CN113416747B - Method for creating temperature-sensitive male sterile plant - Google Patents

Abstract

The present invention discloses a method of creating a temperature sensitive male sterile plant comprising the step of reducing expression or activity of a Rboh gene or a protein encoded thereby in said plant, and a method of transforming a plant from sterile to fertile. In addition, the invention provides the application of the Rboh gene or the inhibitor of the coded protein thereof in the cultivation of plant sterile lines or the preparation of a reagent or a kit for the cultivation of plant sterile lines.

Description

Method for creating temperature-sensitive male sterile plant

Technical Field

The present invention relates to the field of agriculture and biotechnology, in particular to a method of creating temperature sensitive male sterile plants.

Background

Reactive Oxygen Species (ROS) are a class of byproducts of the aerobic metabolism of organisms, mainly comprising superoxide anion radicals (O) ₂ ^- Of the formula (I), hydroxyl radicals (. OH) and hydrogen peroxide (H) ₂ O ₂ ) ROS have dual functions: on one hand, the polypeptide can be used as a toxic secondary metabolite to destroy normal molecules in the body; on the other handAs important signal molecules, are involved in the growth and development of organisms. It has been found that sources of ROS include plasma membrane NADPH oxidase, peroxidase and amine oxidase, with ROS produced mediated by NADPH oxidase being of greatest concern.

Male sterility refers to the phenomenon in which the female organs of a plant function normally while the male organs (degenerated, pollen-free or pollen not viable) lose pollination function. Male organs of the sterile line are abnormal in development and cannot self-fertilize and set; female organs develop normally, have fertilization ability, and can accept fertilization and fructification of external pollen. Male sterility can be classified into: nuclear male sterility, cytoplasmic male sterility, and nuclear-cytoplasmic male sterility. The male sterile line is used as female parent and the restoring line is used as male parent, so that high-quality hybrid can be obtained.

Male sterile lines are widely used in rice breeding, and the hybrid rice breeding currently mainly comprises a three-line method and a two-line method. The three-line hybridization method comprises the following three-line hybridization breeding processes of a maintainer line, a restorer line and a sterile line, such as rice: the male sterile line is remained by hybridization with the sterile line as a parent and the maintainer line as a male parent. And then the sterile line is used as a female parent and the restorer line is used as a male parent for hybridization to obtain hybrid seeds. The method is a method commonly adopted in the history of popularization of thirty calendar of hybrid rice in China, and is still used in a large amount at present. The three-line method has the advantages of long period, low efficiency, more popularization links, low speed, high seed cost and high price because of complex breeding procedures and production links. The two-line hybrid rice comprises a sterile line and a restorer line, the sterile line can be influenced by temperature and illumination to restore fertility, and the two-line hybrid rice is another important technological innovation in genetic breeding of rice after the three-line hybrid rice. Compared with the three-line hybrid rice, the two-line hybrid rice has obvious advantages: firstly, the sterile line and the restorer line are freely matched, so that the probability of breeding excellent combinations is increased; and the sterile line is two-purpose, so that a maintainer line is omitted, and one link is reduced in the seed production process, thereby reducing the seed production cost. And secondly, the composition is free, and almost any variety can be used as a restorer. Thirdly, because the genetic inheritance of the photo-thermo-sensitive sterile nuclear gene is irrelevant to cytoplasm, the negative effect of cytoplasm in the sterile line of the three-line method can be overcome. And fourthly, the breeding of the conventional rice can be followed, a new field of hybrid vigor of indica-japonica subspecies is opened up, and the rice yield can achieve a higher yield target on the basis of the existing hybrid rice. The advantages of the two-line method make the development of photo-thermo-sensitive male sterile line one of the targets for realizing heterosis.

The control gene of the male sterile line capable of restoring fertility is screened, so that the preparation of the sterile line can be rapidly and efficiently realized, and the method is popularized and used among different species. However, at present, the known genes for controlling the male sterile line of plants are not very many, so that the development of related functional genes has important significance for rapid breeding to prepare the male sterile line and promoting the utilization of the advantages of hybridization breeding.

Disclosure of Invention

The invention aims to provide a gene for controlling a plant male sterile line, so that the male sterile line can be quickly bred and prepared, and the utilization of the advantages of hybridization breeding is promoted.

In a first aspect of the present invention, there is provided a method of breeding a plant sterile line comprising the steps of: reducing expression or activity of an Rboh gene or a protein encoded thereby in said plant.

In another preferred embodiment, the method is carried out under conditions at least during the anther development period, preferably the flowering period, with a maximum temperature in the growing environment of greater than or equal to 25 ℃, preferably greater than or equal to 28 ℃, preferably greater than or equal to 29 ℃, preferably greater than or equal to 30 ℃, preferably greater than or equal to 31 ℃, preferably greater than or equal to 32 ℃, preferably greater than or equal to 33 ℃, preferably greater than or equal to 34 ℃, preferably greater than or equal to 35 ℃.

In another preferred embodiment, the method is carried out at a maximum temperature in the growing environment of 25-35 ℃, preferably 26-32 ℃, more preferably 28-30 ℃ at least during the anther development period, preferably the plant flowering period.

In another preferred embodiment, the method is carried out under conditions of a plant anther development period, preferably a flowering period, controlling the temperature fluctuation of the growing environment to a range of 12 ℃ to 35 ℃, preferably 16 ℃ to 30 ℃, more preferably 18 ℃ to 30 ℃, still more preferably 20 ℃ to 30 ℃.

In another preferred embodiment, the Rboh gene is a gene that is specifically expressed during plant anther development.

In another preferred embodiment, the Rboh gene is from one or more plants selected from the group consisting of: cruciferae, gramineae, leguminosae, solanaceae, umbelliferae, chenopodiaceae, actinidiaceae, moraceae, malvaceae, paeoniaceae, rosaceae, liliaceae, or combinations thereof.

In another preferred embodiment, the Rboh gene is derived from one or more plants selected from the group consisting of: arabidopsis thaliana, potato, sweet potato, purple potato, yam, taro, cassava, chrysanthemum, rice, wheat, barley, corn, sorghum, soybean, peanut, millet, quinoa, tomato, tobacco, canola, capsicum, cotton, cabbage, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, onion, lettuce, millet, peanut, kiwi, cotton, strawberry, peony, lily, tulip, mulberry, apple, pear, peach, cherry, and pomegranate.

In another preferred embodiment, the Rboh gene comprises: osRBOHA, osRBOHB, osRBOHE of rice, or a combination thereof; leRBOHE, leRBOHB, leRBOH of tomato, or a combination thereof; atRBOHE of Arabidopsis thaliana; preferably, the RboH gene comprises OsRBOHA, osRBOHB, osRBOHE of rice, or a combination thereof; leRBOHE, leRBOHB, leRBOH from tomato, or a combination thereof.

In another preferred embodiment, the Rboh gene includes a wild-type Rboh gene and a mutant Rboh gene.

In another preferred embodiment, the mutant comprises a mutant form having an unchanged, enhanced and/or reduced function (i.e., the same or substantially the same function as the wild-type encoded protein) function of the encoded protein after mutation.

In another preferred embodiment, the mutant Rboh gene encodes a polypeptide that is the same or substantially the same as the polypeptide encoded by the wild-type Rboh gene.

In another preferred embodiment, the mutant Rboh gene comprises a polynucleotide having a homology of 80% (preferably 90%, more preferably 95%, more preferably 98% or 99%) as compared to the wild-type Rboh gene.

In another preferred embodiment, the mutant Rboh gene comprises a polynucleotide truncated or added with 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5 'and/or 3' end of the wild-type Rboh gene.

In another preferred embodiment, the nucleotide sequence of the Rboh gene is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in any one of SEQ ID NOs.1-7;

(b) A polynucleotide having a sequence as set forth in any one of SEQ ID NOs.8-14;

(c) A polynucleotide having a nucleotide sequence which has a homology of greater than or equal to 50% (preferably greater than or equal to 60%, more preferably greater than or equal to 75%, more preferably greater than or equal to 85%, more preferably greater than or equal to 90% or greater than or equal to 95% or greater than or equal to 98%, such as 99%) to any of the sequences set forth in SEQ ID No. 8-14;

(d) A polynucleotide truncated or added at the 5 'and/or 3' end of the polynucleotide shown in any one of SEQ ID nos. 8 to 14 of 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides;

(e) A polynucleotide complementary to the polynucleotide of any one of (a) - (d).

In another preferred embodiment, the amino acid sequence of the Rboh protein is selected from the group consisting of:

(i) A polypeptide having the amino acid sequence set forth in any one of SEQ ID nos. 1-7;

(ii) A polypeptide derived from (i) having the plant trait regulatory function, which is formed by substitution, deletion or addition of one or more (e.g., 1 to 10) amino acid residues to an amino acid sequence as shown in any one of SEQ ID NO. 1 to 7; or (b)

(iii) The amino acid sequence has a homology of 60% or more (preferably 80% or more, preferably 90% or more, more preferably 95% or 98% or more, such as 99%) with any of the amino acid sequences shown in SEQ ID No. 1-7, and a polypeptide having said Rboh activity.

In another preferred embodiment, the "decrease" means that the expression or activity of the Rboh gene or a protein encoded thereby in the plant is decreased by:

the ratio A1/A0 is less than or equal to 80%, preferably less than or equal to 60%, more preferably less than or equal to 40%, most preferably 0-30%;

wherein A1 is the expression or activity of a Rboh gene or a protein encoded by the Rboh gene in the plant; a0 is the same Rboh gene or the expression or activity of the coded protein in the wild type plant of the same type.

In another preferred embodiment, the method for reducing the Rboh gene or its encoded protein in a plant comprises: the expression level of the Rboh gene is reduced, and/or the activity of the protein encoded by the Rboh gene is reduced.

