 1617 nucleotides (nucleotides 4731-6347 of SEQ ID NO: 4) of the 3 ′ flanking sequence were cloned by the above method. The nucleic acid sequence of the 3 ′ UTR region is shown in FIG.

Screening of Nicotiana sp. For presence or absence of nicotine demethylase gene As shown in Table 8 below, 43 Nicotiana species, 49 Nicotiana rustica lines and about 600 Nicotiana tabacum were seeded in pots and produced Plants were grown in the greenhouse.

  Leaf samples were taken from 6 week old plants. DNA extraction from leaves was performed using DNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol.

  Primers were designed based on the 5 ′ promoter and 3 ′ UTR region described herein. The forward primer is 5'-GGC TCT AGA TAA ATC TCT TAA GTT ACT AGG TTC TAA-3 '(SEQ ID NO: 2290), and the reverse primer is 5'-GGC TCT AGA AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC -3 ′ (SEQ ID NO: 2291) (from −750 to 180 nt 3 ′ UTR of the 5 ′ flanking region). Genomic DNA extracted from all the Nicotiana strains was used for PCR analysis. 100 μl of reaction mixture and Pfx high fidelity enzyme were used for PCR amplification. Due to the lower homology between species, the annealing temperature used was 54 ° C. (this temperature is 2 ° C. lower than the temperature used for cloning the genomic sequence from the 4407 transformant tobacco). After electrophoresis, PCR products were visualized on a 0.8% agarose gel. A single band with a molecular weight of about 3.5 kb was present or absent on the gel. This line with a positive band was assessed to have the target gene. For lines lacking positive bands, four additional PCR reactions were performed using four additional primer sets. These primer sets were selected from different regions of the gene. Those primer sets were as follows.

(1) From the start codon to (5'-GCC CAT CCT ACA GTT ACC TAT AAA AAG GAA G -3 ') to the stop codon 5'-ACC AAG ATG AAA GAT CTT AGG TTT TAA -3') (SEQ ID NO: 2293),
(2) From 570 nt downstream of the start codon to (5′-TCG ATC GTG AAG ATG A -3 ′) (SEQ ID NO: 2294) to the end of the intron (5′-TCC TGC ATC CAA GAC CA -3 ′) (SEQ ID NO: 2295 ),
(3) 300 nt downstream from the beginning of the intron (5'-GGG CTA TAT GGA TTC GC -3 ') (SEQ ID NO: 2296) to the end of the intron (5'-TGC TGC ATC CAA GAC CA -3') (SEQ ID NO: 2295), and (4) 300 nt downstream from the beginning of the intron (5'-GGG CTA TAT GGA TTC GC -3 ') (SEQ ID NO: 2296) up to 3'UTR (5'- AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3 ') (SEQ ID NO: 2195).

  If all five of the PCR reactions showed the correct band, the line was assessed as lacking the target gene. Specific examples of the amount of genomic DNA and PCR products related to the target nicotine demethylase gene are shown in FIGS.

Germplasm identified as lacking the nicotine demethylase gene is used as a raw material for breeding with cultivated tobacco. However, any nucleic acid sequence shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or fragments thereof may be used as well. Abnormal or absent nicotine demethylase gene or any nucleic acid sequence shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or fragments thereof To transfer tobacco from donor sources into cultivated tobacco, interspecific or intraspecific crossing methods combined with standard breeding methods such as backcrossing or line breeding methods can be used. The results of screening experiments for nicotine demethylase are listed in Table 9 below. A strain that is negative for nicotine demethylase can be crossed with itself or another negative strain (eg, Nicotiana africana x Nicotiana africana, or Nicotiana africana x Nicotiana amplexicaulis, or any suitable mating combination). It is also possible to cross negative strains with any commercial variety of tobacco by standard tobacco breeding techniques known in the art. It is possible to cross a tobacco line with any other compatible plant by standard methods in the art.