In another preferred embodiment, the decrease is that the expression level E1 of the Rboh gene or its encoded protein in the plant is 0-80%, preferably 0-60%, preferably 0-40%, preferably 0-30%, preferably 0-20%, preferably 0-10%, more preferably 0-5% of the wild type compared to the expression level E0 of the wild type Rboh gene or its encoded protein; and/or the activity A1 of the Rboh gene or its encoded protein in said plant is 0-80%, preferably 0-60%, preferably 0-40%, preferably 0-30%, preferably 0-20%, preferably 0-10%, more preferably 0-5% of the wild type compared to the activity A0 of the wild type Rboh gene or its encoded protein.

In another preferred embodiment, said reducing expression or activity of a Rboh gene or a protein encoded thereby in a plant is achieved by a means selected from the group consisting of: gene mutation, gene knockout, gene disruption, gene editing techniques, natural mutagenesis, artificial mutagenesis (including physical, chemical, biological methods, etc.), inhibitors (e.g., small molecule compounds, nucleic acid molecules (RNAi), or combinations thereof), regulatory gene expression control elements, or combinations thereof.

In another preferred embodiment, the expression control elements include cis-acting elements and trans-acting factors.

In another preferred embodiment, the gene editing technique comprises TALEN, ZIN, CRISPR.

In another preferred embodiment, the plant is an ampholytic or hermaphroditic plant.

In another preferred embodiment, the plant comprises a dicotyledonous plant and a monocotyledonous plant.

In another preferred example, the plant comprises a crop, a forestry plant, a vegetable, a melon, a flower, a pasture (including turf grass).

In another preferred embodiment, the plant is selected from the group consisting of: cruciferae, gramineae, leguminosae, solanaceae, umbelliferae, chenopodiaceae, actinidiaceae, moraceae, malvaceae, paeoniaceae, rosaceae, liliaceae, or combinations thereof.

In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis thaliana, potato, sweet potato, purple potato, yam, taro, cassava, chrysanthemum, rice, wheat, barley, corn, sorghum, soybean, peanut, millet, quinoa, tomato, tobacco, canola, pepper, cotton, cabbage, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, onion, lettuce, millet, peanut, kiwi, cotton, strawberry, peony, lily, tulip, mulberry, apple, pear, peach, cherry, pomegranate, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of: rice, wheat, corn, tomato, canola, capsicum, cotton, soybean, or a combination thereof.

In another preferred embodiment, the rice is selected from the group consisting of: indica rice, japonica rice, or combinations thereof.

In another preferred embodiment, the plant sterile line has one or more characteristics selected from the group consisting of:

(1) Female organs in hermaphrodite plants shrink, malform or disappear.

(2) Failure to form normal microsporidian tissue;

(3) Microspores are abnormal, forming imperfect, nonviable, malformed or aborted flowers;

(4) Pollen is not mature or has no germination capacity;

(5) Viable pollen is formed but anthers do not crack.

In another preferred embodiment, the plant sterile line is a high temperature sensitive plant sterile line.

In another preferred embodiment, the plant sterile line is a high temperature sensitive male sterile line.

In another preferred embodiment, the plant sterile line comprises nuclear male sterility.

In another preferred embodiment, the elevated temperature is not less than 25 ℃, preferably not less than 28 ℃, preferably not less than 29 ℃, preferably not less than 30 ℃, preferably not less than 31 ℃, preferably not less than 32 ℃, preferably not less than 33 ℃, preferably not less than 34 ℃, preferably not less than 35 ℃.

In another preferred embodiment, the elevated temperature is 25-35 ℃, more preferably 26-32 ℃, still more preferably 28-30 ℃.

In a second aspect, the invention provides the use of an inhibitor of the Rboh gene or its encoded protein for the cultivation of a plant sterile line, or for the preparation of a reagent or kit for the cultivation of a plant sterile line.

In another preferred embodiment, the inhibitor is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, siRNA, RNAi, crispr agent, or a combination thereof.

In a third aspect, the present invention provides a method for restoring fertility to a plant sterile line, comprising the steps of: cultivating the plant sterile line according to the first aspect of the invention under low temperature conditions.

In another preferred embodiment, the low temperature condition is a growth environment maximum temperature of < 25 ℃, preferably equal to or less than 24 ℃, preferably equal to or less than 23 ℃, preferably equal to or less than 22 ℃, preferably equal to or less than 21 ℃, preferably equal to or less than 20 ℃.

In another preferred embodiment, the low temperature condition is a growth environment temperature fluctuation range of 12 ℃ to 24 ℃, preferably 16 ℃ to 22 ℃, and more preferably 18 ℃ to 22 ℃.

In another preferred embodiment, the ambient temperature comprises the ambient temperature during the bolting phase of plant growth, preferably the ambient temperature during the anther development phase, preferably the ambient temperature during the flowering phase.

In another preferred embodiment, said restoring fertility comprises pollination of a plant to produce seeds with reproductive capacity.

In a fourth aspect the invention provides a method of converting a plant from sterile to fertile comprising the steps of: culturing a plant sterile line cultivated by the method according to the first aspect of the invention under low temperature conditions.

In another preferred embodiment, the low temperature condition is a growth environment maximum temperature of < 25 ℃, preferably equal to or less than 24 ℃, preferably equal to or less than 23 ℃, preferably equal to or less than 22 ℃, preferably equal to or less than 21 ℃, preferably equal to or less than 20 ℃.

In another preferred embodiment, the low temperature condition is a growth environment temperature fluctuation range of 12 ℃ to 24 ℃, preferably 16 ℃ to 22 ℃, and more preferably 18 ℃ to 22 ℃.

In another preferred embodiment, the ambient temperature comprises the ambient temperature during the bolting period of the plant growth, preferably the ambient temperature during the anther development period, more preferably the ambient temperature during the anther development period.

In another preferred embodiment, said restoring fertility comprises pollination of a plant to produce seeds with reproductive capacity.

In a fifth aspect, the present invention provides a plant breeding method comprising the steps of: crossing the plant sterile line according to the first aspect of the invention with another parent plant to obtain a hybrid.

In a sixth aspect, the present invention provides a method of plant breeding comprising the step of maintaining sterility of a plant; a step of changing the plants from sterile to fertile; and/or maintaining the plant fertility and breeding steps;

in said step of maintaining sterility of the plant, comprising maintaining a sterile line of the plant cultivated according to the method of the first aspect of the invention;

in the step of changing the plant from sterile to fertile, the method of the third aspect of the invention or the fourth aspect of the invention is used to change the plant from sterile to fertile.

In a seventh aspect, the invention provides a plant cell, the plant cell and/or plant from which it has been developed having reduced expression or activity of a Rboh gene or a protein encoded thereby.

In an eighth aspect, the present invention provides a method of identifying a plant sterile line or propagation material thereof, comprising the steps of: detecting expression or activity of the Rboh gene or its encoded protein in the plant sample.

In another preferred embodiment, the plant is a plant sterile line or propagation material thereof when the expression or activity of the Rboh gene or its encoded protein in the plant sample satisfies the following conditions:

the ratio A1/A0 is less than or equal to 80%, preferably less than or equal to 60%, more preferably less than or equal to 40%, most preferably 0-30%;

wherein A1 is the expression or activity of a Rboh gene or a protein encoded thereby in the plant sample; a0 is the same Rboh gene or its encoded protein expression or activity in a wild-type isotype plant sample.

In another preferred embodiment, when the expression level E1 of the Rboh gene or its encoded protein in said plant sample is 0-80%, preferably 0-60%, preferably 0-40%, preferably 0-30%, preferably 0-20%, preferably 0-10%, more preferably 0-5% of the wild type; and/or the plant is a plant sterile line or propagation material thereof when the activity A1 of the Rboh gene or its encoded protein in the plant sample is 0-80%, preferably 0-60%, preferably 0-40%, preferably 0-30%, preferably 0-20%, preferably 0-10%, more preferably 0-5% of the activity A0 of the wild-type Rboh gene or its encoded protein.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows seed growth of Arabidopsis rbohe mutants at temperatures ranging from 18℃to 30℃and temperatures ranging from 20℃to 22 ℃.

FIG. 2 shows pollen production of Arabidopsis rbohe mutants at temperatures ranging from 18℃to 30℃and temperatures ranging from 20℃to 22 ℃.

FIG. 3 shows pollen development of Arabidopsis rbohe mutants at temperatures ranging from 18℃to 30℃and temperatures ranging from 20℃to 22 ℃.

Detailed Description

The inventors have conducted extensive and intensive studies and have unexpectedly found for the first time that, for certain specific plant sterile lines, the fertility of the plants can be regulated by regulating the expression or activity of the Rboh gene or its encoded protein, and a controllable switch between sterility and fertility can be achieved. The inventor also develops a plurality of technologies with wide application value in agricultural breeding and the like for correspondingly cultivating plant sterile lines and the like. The present invention has been completed on the basis of this finding.

Rboh gene

Rboh, a plant NADPH oxidase also known as respiratory burst oxidase (Respiratoryburstoxidase homologue, rboh), is the mammalian macrophage NADPH oxidase catalytic subunit gp91 ^phox Is a homolog of (C). The plant NADPH oxidase comprises an NADPH binding domain, a FAD binding domain and 6 conserved transmembrane domains, which are identical to mammalian gp91 ^phox Is identical to the conserved domain of (a). Unlike mammals, all plant Rboh proteins contain an N-terminal extension of 300 amino acids in size, which contains 2 conserved ca2+ binding EF chiral structures. The positioning mode of NADPH oxidase in specific areas in different types of cells can enable the NADPH oxidase to generate ROS in specific areas of the cells and play a special biological function.

OsRbohA in rice is the first cloned Rboh gene in plants, and then the Rboh gene is cloned from various crops such as Arabidopsis, potato, tomato, tobacco and the like. Studies have shown that the Rboh genes in plants are generally present in the form of a gene family, 10 Rboh genes (AtRbohA-J) are included in the Arabidopsis genome, and 7 Rboh genes (AtRbohA-G) are included in rice. The Rboh gene refers to Rboh subtype genes related to plant pollen development, and can be one or more Rboh subtype genes or a combination of a plurality of subtype genes.