Screening for the creation or generation of mutations and genetic mutations within the nicotine demethylase gene Sequences encoding nicotine demethylase or FIGS. 1, 3-7, 10-158, 162-170, 172-1-172-19 and 173- Pre-existing genetic mutations or mutations in any other gene represented by the nucleic acid sequence shown in 1-173-294 or fragments thereof are targeted induced local lesions in genomes (TILLING) ), Screening using molecular fingerprinting techniques such as amplified fragment length polymorphism (AFLP) and single nucleotide polymorphism (SNP). In practice, chemical mutagenesis, eg, alkylation, of a plant population that represents an existing genetic variation, eg, a transgenic plant (eg, any transgenic plant described herein) or a reproductive tissue or other plant tissue. Plants made by exposure to agents such as ethane methyl sulfonate (EMS) or radiation such as X-rays or gamma rays are used. In order to obtain a mutagenized population, the amount of mutagenic compound or radiation is experimentally determined for each type of plant tissue so that a mutation frequency is obtained that is below the threshold level characterized by lethality or reproductive sterility. Is done. The number of M1 generation seeds or the size of the M1 plant population resulting from the mutagenesis treatment is estimated based on the expected frequency of mutation. The M2 generation, the progeny of M1 plants, desirably corresponds to a population that is evaluated for mutations in a gene (eg, nicotine demethylase gene).

  Tilling, DNA fingerprinting, SNP or similar techniques can be used to detect induced or naturally occurring genetic variations within the desired gene (eg, nicotine demethylase gene). The mutation can result from a deletion, substitution, point mutation, translocation, inversion, duplication, insertion or complete null mutation. These techniques involve the nicotine demethylase gene or any nucleic acid sequence shown in FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or fragments thereof. Could be used in a marker-assisted selection (MA breeding program) to transfer or breed other null or dissimilar alleles into other tobacco. Breeders could create a segregating population by crossing a genotype containing a null or heterologous allele with a genotype desirable for agriculture. Using one of the techniques already mentioned, the nicotine demethylase sequence or the nucleic acid sequence shown in Figures 1, 3-7, 10-158, 162-170, 172-1-172-19 and 173-1-173-294 Alternatively, markers generated from these fragments could be used to screen plants in F2 or backcross generations. Plants identified as having null or conservative alleles could be backcrossed or self-pollinated to produce the next generation that could be screened. Depending on the expected genetic pattern or the MAS technique used, it may be necessary to self-pollinate the selected plants prior to each backcross cycle to help identify the desired individual plants. Backcrossing or other breeding methods can be repeated until the desired phenotype of the recurrent parent is restored.

Mating or transferring mutant nicotine demethylase gene expression into cultivated tobacco
A. Selection of parental line A donor tobacco line with mutated nicotine demethylase gene expression (eg, using a PCR-based method, nicotine lacking the nicotine demethylase gene or null with respect to nicotine demethylase, or with altered enzyme activity Tobacco lines identified as expressing demethylase, or tobacco lines expressing a transgene that alters or silences gene expression are also considered variants for nicotine demethylase gene expression) or FIG. Any nucleic acid sequence shown in -7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or a fragment thereof is identified and selected for use as a donor parent. Such plants are produced by standard methods known in the art (eg, those described herein). Other donor parents are mutagenized and then mutated nicotine demethylase gene activity or shown in Figures 1, 3-7, 10-158, 162-170, 172-1-172-19 and 173-1-173-294 Included are tobacco plants identified as having the mutating activity of the gene product encoded by any nucleic acid sequence or fragment thereof. One typical donor is the ne tobacco line Nicotiana rustica.

The recipient (recipient) tobacco line is typically any commercial tobacco variety, such as Nicotiana tabacum TN 90. Other useful Nicotiana tabacum varieties include BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KT 200, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 160, Little Crittenden, McNair 373, McNair 944, msKY 14 x L8, Narrow Leaf Madole, NC 100, NC 102 , NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC 606, NC 71, NC 72, NC 810, NC BH 129, OXFORD 207, 'Perique' tobacco, PVH03 , PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H4, RG H51, RGH 4, RGH 51, RS 1410, SP 168, SP 172, SP 179, SP 210, SP 220, SP G-28, SP G-70, SP H20, SP NF3, TN 86, TN 97, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA 309 or VA 359 is included. Seeds from such varieties can also be derived from sources obtained by screening for the absence or presence of nicotine conversion using standard chemical or molecular methods. Such commercial varieties may also provide material for modifying nicotine demethylase activity by the methods described herein. Other null lines and acceptor or donor lines known in the art are also useful, and lines identified as being distinct from the nicotine demethylase genes described herein are also used as donor parents. Also, the recipient line may be selected from any tobacco variety for fire-curing, burley, dark tobacco, Virginia or Oriental tobacco. Table 10 shows typical Nicotiana species that are breeding compatible with Nicotiana tabacum (see also, for example, Compendium of Tobacco Diseases published by APS or The Genus Nicotiana Illustrated published by Japan Tobacco Industry). Wanna)