The plant source of the Rboh gene or protein encoded thereby suitable for use in the present invention is not particularly limited and may be derived from any plant variety, and it is well known to those skilled in the art that the Rboh family in different crops contains varying numbers of subtype genes, preferably the Rboh gene of the present invention is a Rboh subtype gene which is related to plant male organ (e.g.anther, pollen) development or which is specifically expressed during male organ development or in a tissue related to male organ. Representative plant and functional genes include, but are not limited to:

OsRBOHA, osRBOHB, osRBOHE of rice, or a combination thereof; leRBOHE, leRBOHB, leRBOH of tomato, or a combination thereof; atRBOHE of Arabidopsis thaliana; preferably, the RboH gene comprises OsRBOHA, osRBOHB, osRBOHE of rice, or a combination thereof; leRBOHE, leRBOHB, leRBOH from tomato, or a combination thereof.

In a preferred embodiment, the protein sequence of OsRBOHA is shown as SEQ ID NO. 1, the protein sequence of OsRBOHB is shown as SEQ ID NO. 2, the protein sequence of OsRBOHE is shown as SEQ ID NO. 3, the protein sequence of AtRBOHE is shown as SEQ ID NO. 4, the protein sequence of LeRBOHE is shown as SEQ ID NO. 5, the protein sequence of LeRBOHE is shown as SEQ ID NO. 6, and the protein sequence of LeRBOHE is shown as SEQ ID NO. 7;

The nucleotide sequence of OSRBOHA is shown as SEQ ID NO. 8, the nucleotide sequence of OSRBOHB is shown as SEQ ID NO. 9, the nucleotide sequence of OSRBOHB is shown as SEQ ID NO.10, the nucleotide sequence of LeRBOHB is shown as SEQ ID NO. 11, the nucleotide sequence of LeRBOHI is shown as SEQ ID NO. 12, the nucleotide sequence of LeRBOH is shown as SEQ ID NO. 13, and the nucleotide sequence of AtRBOHI is shown as SEQ ID NO. 14.

As used herein, the terms "Rboh gene of the invention", "gene of NADPH oxidase", "gene of respiratory burst oxidase" are used interchangeably and refer to a Rboh gene derived from a plant (e.g. arabidopsis, rice, tomato) or a variant thereof. In a preferred embodiment, the nucleotide sequence of the Rboh gene of the invention is set forth in SEQ ID NO. 8-14. Variants of the gene may be obtained by random or site-directed mutagenesis or the like by insertion, substitution or deletion of nucleotides.

The invention also includes nucleic acids having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology to the preferred gene sequences of the invention (SEQ ID NO.: 8-14), which nucleic acids are also effective in regulating traits in plants (e.g., arabidopsis, rice, tomato) such as growing plant sterile lines by reducing expression or activity of the gene or its encoded protein. "homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids in terms of percentage of positional identity.

In the present invention, the nucleotide sequence of SEQ ID Nos. 8-14 may be substituted, deleted or added by one or more (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotides, and several (usually 60 or less, preferably 30 or less, more preferably 10 or less, most preferably 5 or less) nucleotides at the 5 'and/or 3' end, resulting in a derivative sequence of SEQ ID Nos. 8-14, which, due to the degeneracy of the codons, substantially encodes the amino acid sequence as shown in SEQ ID Nos. 1-7, even though the homology to SEQ ID Nos. 8-14 is low.

In addition, the meaning of "the nucleotide sequence in SEQ ID NO. 8-14 is substituted, deleted or added with at least one nucleotide derivative sequence" also includes nucleotide sequences which hybridize under moderately stringent conditions, more preferably under highly stringent conditions, to the nucleotide sequences shown in SEQ ID NO. 8-14. These variants include (but are not limited to): deletions, insertions and/or substitutions of several (typically 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotides, and additions of several (typically within 60, more preferably within 30, more preferably within 10, most preferably within 5) nucleotides at the 5 'and/or 3' end.

It is to be understood that although the genes provided in the examples of the present application are derived from Arabidopsis, rice, tomato, gene sequences derived from other similar plants (especially plants belonging to the same family or genus as Arabidopsis, rice or tomato or plants belonging to other families or genera having a relatively high homology to Arabidopsis, rice or tomato) having a certain homology (preferably, sequences as shown in SEQ ID Nos. 8-14) to the sequences of the present application, such as gene sequences of Rboh having more than 50%, such as 60%,70%,80%,85%,90%,95% or even 98% sequence identity, are also included in the scope of the present application, as long as the sequences can be conveniently isolated from other plants by the information provided according to the present application after reading the present application, methods and means for aligning sequence identity are also well known in the art, such as BLAST.

The polynucleotides of the application may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or synthetic DNA, which may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence set forth in SEQ ID No. 8-14 or a degenerate variant.

Polynucleotides encoding the mature polypeptide include coding sequences encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences. The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polyglycosides or polypeptides having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant such as 50% (v/v) citalopram, 0.1% calf serum/0.1% Ficoll,42 ℃ and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

The full-length sequence of the Rboh nucleotide or a fragment thereof of the present invention can be generally obtained by PCR amplification, recombinant or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available DNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. It is usually cloned into a vector, transferred into a cell, and then isolated from the proliferated host cell by a conventional method to obtain the relevant sequence.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

Polypeptides encoded by Rboh genes

As used herein, the terms "polypeptide of the invention", "protein encoded by a Rboh gene", and interchangeably refer to polypeptides of plant origin and variants thereof. In a preferred embodiment, a typical amino acid sequence of a polypeptide of the invention is shown in SEQ ID NO. 1-7.

The invention relates to a Rboh polypeptide for regulating plant traits and a variant thereof, and in a preferred embodiment of the invention, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1-7. The polypeptide of the invention can effectively regulate and control the characters of plants (such as arabidopsis, rice or tomatoes) (such as by reducing the activity of Rboh protein, thereby cultivating a plant sterile line).

The invention also includes polypeptides or proteins having the same or similar function that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology to the sequences set forth in SEQ ID No. 1-7 of the invention.

The term "same or similar functions" mainly means: "control the traits of a plant or crop (e.g., arabidopsis, rice, or tomato) (e.g., by decreasing the activity of a Rboh protein, thereby breeding a plant sterile line)".

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments and analogs of the Rboh proteins that have Rboh protein activity. As used herein, the terms "fragment" and "analog" refer to polypeptides that retain substantially the same biological function or activity of the native Rboh proteins of the invention.

The polypeptide fragment, derivative or analogue of the invention may be: (i) Polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, which may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent in one or more amino acid residues; or (iii) a polypeptide formed by fusion of the mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide (such as a leader or secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein) formed by fusing an additional amino acid sequence to the polypeptide sequence. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the definitions herein.

In the present invention, the polypeptide variants are derived sequences of amino acid sequences as shown in SEQ ID No. 1-7, obtained by substitution, deletion or addition of at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal end. For example, in such proteins, substitution with similar or similar amino acids will not generally alter the function of the protein, nor will addition of one or more amino acids at the C-terminus and/or/terminus. These conservative variations are best made by making substitutions according to table 1.

TABLE 1

The invention also includes analogs of the claimed proteins. The difference between these analogs and the native SEQ ID No. 1-7 may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (typically without altering the primary structure) forms include: chemically derivatized forms of proteins such as ethylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those that are glycosylation modified during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

Plant sterile line and cultivation method thereof

The invention also provides a method for cultivating the plant sterile line, which comprises the following steps: reducing expression or activity of an Rboh gene or a protein encoded thereby in said plant.

The method of the invention further comprises controlling the ambient temperature during plant growth. At least during the anther development period, preferably the plant flowering period, the maximum temperature of the growing environment is greater than or equal to 25 ℃, preferably greater than or equal to 28 ℃, preferably greater than or equal to 29 ℃, preferably greater than or equal to 30 ℃, preferably greater than or equal to 31 ℃, preferably greater than or equal to 32 ℃, preferably greater than or equal to 33 ℃, preferably greater than or equal to 34 ℃, preferably greater than or equal to 35 ℃. Alternatively, during the plant anther development period, preferably the flowering period, the growth environment temperature fluctuation range is controlled to be 12 ℃ to 35 ℃, preferably 16 ℃ to 30 ℃, more preferably 18 ℃ to 30 ℃, still more preferably 20 ℃ to 30 ℃.

The plant sterile line refers to a plant line with normal female organ development and abnormal male organ (such as anther and pollen) function. Preferably, the plant sterile line is high temperature sensitive male sterile, i.e. the male gamete development of the plant is affected by the ambient temperature. Growing under high temperature conditions, the male gametes become inactive or inactive and unable to pollinate normally. The plant sterile line (or material) comprises plant cells, tissues, propagation materials, seedlings, mature plants and the like.

In a preferred embodiment, the Rboh proteins of the invention may comprise the sequence of any of the amino acids shown in SEQ ID NOs 1 to 7. However, it is not limited thereto, since the amino acid sequence of the protein may be different depending on the kind or variety of the plant. In other words, it may be a mutant protein or an artificial variant whose amino acid sequence comprises one or several amino acid substitutions, deletions, insertions or additions at one or more positions of the amino acid sequence shown in any of SEQ ID NOS: 1-7, as long as it contributes to the cultivation of a plant sterile line by attenuating the activity of the protein. The "several" herein may vary depending on the position or type of the three-dimensional structure of the amino acid residues in the protein, but especially means 2-20, especially 2-10, more especially 2-5. Furthermore, substitution, deletion, insertion, addition, or inversion of amino acids include those caused by artificial variants or natural mutations, depending on the individual or species of the plant.

The activity of the Rboh proteins of the invention can be reduced (attenuated) by: 1) a partial or complete deletion of a polynucleotide encoding the protein, or substitution of one or more nucleotides or nucleotide fragments, or insertion of one or more nucleotides or nucleotide fragments, 2) modification of an expression control sequence to reduce expression of the polynucleotide, 3) modification of a sequence on a chromosome, or 4) a combination thereof.

In the above, partial or complete deletion of the polynucleotide encoding the protein can be carried out by replacing the polynucleotide encoding the endogenous target protein with a polynucleotide in which a marker gene or a partial nucleotide sequence is deleted, using a vector in which a chromosomal gene is inserted. The length of the "partial" deletion may vary depending on the type of polynucleotide, but is in particular from 2bp to 300bp, more particularly from 2bp to 100bp, more particularly from 1bp to 5bp. Substitution of single or multiple nucleotides can be achieved using mutation techniques and insertion or substitution of nucleotide fragments can be achieved using homologous recombination techniques.

Expression control sequences may also be modified to reduce polynucleotide expression by: mutations are induced in the expression control sequences by deletions, insertions, conservative or non-conservative substitutions or combinations thereof of nucleotide sequences to further attenuate the activity of the expression control sequences or to replace the expression control sequences with less active sequences. Expression control sequences include sequences encoding promoters, operator sequences, ribosome binding sites and sequences which control transcription and translation termination.

In addition, polynucleotide sequences on chromosomes can be modified to attenuate protein activity by: mutations are induced in the sequence by deletions, insertions, conservative or non-conservative substitutions or combinations thereof of the nucleotide sequence to further attenuate the activity of the sequence, or the polynucleotide sequence is replaced with a modified sequence in order to obtain weaker protein activity.

The invention also relates to vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors or the coding sequences of the Rboh proteins of the invention, and methods for producing the polypeptides of the invention by recombinant techniques. The polynucleotide sequences of the present invention may be used to express or produce recombinant Rboh proteins by conventional recombinant DNA techniques (Science, 1984; 224:1431). Generally, there are the following steps: (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a Rboh protein of the invention, or with a recombinant expression vector comprising the polynucleotide; (2) host cells cultured in a suitable medium; (3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the Rboh protein polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding an Rboh protein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli. Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: coli, streptomyces and agrobacterium; fungal cells such as yeast; plant cells, and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformed plants may also be transformed using Agrobacterium transformation or gene gun transformation, for example leaf disk. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain plants with altered tolerance.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as a "gene chip") for analysis of differential expression of genes in tissues. Transcription products of GDSL protein can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for GDSL protein.

The invention further provides a method for restoring fertility of a plant sterile line according to the invention, comprising the step of controlling the ambient temperature of the sterile line propagation material during growth. At least during the anther development period, preferably the anther flowering period, the highest ambient temperature is < 25 ℃, preferably less than or equal to 24 ℃, preferably less than or equal to 23 ℃, preferably less than or equal to 22 ℃, preferably less than or equal to 21 ℃, preferably less than or equal to 20 ℃, or during the anther development period, preferably the anther flowering period, the fluctuation range of the growing ambient temperature is controlled between 12 ℃ and 24 ℃, preferably between 16 ℃ and 22 ℃, preferably between 18 ℃ and 22 ℃. Fertility restoration refers to the fact that in the growth process of propagation materials, male gametes can basically develop normally, and fertilize with female gametes (restorer lines) to produce seeds, and the seeds still maintain the high-temperature sterile characteristic.

The temperature range of the invention is the temperature fluctuation range in the growth process of crops, and the fluctuation mode can be instant heating or cooling or gradient slow heating or cooling. Preferably, it simulates or is equivalent to the natural temperature change range and law, and gradient heating and cooling is completed within a certain period of time, for example, heating or cooling is performed at 0.5-5 ℃ per hour, specifically, for example, 0.5 ℃ or 1 ℃ or 1.5 ℃ or 2 ℃ or 3 ℃ or 4 ℃, and the temperature change mode can be, for example, that six-point temperature in the morning is 16 ℃, 12-noon is heated to 22 ℃, 14-noon is cooled to 20 ℃, and six-point afternoon is cooled to 16 ℃. Preferably, the pollen is maintained at the highest temperature (25-35 ℃, preferably 26-32 ℃, more preferably 28-30 ℃) for 1-3 hours, preferably 2 hours, without affecting plant growth. The temperature range of the invention can fluctuate up and down by +/-5 ℃, preferably +/-3 ℃, without greatly affecting the technical effect of the invention. In addition, the low temperature and the high temperature which are suitable for different crops are different, based on the growth and development characteristics of the crops, the technical effect of the invention can be obtained by adjusting the temperature range of the invention up and down appropriately by a person skilled in the art, and the technical scheme obtained based on the growth characteristics of the different crops is also included in the scope of the invention.

The invention also provides a method for identifying a male sterile line or propagation material thereof, comprising the step of detecting expression or activity of a Rboh gene or protein thereof. The propagation material includes callus, roots, stems, leaves, flowers, seeds, etc. The identification method can be a gene and protein detection method commonly used in the field, including Northern hybridization, RT-PCR (reverse transcribed PCR), biochemical reaction detection method, immunological detection method and the like.

The main advantages of the invention include:

(a) A method for creating a plant sterile line by reducing expression or activity of an Rboh gene or a protein encoded by the Rboh gene in a plant is provided, by which the diversity of male sterile resources can be greatly enriched.

(b) The invention provides a method for transforming the characteristics of sterile plants into fertility by culturing the sterile line under the low-temperature condition, so that the sterile line can be maintained, and the production cost of seeds is reduced.

(c) The obtained male sterile plant can be used as a female parent in two-line hybrid seed production, and can maintain hybrid vigor in hybrid breeding.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples were all commercially available products.

Example 1 Effect of OsRBOH knockout on Rice development

1. Design of Gene editing sites

Designing a gene editing vector for simultaneously knocking out OsRBOHA, osRBOHB, osRBOHE, respectively selecting target sites of OsRBOHA, osRBOHB, osRBOHE genes, and designing sgRNA sequences aiming at the target sites:

OsRBOHA：TCCGACTTCGCCGAATGCAT(SEQ ID NO.:15)

OsRBOHB：GCAATCTGGGCTCGAGCAAC(SEQ ID NO.:16)

OsRBOHE：GGTGAGGCAGTTCGCGTCGA(SEQ ID NO.:17)

2. gene editing vector construction

A) The target fragment was amplified using the SF55-scaffold-tRNA plasmid as template and the forward/reverse primer tRNA-gRBOHA-F/tRNA-RBOHB-R to give PCR product (length about 230bp, primer annealing temperature 58).

The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 98℃for 30 sec, annealing at 58℃for 30 sec, extension at 72℃for 10s,35 cycles, extension at 72℃for 5 min

B) The target fragment was amplified with the forward/reverse primer gRBOHB-gRNA-F/tRNA-gRBOHE-gRNA-R using the SF55-scaffold-tRNA plasmid as template to obtain PCR product (length about 230bp, primer annealing temperature 58).

The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 98℃for 30 sec, annealing at 58℃for 30 sec, extension at 72℃for 10s,35 cycles, extension at 72℃for 5 min

C) CRISPR-Cas9 vector (35S-OsU 6-tRNA) was digested with BsaI

D) Respectively carrying out gel running recovery on each segment

E) Homologous recombination is carried out on cloned fragments and vector enzyme digestion recovery fragments

The PCR reaction conditions were: 50 ℃ for 30min

F) E.coli was transformed and the monoclonal M13F/T6-R sequencing was chosen to verify that the fragment was successfully ligated into the vector.

3. Genetic transformation

(A) The above constructed plasmid was transformed directly into agrobacterium EHA105:

(1) Adding plasmid DNA into Agrobacterium competent cells, ice-bathing for 30min, placing into liquid nitrogen for 5min, immediately placing into 37 deg.C water bath for 5min, and placing on ice for 5min

(2) Taking out the centrifuge tube, adding 700ul of YEP culture medium, and shake culturing for 2-4 hr.

(3) And (3) taking out the bacterial liquid and a coating plate on a YEP culture medium flat plate containing corresponding antibiotics, and culturing the bacterial liquid and the YEP culture medium flat plate in an incubator in an inverted mode, wherein colonies are visible about 2 days.

(B) Transgenic rice:

(1) Callus induction, soaking and sterilizing the shelled seeds with NaClO, washing with sterile water, inoculating into NB induction medium, and culturing in an incubator for 10-15 days.

(2) And (3) subculturing the callus, cutting the induced callus by a single-sided knife, and placing the callus into a subculture medium for culture under the same conditions.

(3) Agrobacteria dip-dyeing and screening resistant callus, propagating agrobacteria EH105 strain transferred into target carrier, and soaking callus with good state.

(4) Sucking out or pouring out the bacterial liquid, and placing the callus in a box for dark culture for 48-72h.

(5) The callus after the end of co-culture was rinsed by immersing in a Carbenicillin-resistant sterile water to remove Agrobacterium.

(6) The callus was blotted dry and inoculated onto a screening medium containing antibiotics and incubated with light for two weeks.

(7) Callus differentiation culture, namely selecting callus with vigorous growth (resistant callus) and transferring the callus to a differentiation culture medium containing antibiotics. Most of the calli grow rapidly within a week, and the greenish calli differentiate into seedlings rapidly when the calli indicate green spots.

4. Plant culture and mutant screening

(1) Transferring the differentiated robust seedlings to a rooting culture medium containing antibiotics for rooting culture for one week, performing seedling training at room temperature for 2-3 days, performing greenhouse substrate culture for 15-20 days, and transplanting into a field.

(2) And taking leaves of each plant, extracting genome DNA, and designing primers at two sides of a target site. The amplified fragments were subjected to Sanger sequencing to determine the genotype of each plant.

(3) Detecting OsRBOHA, osRBOHB, osRBOHE gene mutation type, screening mutation type knocked out OsRBOHA, osRBOHB, osRBOHE gene in T0 generation, and continuously propagating for 4-5 generation to continuously increase mutation type population.

(4) The mutants were treated at different temperatures and fertility of the mutants was observed at high temperatures.

5. Experimental results

Under the high temperature condition, the pollen of the mutant rice is abortive relative to the wild type, and normal pollination is not possible.