B. Gene transfer The donor parent is repeatedly mated or hybridized with the donor parent using standard breeding methods. A successful cross, identified by standard methods, gives F1 plants that are fertile or optionally backcrossed with recurrent parents. Plant populations in the F2 generation derived from F1 plants can be mutated for mutant nicotine demethylase gene expression (eg, using standard methods, eg, primers using primers based on nucleotide sequence information for nicotine demethylase described herein) Identifies plants that do not express nicotine demethylase due to the absence of the nicotine demethylase gene) or FIGS. 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to Screen for mutant expression of any of the nucleic acid sequences shown in 173-294 or fragments thereof. Alternatively, any standard screening method known in the art for assessing plant alkaloid content is used to identify plants that do not convert nicotine to nornicotine. The selected plant is then crossed with the recipient parent, and the first backcross (BC1) generation plant is self-pollinated to obtain the BC1F2 population, which is mutated nicotine demethylase gene expression (eg, nicotine with the null form of the methylase gene) Screen again for. The process of backcrossing, self-pollination and screening is repeated, for example, at least four times until the final screening gives fertile and reasonably similar plants to the recipient parent. If desired, the plant is self-pollinated and then its progeny are screened again so that the plant exhibits mutant nicotine demethylase gene expression (eg, a plant that exhibits a null state for nicotine demethylase) or FIG. 1, 3-7, 10 It is confirmed to show mutant expression of any of the nucleic acid sequences shown in 158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or fragments thereof. If desired, cytogenetic analysis of selected plants is performed to confirm chromosomal complementation and chromosome pairing relationships. Standard methods such as field tests, null status for nicotine demethylase or any of those shown in Figures 1, 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 Confirmation of the null or accelerated state of the polypeptide encoded by the nucleic acid sequences or fragments thereof, and chemical analysis of dried leaves (alkaloid levels, particularly nornicotine content and nornicotine / nicotine + nornicotine ratio, or FIG. Any of the nucleic acid sequences shown in 3-7, 10-158, 162-170, 172-1 to 172-19 and 173-1 to 173-294 or other gene sequences thereof found in their fragments For measuring the desired characteristics as described above) to obtain seeds for breeding the selected plant.

  If the original F1 hybrid obtained from a cross between a recipient plant (eg N. rustica) and a donor parent (eg TN90) is crossed or backcrossed to the donor plant (eg TN90), this The progeny of backcrossing is self-pollinated to obtain the BC1F2 generation, which is a null or atypical form of nicotine demethylase or Figure 1, 3-7, 10-158, 162-170, 172-1-172-19 and 173 Screening for null or facilitating states of polypeptides encoded by any of the nucleic acid sequences shown in -1 to 173-294 or fragments thereof. Other aspects of the breeding operation are as described in the previous paragraph.

C. Testing agricultural characteristics and confirming the phenotype The breeding and mutant nicotine demethylase gene expression (eg, null status for nicotine demethylase, or FIG. 1, 3-7, 10-158, 162-170, 172-1 Lines obtained from screening for any nucleic acid sequences shown in ˜172-19 and 173-1 to 173-294 or the null state thereof are evaluated in the field using standard field methods. A control genotype containing the original recipient parent (eg, TN90) is added and the registrant is placed in the field in a fully random block design or other suitable field design. Standard agricultural practices for tobacco are performed, for example, the tobacco is harvested, weighed, and sampled for chemical and other common tests before and during drying (curing). Statistical analysis of the data is performed to confirm the similarity of the selected line to the recipient plant (eg, parent line TN90).