Example 2 Effect of knockout of LeRBOHE, leRBOHB, leRBOH Gene on tomato development

1. Gene editing site design

Designing a gene editing vector for simultaneously knocking out LeRBOHE, leRBOHB, leRBOH, respectively selecting target sites of LeRBOHE, leRBOHB, leRBOH genes, and designing sgRNA sequences aiming at the target sites:

LeRBOHE：TCTAGCAAGTAATCCGTCTT(SEQ ID NO.:18)

LeRBOHB：TAAGCACAACCACTGTCGAC(SEQ ID NO.:19)

LeRBOH：TAGCTAGCAAGCTCGAAAAG(SEQ ID NO.:20)

2. construction of CRISPR-Cas9 Gene editing tools

A) The target fragment was amplified with the forward/reverse primer gRBOHE-F/gRBOHB-R using the SF55-scaffold-tRNA plasmid as template to obtain a PCR product (length about 230bp, primer annealing temperature 58).

The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 98℃for 30 sec, annealing at 58℃for 30 sec, extension at 72℃for 10s,35 cycles, extension at 72℃for 5 min

B) The target fragment was amplified using the scafold-tRNA plasmid as template and the forward/reverse primer gRBOHB-F/gRBOH-R to give PCR product (length about 230bp, primer annealing temperature 58).

The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 98℃for 30 sec, annealing at 58℃for 30 sec, extension at 72℃for 10s,35 cycles, extension at 72℃for 5 min

C) CRISPR-Cas9 vector (35S-AtU 6-tRNA) was digested with BsaI

D) Respectively carrying out gel running recovery on each segment

E) Homologous ligation of cloned fragments and vector cleavage recovery fragments

The PCR reaction conditions were: 50 ℃ for 30min

F) E.coli was transformed and the monoclonal M13F/T6-R sequencing was chosen to verify that the fragment was successfully ligated into the vector.

3. Genetic transformation of vectors

A) The above constructed plasmid was transformed directly into agrobacterium EHA105:

(1) Plasmid DNA is added into the agrobacteria competent cells, then the agrobacteria competent cells are subjected to ice bath for 30min, placed into liquid nitrogen for 5min, then immediately placed into a water bath kettle at 37 ℃ for 5min, and placed on ice for 5min.

(2) Taking out the centrifuge tube, adding 700ul of YEP culture medium, and shake culturing for 2-4 hr.

(3) And (3) taking out the bacterial liquid and a coating plate on a YEP culture medium flat plate containing corresponding antibiotics, and culturing the bacterial liquid and the YEP culture medium flat plate in an incubator in an inverted mode, wherein colonies are visible about 2 days.

B) Tomato transgene:

(1) Aseptic seedling obtaining: sterilizing with 75% ethanol for 30-60s, washing with sterile water for 1-3 times, sterilizing with NaClO (effective chlorine 1%) for 15min, and washing with sterile water for 5 times. Seed soaking with sterile water for 6 hr, and sowing and seed culture medium. Dark culture at 25℃for 2-3d, and then transfer to light for culture.

(2) Pretreatment of explants: when the cotyledon is fully unfolded and the first true leaf is slightly exposed, the cotyledon is cut into 5mm square leaves, and the leaves are placed in a preculture medium and are subjected to dark culture at 25 ℃ for 2d.

(3) And (3) agrobacterium activation and bacterial liquid preparation: shaking culture at 28deg.C until OD600 = 0.6-0.8. After centrifugation, the cells were resuspended with an equal volume of the invasion solution.

(4) Infection and co-cultivation: after 15min of infection, the excess bacterial liquid is sucked by sterile filter paper and placed on a co-culture medium for 2d of dark culture at 25 ℃.

(5) Screening and culturing: cotyledons were transferred to A2 resistant medium. The first 2 times of subculture are performed once every 5d or so, and the later times of subculture are performed once every 14d or so.

(6) And (3) differentiation culture: after callus formation, it was transferred to A3 medium to induce sprouting. And subculturing every 14d or so.

4. Plant culture and mutant screening

(1) When the differentiated seedlings grow to 1-2cm, cutting the seedlings, and transferring the seedlings into a rooting culture medium. Sampling and detecting after the seedlings grow out of roots.

(2) And taking leaves of each plant, extracting genome DNA, and designing primers at two sides of a target site. The amplified fragments were subjected to Sanger sequencing to determine the genotype of each plant.

(3) Detecting LeRBOHE, leRBOHB, leRBOHA gene mutation types, screening mutation types knocked out LeRBOHE, leRBOHB, leRBOHA in the T0 generation, and continuously propagating the mutation types through 4-5 generations to continuously increase mutation type populations.

(4) The mutants were treated at different temperatures and fertility of the mutants was observed at high temperatures.

5. Experimental results

Under the high temperature condition, pollen of the mutant tomato is abortive relative to wild type, and normal pollination is not possible.

Example 3 Effect of Arabidopsis rboh mutations on plant development

Obtaining a functional deletion mutant rbohe caused by the T-DNA insertion of an arabidopsis respiratory burst peroxidase gene, and processing under different temperature conditions to obtain a temperature-sensitive male sterile plant.

The T-DNA insertion mutant used in this study, rbohe (SALK_ 150096C), was obtained from ABRC with a background of Col-0 ecotype.

The planting method of the arabidopsis materials in the experiment is as follows:

(1) Arabidopsis thaliana Col-0 and mutant rbohe seed surface were sterilized and plated on 1/2MS medium. Transferring to a 20-22 ℃ long illumination (16 h) bar incubator for 4-5d at the temperature of 4 ℃ below Wen Chunhua d.

(2) Transplanting the seedlings in the culture dish into the green manure-poured soil, transferring the seedlings into a greenhouse for culturing, moisturizing the seedlings by using a plastic film, and simultaneously cutting a few ventilation holes on the plastic film.

(3) Removing the plastic film for about one week, and watering nutrient solution once every week for bolting the plants.

(4) Placing a part of Col-0 and mutant rbohe which are about to be bolting in a variable-temperature illumination incubator, wherein the set temperature is as follows:

Time	temperature (temperature)	Lighting device
			9:00	22℃	Illumination of
12:00	26℃	Illumination of
			14:00	30℃	Illumination of
16:00	26℃	Illumination of
			19:00	22℃	Illumination of
22:00-9：00	18℃	Darkness

(5) The rest Arabidopsis thaliana is placed in an illumination bar incubator at 20-22 ℃ for continuous growth.

(6) Alexander staining observed pollen development: taking Arabidopsis anther (bud dew white period) which is not dispersed, immersing in Alexander dye solution, standing at room temperature for 4-8 hours, and observing and photographing by a microscope.

(7) The treatment was continued at different temperatures until the setting rate was observed after setting of Arabidopsis.

5. Experimental results

(1) Arabidopsis rbohe mutant plants were unable to seed under continuously varying temperature conditions of 18℃to 30℃and were able to seed normally when the temperature was reduced to a temperature of 20℃to 22℃while wild type plants were able to seed normally under both conditions (see FIG. 1).

(2) Alexander staining showed that Arabidopsis rbohe mutants failed to produce normal pollen at continuously varying temperature swings of 18℃to 30℃and that normal pollen was produced when the temperature was reduced to a temperature swing of 20℃to 22 ℃.

(3) The scanning electron microscope result shows that the arabidopsis rbohe mutant can produce normal pollen under the temperature changing condition of 18-30 ℃ and the temperature is reduced to 20-22 ℃ when the pollen is aborted under the temperature changing condition of continuous change (see figure 3).

Conclusion of the experiment

The research of the invention finds that the respiratory burst peroxidase gene RBOH specifically expressed in the plant anther is reduced, inhibited or knocked out, so that the plant can be rendered male sterile under the high temperature condition, and the plant can be used as a parent to prepare hybrid seeds. Can restore fertility at low temperature and normally seed.

Sequence listing

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Ile Leu Arg Phe Phe Arg Ser Gly Ser Tyr Ser Val Arg Leu Leu Lys

610 615 620

Val Ala Ile Tyr Pro Gly Asn Val Leu Thr Leu Gln Met Ser Lys Pro

625 630 635 640

Pro Thr Phe Arg Tyr Lys Ser Gly Gln Tyr Met Phe Val Gln Cys Pro

645 650 655

Ala Val Ser Pro Phe Glu Trp His Pro Phe Ser Ile Thr Ser Ala Pro

660 665 670

Gly Asp Asp Tyr Leu Ser Ile His Val Arg Gln Leu Gly Asp Trp Thr

675 680 685

Arg Glu Leu Lys Arg Val Phe Ala Ala Ala Cys Glu Pro Pro Ala Gly

690 695 700

Gly Lys Ser Gly Leu Leu Arg Ala Asp Glu Thr Thr Lys Lys Ile Leu

705 710 715 720

Pro Lys Leu Leu Ile Asp Gly Pro Tyr Gly Ser Pro Ala Gln Asp Tyr

725 730 735

Ser Lys Tyr Asp Val Leu Leu Leu Val Gly Leu Gly Ile Gly Ala Thr

740 745 750

Pro Phe Ile Ser Ile Leu Lys Asp Leu Leu Asn Asn Ile Ile Lys Met

755 760 765

Glu Glu Glu Glu Asp Ala Ser Thr Asp Leu Tyr Pro Pro Met Gly Arg

770 775 780

Asn Lys Pro His Val Asp Leu Gly Thr Leu Met Thr Ile Thr Ser Arg

785 790 795 800

Pro Lys Lys Ile Leu Lys Thr Thr Asn Ala Tyr Phe Tyr Trp Val Thr

805 810 815

Arg Glu Gln Gly Ser Phe Asp Trp Phe Lys Gly Val Met Asn Glu Ile

820 825 830

Ala Asp Leu Asp Gln Arg Asn Ile Ile Glu Met His Asn Tyr Leu Thr

835 840 845

Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala Leu Ile Thr Met Leu

850 855 860

Gln Ala Leu Asn His Ala Lys Asn Gly Val Asp Ile Val Ser Gly Thr

865 870 875 880

Lys Val Arg Thr His Phe Ala Arg Pro Asn Trp Arg Lys Val Leu Ser

885 890 895

Lys Ile Ser Ser Lys His Pro Tyr Ala Lys Ile Gly Val Phe Tyr Cys

900 905 910

Gly Ala Pro Val Leu Ala Gln Glu Leu Ser Lys Leu Cys His Glu Phe

915 920 925

Asn Gly Lys Cys Thr Thr Lys Phe Glu Phe His Lys Glu His Phe

930 935 940

<210> 2

<211> 905

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Ala Asp Leu Glu Ala Gly Met Val Ala Ala Ala Thr Asp Gln Gly

1 5 10 15

Asn Ser Thr Arg Ser Gln Asp Asp Ala Ala Thr Leu Ile Pro Asn Ser

20 25 30

Gly Asn Leu Gly Ser Ser Asn Arg Ser Thr Lys Thr Ala Arg Phe Lys

35 40 45

Asp Asp Asp Glu Leu Val Glu Ile Thr Leu Asp Val Gln Arg Asp Ser

50 55 60

Val Ala Ile Gln Glu Val Arg Gly Val Asp Glu Gly Gly Ser Gly His

65 70 75 80

Gly Thr Gly Phe Asp Gly Leu Pro Leu Val Ser Pro Ser Ser Lys Ser

85 90 95

Gly Lys Leu Thr Ser Lys Leu Arg Gln Val Thr Asn Gly Leu Lys Met

100 105 110

Lys Ser Ser Ser Arg Lys Ala Pro Ser Pro Gln Ala Gln Gln Ser Ala

115 120 125

Lys Arg Val Arg Lys Arg Leu Asp Arg Thr Lys Ser Ser Ala Ala Val

130 135 140

Ala Leu Lys Gly Leu Gln Phe Val Thr Ala Lys Val Gly Asn Asp Gly

145 150 155 160

Trp Ala Ala Val Glu Lys Arg Phe Asn Gln Leu Gln Val Asp Gly Val

165 170 175

Leu Leu Arg Ser Arg Phe Gly Lys Cys Ile Gly Met Asp Gly Ser Asp

180 185 190

Glu Phe Ala Val Gln Met Phe Asp Ser Leu Ala Arg Lys Arg Gly Ile

195 200 205

Val Lys Gln Val Leu Thr Lys Asp Glu Leu Lys Asp Phe Tyr Glu Gln

210 215 220

Leu Thr Asp Gln Gly Phe Asp Asn Arg Leu Arg Thr Phe Phe Asp Met

225 230 235 240

Val Asp Lys Asn Ala Asp Gly Arg Leu Thr Ala Glu Glu Val Lys Glu

245 250 255

Ile Ile Ala Leu Ser Ala Ser Ala Asn Lys Leu Ser Lys Ile Lys Glu

260 265 270

Arg Ala Asp Glu Tyr Thr Ala Leu Ile Met Glu Glu Leu Asp Pro Thr

275 280 285

Asn Leu Gly Tyr Ile Glu Met Glu Asp Leu Glu Ala Leu Leu Leu Gln

290 295 300

Ser Pro Ser Glu Ala Ala Ala Arg Ser Thr Thr Thr His Ser Ser Lys

305 310 315 320

Leu Ser Lys Ala Leu Ser Met Lys Leu Ala Ser Asn Lys Glu Met Ser

325 330 335

Pro Val Arg His Tyr Trp Gln Gln Phe Met Tyr Phe Leu Glu Glu Asn

340 345 350

Trp Lys Arg Ser Trp Val Met Thr Leu Trp Ile Ser Ile Cys Ile Ala

355 360 365

Leu Phe Ile Trp Lys Phe Ile Gln Tyr Arg Asn Arg Ala Val Phe Gly

370 375 380

Ile Met Gly Tyr Cys Val Thr Thr Ala Lys Gly Ala Ala Glu Thr Leu

385 390 395 400

Lys Phe Asn Met Ala Leu Val Leu Leu Pro Val Cys Arg Asn Thr Ile

405 410 415

Thr Trp Ile Arg Ser Lys Thr Gln Val Gly Ala Val Val Pro Phe Asn

420 425 430

Asp Asn Ile Asn Phe His Lys Val Ile Ala Ala Gly Val Ala Val Gly

435 440 445

Val Ala Leu His Ala Gly Ala His Leu Thr Cys Asp Phe Pro Arg Leu

450 455 460

Leu His Ala Ser Asp Ala Gln Tyr Glu Leu Met Lys Pro Phe Phe Gly

465 470 475 480

Glu Lys Arg Pro Pro Asn Tyr Trp Trp Phe Val Lys Gly Thr Glu Gly

485 490 495

Trp Thr Gly Val Val Met Val Val Leu Met Ala Ile Ala Phe Thr Leu

500 505 510

Ala Gln Pro Trp Phe Arg Arg Asn Lys Leu Lys Asp Ser Asn Pro Leu

515 520 525

Lys Lys Met Thr Gly Phe Asn Ala Phe Trp Phe Thr His His Leu Phe

530 535 540

Val Ile Val Tyr Thr Leu Leu Phe Val His Gly Thr Cys Leu Tyr Leu

545 550 555 560

Ser Arg Lys Trp Tyr Lys Lys Thr Thr Trp Met Tyr Leu Ala Val Pro

565 570 575

Val Val Leu Tyr Val Ser Glu Arg Ile Leu Arg Leu Phe Arg Ser His

580 585 590

Asp Ala Val Gly Ile Gln Lys Val Ala Val Tyr Pro Gly Asn Val Leu

595 600 605

Ala Leu Tyr Met Ser Lys Pro Pro Gly Phe Arg Tyr Arg Ser Gly Gln

610 615 620

Tyr Ile Phe Ile Lys Cys Thr Ala Val Ser Pro Tyr Glu Trp His Pro

625 630 635 640

Phe Ser Ile Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His Ile

645 650 655

Arg Thr Arg Gly Asp Trp Thr Ser Arg Leu Arg Thr Val Phe Ser Glu

660 665 670

Ala Cys Arg Pro Pro Thr Glu Gly Glu Ser Gly Leu Leu Arg Ala Asp

675 680 685

Leu Ser Lys Gly Ile Thr Asp Glu Lys Ala Arg Phe Pro Lys Leu Leu

690 695 700

Val Asp Gly Pro Tyr Gly Ala Pro Ala Gln Asp Tyr Arg Glu Tyr Asp

705 710 715 720

Val Leu Leu Leu Ile Gly Leu Gly Ile Gly Ala Thr Pro Leu Ile Ser

725 730 735

Ile Val Lys Asp Val Leu Asn His Ile Gln Gly Glu Gly Ser Val Gly

740 745 750

Thr Thr Glu Pro Glu Ser Ser Ser Lys Ala Lys Lys Lys Pro Phe Met

755 760 765

Thr Lys Arg Ala Tyr Phe Tyr Trp Val Thr Arg Glu Glu Gly Ser Phe

770 775 780

Glu Trp Phe Arg Gly Val Met Asn Glu Val Ser Glu Lys Asp Lys Asp

785 790 795 800

Gly Val Ile Glu Leu His Asn His Cys Ser Ser Val Tyr Gln Glu Gly

805 810 815

Asp Ala Arg Ser Ala Leu Ile Val Met Leu Gln Glu Leu Gln His Ala

820 825 830

Lys Lys Gly Val Asp Ile Leu Ser Gly Thr Ser Val Lys Thr His Phe

835 840 845

Ala Arg Pro Asn Trp Arg Ser Val Phe Lys Lys Val Ala Val Ser His

850 855 860

Glu Asn Gln Arg Val Gly Val Phe Tyr Cys Gly Glu Pro Val Leu Val

865 870 875 880

Pro Gln Leu Arg Gln Leu Ser Ala Asp Phe Thr His Lys Thr Asn Thr

885 890 895

Arg Phe Asp Phe His Lys Glu Asn Phe

900 905

<210> 3

<211> 843

<212> PRT

<213> Rice (Oryza sativa)