Crossing (breeding) or transferring modified attributes to cultivated tobacco Any gene described herein, for example, FIG. 1, FIG. 3-7, FIG. 10-158, FIG. 162-170, FIG. And the expression of any of the nucleic acid sequences shown in FIGS. 173-1 to 173-294 can be modified by the methods described herein. Such genes are the basis for modifying the phenotype of plants, for example improving flavor or fragrance or both, improving sensory stimulating properties, or improving dryness . The plants identified as having a modified phenotype are then used in a breeding protocol by standard methods known in the art (eg, the methods described herein).

Using the manufacturing protoplast fusion hybrid plants, standard protoplast culture methods developed for the production of hybrid plants also apply, mutant gene expression (e.g., mutant nicotine demethylase gene expression) to produce a plant having Useful for. Accordingly, protoplasts are produced from the first and second tobacco plants having mutant gene expression. Callus is cultured from the successfully obtained protoplast fusion, and then the plant is regenerated. The resulting progeny hybrid plants can be identified, selected for mutant gene expression by standard methods, and used in any standard breeding protocol if desired.

  WO 03/078577, WO 2004/035745, PCT / US / 2004/034218 and PCT / US / 2004/034065 and all other references, patents, patent publications and patent applications mentioned in this specification Are hereby incorporated by reference as if each of these references, patents, patent application publications and patent applications were individually incorporated herein by reference.

  In view of the foregoing detailed description of the invention, many modifications and changes in the practice of the invention are expected to occur to those skilled in the art. Accordingly, such modifications and variations are intended to be included within the scope of the claims.

Claims (14)

A method of reducing the expression of nicotine demethylase in a tobacco plant comprising the step of expressing a transgene encoding a double stranded RNA molecule that inhibits expression of nicotine demethylase in the plant, The strand RNA molecule comprises at least 25 contiguous nucleotides having 100% sequence identity with SEQ ID NO: 7 or 9, and the tobacco plant lacking the transgene has expression of nicotine demethylase in the plant. Said method being inhibited. 2. The method of claim 1, wherein the double stranded RNA molecule comprises at least 100 nucleotides having 100% sequence identity with SEQ ID NO: 7 or 9. 2. The method of claim 1, wherein the double stranded RNA molecule comprises at least 250 nucleotides having 100% sequence identity with SEQ ID NO: 7 or 9.   2. The method of claim 1, wherein the tobacco plant is derived from a species selected from the group consisting of Nicotiana tabacum, Nicotiana sylvestris, Nicotiana tomentosiformis, and Nicotiana glauca.   2. The method of claim 1, wherein the tobacco plant is Nicotiana tabacum.   2. The method of claim 1, wherein the transgene comprises a constitutive promoter.   2. The method of claim 1, wherein the transgene comprises an inducible promoter. A method of producing a tobacco plant with reduced expression of nicotine demethylase comprising the steps of:
a) mutagenizing a plurality of tobacco plant cells;
b) screening progeny of plants made from the mutagenized cells for reduced nicotine demethylase expression; and
c) a step of selecting one or more progeny having an endogenous nucleic acid containing a mutation in the nucleotide sequence represented by SEQ ID NO: 4 and having reduced expression of nicotine demethylase. Tobacco plant cell is a Nicotiana tabacum cells The method of claim 8. 9. The method according to claim 8, wherein the tobacco plant cell is a seed cell. 9. The method of claim 8 , wherein the mutation is selected from the group consisting of point mutations, deletions, insertions, duplications and inversions. 9. The method according to claim 8 , wherein the mutation is a point mutation or a deletion.   A tissue culture of a reproducible tobacco cell having a mutation in the endogenous nicotine demethylase gene having the sequence represented by SEQ ID NO: 4, wherein all the physiological and morphological characteristics of the tobacco plant having the mutation are Said tissue culture regenerating tobacco plants that can be expressed. 14. Tissue culture according to claim 13 , wherein the renewable cells are embryos, dividing cells, seeds, pollen, leaves, roots, root tips or flowers, or protoplasts or callus derived therefrom.

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