<400> 3

Met Ala Ser Pro Tyr Asp His Gln Ser Pro His Ala Gln His Pro Ser

1 5 10 15

Gly Leu Pro Arg Pro Pro Gly Ala Gly Ala Gly Ala Ala Ala Gly Gly

20 25 30

Phe Ala Arg Gly Leu Met Lys Gln Pro Ser Arg Leu Ala Ser Gly Val

35 40 45

Arg Gln Phe Ala Ser Arg Val Ser Met Lys Val Pro Glu Gly Val Gly

50 55 60

Gly Met Arg Pro Gly Gly Gly Arg Met Thr Arg Met Gln Ser Ser Ala

65 70 75 80

Gln Val Gly Leu Arg Gly Leu Arg Phe Leu Asp Lys Thr Ser Gly Gly

85 90 95

Lys Glu Gly Trp Lys Ser Val Glu Arg Arg Phe Asp Glu Met Asn Arg

100 105 110

Asn Gly Arg Leu Pro Lys Glu Ser Phe Gly Lys Cys Ile Gly Met Gly

115 120 125

Asp Ser Lys Glu Phe Ala Gly Glu Leu Phe Val Ala Leu Ala Arg Arg

130 135 140

Arg Asn Leu Glu Pro Glu Asp Gly Ile Thr Lys Glu Gln Leu Lys Glu

145 150 155 160

Phe Trp Glu Glu Met Thr Asp Gln Asn Phe Asp Ser Arg Leu Arg Ile

165 170 175

Phe Phe Asp Met Cys Asp Lys Asn Gly Asp Gly Met Leu Thr Glu Asp

180 185 190

Glu Val Lys Glu Val Ile Ile Leu Ser Ala Ser Ala Asn Lys Leu Ala

195 200 205

Lys Leu Lys Gly His Ala Ala Thr Tyr Ala Ser Leu Ile Met Glu Glu

210 215 220

Leu Asp Pro Asp Asp Arg Gly Tyr Ile Glu Ile Trp Gln Leu Glu Thr

225 230 235 240

Leu Leu Arg Gly Met Val Ser Ala Gln Ala Ala Pro Glu Lys Met Lys

245 250 255

Arg Thr Thr Ser Ser Leu Ala Arg Thr Met Ile Pro Ser Arg Tyr Arg

260 265 270

Ser Pro Leu Lys Arg His Val Ser Arg Thr Val Asp Phe Val His Glu

275 280 285

Asn Trp Lys Arg Ile Trp Leu Val Ala Leu Trp Leu Ala Val Asn Val

290 295 300

Gly Leu Phe Ala Tyr Lys Phe Glu Gln Tyr Glu Arg Arg Ala Ala Phe

305 310 315 320

Gln Val Met Gly His Cys Val Cys Val Ala Lys Gly Ala Ala Glu Val

325 330 335

Leu Lys Leu Asn Met Ala Leu Ile Leu Leu Pro Val Cys Arg Asn Thr

340 345 350

Leu Thr Thr Leu Arg Ser Thr Ala Leu Ser His Val Ile Pro Phe Asp

355 360 365

Asp Asn Ile Asn Phe His Lys Val Ile Ala Ala Thr Ile Ala Ala Ala

370 375 380

Thr Ala Val His Thr Leu Ala His Val Thr Cys Asp Phe Pro Arg Leu

385 390 395 400

Ile Asn Cys Pro Ser Asp Lys Phe Met Ala Thr Leu Gly Pro Asn Phe

405 410 415

Gly Tyr Arg Gln Pro Thr Tyr Ala Asp Leu Leu Glu Ser Ala Pro Gly

420 425 430

Val Thr Gly Ile Leu Met Ile Ile Ile Met Ser Phe Ser Phe Thr Leu

435 440 445

Ala Thr His Ser Phe Arg Arg Ser Val Val Lys Leu Pro Ser Pro Leu

450 455 460

His His Leu Ala Gly Phe Asn Ala Phe Trp Tyr Ala His His Leu Leu

465 470 475 480

Val Leu Ala Tyr Val Leu Leu Val Val His Ser Tyr Phe Ile Phe Leu

485 490 495

Thr Arg Glu Trp Tyr Lys Lys Thr Thr Trp Met Tyr Leu Ile Val Pro

500 505 510

Val Leu Phe Tyr Ala Cys Glu Arg Thr Ile Arg Lys Val Arg Glu Asn

515 520 525

Asn Tyr Arg Val Ser Ile Val Lys Ala Ala Ile Tyr Pro Gly Asn Val

530 535 540

Leu Ser Leu His Met Lys Lys Pro Pro Gly Phe Lys Tyr Lys Ser Gly

545 550 555 560

Met Tyr Leu Phe Val Lys Cys Pro Asp Val Ser Pro Phe Glu Trp His

565 570 575

Pro Phe Ser Ile Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His

580 585 590

Ile Arg Thr Leu Gly Asp Trp Thr Thr Glu Leu Arg Asn Leu Phe Gly

595 600 605

Lys Ala Cys Glu Ala Gln Val Thr Ser Lys Lys Ala Thr Leu Ser Arg

610 615 620

Leu Glu Thr Thr Val Val Ala Asp Ala Gln Thr Glu Asp Thr Arg Phe

625 630 635 640

Pro Lys Val Leu Ile Asp Gly Pro Tyr Gly Ala Pro Ala Gln Asn Tyr

645 650 655

Lys Lys Tyr Asp Ile Leu Leu Leu Ile Gly Leu Gly Ile Gly Ala Thr

660 665 670

Pro Phe Ile Ser Ile Leu Lys Asp Leu Leu Asn Asn Ile Lys Ser Asn

675 680 685

Glu Glu Val Glu Ser Ile His Gly Ser Glu Ile Gly Ser Phe Lys Asn

690 695 700

Asn Gly Pro Gly Arg Ala Tyr Phe Tyr Trp Val Thr Arg Glu Gln Gly

705 710 715 720

Ser Phe Glu Trp Phe Lys Gly Val Met Asn Asp Val Ala Glu Ser Asp

725 730 735

His Asn Asn Ile Ile Glu Met His Asn Tyr Leu Thr Ser Val Tyr Glu

740 745 750

Glu Gly Asp Ala Arg Ser Ala Leu Ile Ala Met Val Gln Ser Leu Gln

755 760 765

His Ala Lys Asn Gly Val Asp Ile Val Ser Gly Ser Arg Ile Arg Thr

770 775 780

His Phe Ala Arg Pro Asn Trp Arg Lys Val Phe Ser Asp Leu Ala Asn

785 790 795 800

Ala His Lys Asn Ser Arg Ile Gly Val Phe Tyr Cys Gly Ser Pro Thr

805 810 815

Leu Thr Lys Gln Leu Lys Asp Leu Ser Lys Glu Phe Ser Gln Thr Thr

820 825 830

Thr Thr Arg Phe His Phe His Lys Glu Asn Phe

835 840

<210> 4

<211> 952

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 4

Met Lys Leu Ser Pro Leu Ser Phe Ser Thr Ser Ser Ser Phe Ser His

1 5 10 15

Ala Asp Gly Ile Asp Asp Gly Val Glu Leu Ile Ser Ser Pro Phe Ala

20 25 30

Gly Gly Ala Met Leu Pro Val Phe Leu Asn Asp Leu Ser Arg Asn Ser

35 40 45

Gly Glu Ser Gly Ser Gly Ser Ser Trp Glu Arg Glu Leu Val Glu Val

50 55 60

Thr Leu Glu Leu Asp Val Gly Asp Asp Ser Ile Leu Val Cys Gly Met

65 70 75 80

Ser Glu Ala Ala Ser Val Asp Ser Arg Ala Arg Ser Val Asp Leu Val

85 90 95

Thr Ala Arg Leu Ser Arg Asn Leu Ser Asn Ala Ser Thr Arg Ile Arg

100 105 110

Gln Lys Leu Gly Lys Leu Leu Arg Ser Glu Ser Trp Lys Thr Thr Thr

115 120 125

Ser Ser Thr Ala Gly Glu Arg Asp Arg Asp Leu Glu Arg Gln Thr Ala

130 135 140

Val Thr Leu Gly Ile Leu Thr Ala Arg Asp Lys Arg Lys Glu Asp Ala

145 150 155 160

Lys Leu Gln Arg Ser Thr Ser Ser Ala Gln Arg Ala Leu Lys Gly Leu

165 170 175

Gln Phe Ile Asn Lys Thr Thr Arg Gly Asn Ser Cys Val Cys Asp Trp

180 185 190

Asp Cys Asp Cys Asp Gln Met Trp Lys Lys Val Glu Lys Arg Phe Glu

195 200 205

Ser Leu Ser Lys Asn Gly Leu Leu Ala Arg Asp Asp Phe Gly Glu Cys

210 215 220

Val Gly Met Val Asp Ser Lys Asp Phe Ala Val Ser Val Phe Asp Ala

225 230 235 240

Leu Ala Arg Arg Arg Arg Gln Lys Leu Glu Lys Ile Thr Lys Asp Glu

245 250 255

Leu His Asp Phe Trp Leu Gln Ile Ser Asp Gln Ser Phe Asp Ala Arg

260 265 270

Leu Gln Ile Phe Phe Asp Met Ala Asp Ser Asn Glu Asp Gly Lys Ile

275 280 285

Thr Arg Glu Glu Ile Lys Glu Leu Leu Met Leu Ser Ala Ser Ala Asn

290 295 300

Lys Leu Ala Lys Leu Lys Glu Gln Ala Glu Glu Tyr Ala Ser Leu Ile

305 310 315 320

Met Glu Glu Leu Asp Pro Glu Asn Phe Gly Tyr Ile Glu Leu Trp Gln

325 330 335

Leu Glu Thr Leu Leu Leu Gln Arg Asp Ala Tyr Met Asn Tyr Ser Arg

340 345 350

Pro Leu Ser Thr Thr Ser Gly Gly Val Ser Thr Pro Arg Arg Asn Leu

355 360 365

Ile Arg Pro Arg His Val Val Gln Lys Cys Arg Lys Lys Leu Gln Cys

370 375 380

Leu Ile Leu Asp Asn Trp Gln Arg Ser Trp Val Leu Leu Val Trp Val

385 390 395 400

Met Leu Met Ala Ile Leu Phe Val Trp Lys Phe Leu Glu Tyr Arg Glu

405 410 415

Lys Ala Ala Phe Lys Val Met Gly Tyr Cys Leu Thr Thr Ala Lys Gly

420 425 430

Ala Ala Glu Thr Leu Lys Leu Asn Met Ala Leu Val Leu Leu Pro Val

435 440 445

Cys Arg Asn Thr Leu Thr Trp Leu Arg Ser Thr Arg Ala Arg Ala Cys

450 455 460

Val Pro Phe Asp Asp Asn Ile Asn Phe His Lys Ile Ile Ala Cys Ala

465 470 475 480

Ile Ala Ile Gly Ile Leu Val His Ala Gly Thr His Leu Ala Cys Asp

485 490 495

Phe Pro Arg Ile Ile Asn Ser Ser Pro Glu Gln Phe Val Leu Ile Ala

500 505 510

Ser Ala Phe Asn Gly Thr Lys Pro Thr Phe Lys Asp Leu Met Thr Gly

515 520 525

Ala Glu Gly Ile Thr Gly Ile Ser Met Val Ile Leu Thr Thr Ile Ala

530 535 540

Phe Thr Leu Ala Ser Thr His Phe Arg Arg Asn Arg Val Arg Leu Pro

545 550 555 560

Ala Pro Leu Asp Arg Leu Thr Gly Phe Asn Ala Phe Trp Tyr Thr His

565 570 575

His Leu Leu Val Val Val Tyr Ile Met Leu Ile Val His Gly Thr Phe

580 585 590

Leu Phe Phe Ala Asp Lys Trp Tyr Gln Lys Thr Thr Trp Met Tyr Ile

595 600 605

Ser Val Pro Leu Val Leu Tyr Val Ala Glu Arg Ser Leu Arg Ala Cys

610 615 620

Arg Ser Lys His Tyr Ser Val Lys Ile Leu Lys Val Ser Met Leu Pro

625 630 635 640

Gly Glu Val Leu Ser Leu Ile Met Ser Lys Pro Pro Gly Phe Lys Tyr

645 650 655

Lys Ser Gly Gln Tyr Ile Phe Leu Gln Cys Pro Thr Ile Ser Arg Phe

660 665 670

Glu Trp His Pro Phe Ser Ile Thr Ser Ala Pro Gly Asp Asp Gln Leu

675 680 685

Ser Val His Ile Arg Thr Leu Gly Asp Trp Thr Glu Glu Leu Arg Arg

690 695 700

Val Leu Thr Val Gly Lys Asp Leu Ser Thr Cys Val Ile Gly Arg Ser

705 710 715 720

Lys Phe Ser Ala Tyr Cys Asn Ile Asp Met Ile Asn Arg Pro Lys Leu

725 730 735

Leu Val Asp Gly Pro Tyr Gly Ala Pro Ala Gln Asp Tyr Arg Ser Tyr

740 745 750

Asp Val Leu Leu Leu Ile Gly Leu Gly Ile Gly Ala Thr Pro Phe Ile

755 760 765

Ser Ile Leu Lys Asp Leu Leu Asn Asn Ser Arg Asp Glu Gln Thr Asp

770 775 780

Asn Glu Phe Ser Arg Ser Asp Phe Ser Trp Asn Ser Cys Thr Ser Ser

785 790 795 800

Tyr Thr Thr Ala Thr Pro Thr Ser Thr His Gly Gly Lys Lys Lys Ala

805 810 815

Val Lys Ala His Phe Tyr Trp Val Thr Arg Glu Pro Gly Ser Val Glu

820 825 830

Trp Phe Arg Gly Val Met Glu Glu Ile Ser Asp Met Asp Cys Arg Gly

835 840 845

Gln Ile Glu Leu His Asn Tyr Leu Thr Ser Val Tyr Asp Glu Gly Asp

850 855 860

Ala Arg Ser Thr Leu Ile Lys Met Val Gln Ala Leu Asn His Ala Lys

865 870 875 880

His Gly Val Asp Ile Leu Ser Gly Thr Arg Val Arg Thr His Phe Ala

885 890 895

Arg Pro Asn Trp Lys Glu Val Phe Ser Ser Ile Ala Arg Lys His Pro

900 905 910

Asn Ser Thr Val Gly Val Phe Tyr Cys Gly Ile Gln Thr Val Ala Lys

915 920 925

Glu Leu Lys Lys Gln Ala Gln Asp Met Ser Gln Lys Thr Thr Thr Arg

930 935 940

Phe Glu Phe His Lys Glu His Phe

945 950

<210> 5

<211> 624

<212> PRT

<213> tomato (Lycopersicon esculentum)

<400> 5

Met Val Ser Ala Ser Ala Asn Lys Leu Ser Lys Leu Lys Glu Asn Ala

1 5 10 15

Gly Glu Tyr Ala Cys Leu Ile Met Glu Glu Leu Asp Pro Glu Asn Val

20 25 30

Gly Tyr Ile Glu Ile Trp Gln Leu Glu Thr Leu Leu Leu Gln Arg Asp

35 40 45

Asn Tyr Met Ser His Ser Thr Pro Leu Ser Thr Thr Thr Val Asp Trp

50 55 60

Ser Pro Asn Ile Gly Thr Ser Asn Ala Asn Asn Ile Val Lys Lys Ala

65 70 75 80

Ser Arg Thr Val Lys Cys Leu Val Leu Glu Asn Trp His Arg Gly Leu

85 90 95

Ile Ile Leu Leu Trp Leu Leu Ala Met Ile Gly Leu Phe Ile Trp Lys

100 105 110

Phe Met Gln Tyr Arg Lys Met Glu Ala Phe Gln Val Met Gly Tyr Cys

115 120 125

Leu Ala Thr Ala Lys Gly Ala Ala Glu Thr Leu Lys Phe Asn Met Ala

130 135 140

Leu Ile Leu Leu Pro Val Cys Arg Asn Met Leu Thr Arg Leu Arg Ser

145 150 155 160

Thr Arg Ala Arg Ile Leu Ile Pro Phe Asp Asp Asn Ile Asn Phe His

165 170 175

Lys Ile Ile Ala His Ala Ile Ala Val Ala Ile Ile Leu His Ala Ala

180 185 190

Asn His Leu Ala Cys Asp Phe Pro His Leu Val Asn Ser Ser Pro Glu

195 200 205

Lys Phe Ala Leu Ile Ala Ser Asp Phe Asp Lys Leu Lys Pro Ser Tyr

210 215 220

Ile Ser Leu Leu Thr Gly Val Glu Gly Ile Thr Gly Ile Ser Met Val

225 230 235 240

Ile Leu Met Thr Ile Ala Phe Ile Leu Ala Thr Arg Arg Phe Arg Arg

245 250 255

Ser Val Pro Lys Leu Pro Thr Pro Leu Asn Arg Leu Thr Gly Phe Asn

260 265 270

Ala Phe Trp Tyr Ser His His Leu Leu Ala Phe Val Tyr Ile Leu Leu

275 280 285

Leu Leu His Gly Thr Phe Leu Phe Phe Val His Lys Trp Tyr Gln Lys

290 295 300

Thr Thr Trp Met Tyr Ile Ser Ile Pro Leu Ile Leu Tyr Ile Ala Glu

305 310 315 320

Arg Thr Leu Arg Thr Trp Arg Ser Glu Asp Tyr Ala Val Lys Ile Leu

325 330 335

Lys Val Ser Val Leu Pro Gly Asp Val Leu Ser Leu Ile Met Ser Lys

340 345 350

Pro Ala Gly Phe Lys Tyr Lys Ser Gly Gln Tyr Ile Phe Leu Gln Cys

355 360 365

Pro Thr Ile Ser Ser Phe Glu Trp His Pro Phe Ser Ile Thr Ser Ala

370 375 380

Pro Gly Asp Asp Tyr Ile Ser Val Tyr Ile Arg Ile Val Gly Asp Trp

385 390 395 400

Thr Lys Glu Leu Lys Arg Val Phe Thr Glu Ile Pro Ala Cys Leu Val

405 410 415

Gly Arg Ala Lys Phe Glu Glu Gln Glu Asn Val Asp Gln Arg Gly Leu

420 425 430

Pro Arg Leu Leu Val Asp Gly Pro Tyr Gly Ala Pro Ala Gln Asp Tyr

435 440 445

Gln Asn Tyr Asp Val Leu Leu Leu Val Gly Leu Gly Ile Gly Ala Thr

450 455 460

Pro Phe Ile Ser Ile Leu Lys Asp Leu Leu Asn Asn Thr Arg Ala Asp

465 470 475 480

Glu Asn Val Asp Ser Asn Thr Glu Thr Ser Ala Ser Asp Asp Ser Trp

485 490 495

Thr Ser Phe Gly Ser Ser Ser Ser Ala Ser Ser Val Lys Lys Lys Ser

500 505 510

Gln Arg Ala Arg Ser Ala His Phe Tyr Trp Val Thr Arg Glu Pro Gly

515 520 525

Ser Leu Glu Trp Phe Lys Gly Val Met Asn Glu Val Ala Glu Met Asp

530 535 540

His Lys Gly Gln Ile Glu Met His Asn Tyr Leu Thr Ser Val Tyr Glu

545 550 555 560

Glu Gly Asp Ala Arg Ser Thr Leu Ile Thr Met Leu Gln Ala Leu Asn

565 570 575

His Ala Lys His Gly Val Asp Ile Leu Ser Gly Thr Lys Val Arg Thr

580 585 590

His Phe Ala Arg Pro Lys Trp Thr Glu Val Phe Lys Arg Ile Ala Leu

595 600 605

Lys His Pro Tyr Ser Thr Val Gly Lys Glu Phe Leu Gln Ser Gln Val

610 615 620

<210> 6

<211> 881

<212> PRT

<213> tomato (Lycopersicon esculentum)

<400> 6

Met Asn Glu Gly Ser Glu Arg Gly Glu Glu Glu Ala Pro Pro Asn Gly

1 5 10 15

Phe Leu Ala Arg Ser Ala Ser Ala Ala Ser Lys Leu Arg Arg Lys Phe

20 25 30

Ser Trp Ile Arg Ser Pro Ser Val Met Ser Arg Thr Ser Ala Ala Ala

35 40 45

Ser Glu Val Ser Asp Asp Asn Tyr Gln Leu His His Thr Ser Asn Thr

50 55 60

Leu Ser Ala Arg Glu Glu Met Lys Ser Lys Leu Lys Leu Val Arg Ser

65 70 75 80

Lys Ser Thr Ala Gln Arg Ala Leu Gly Gly Leu Arg Phe Ile Ser Lys

85 90 95

Thr Thr Gly Glu Ser Asp Thr Asn Val Leu Trp Lys Lys Val Glu Ala

100 105 110

Arg Phe Asp Ala Leu Ala Lys Asp Gly Leu Leu Ala Arg Glu Asp Phe

115 120 125

Gly Glu Cys Ile Gly Met Glu Asp Ser Lys Glu Phe Ala Val Gly Val

130 135 140

Phe Asp Ala Leu Ile Arg Arg Arg Arg Gln Lys Ala Ala Lys Ile Thr

145 150 155 160

Lys Ile Glu Leu His Asp Phe Trp Leu Gln Ile Ser Asp Gln Ser Phe

165 170 175

Asp Ala Arg Leu Gln Ile Phe Phe Asp Met Ala Asp Ser Asn Gly Asp

180 185 190

Gly Lys Ile Thr Arg Asp Glu Val Gln Glu Leu Ile Met Leu Ser Ala

195 200 205

Ser Ala Asn Lys Leu Ser Lys Leu Lys Glu Arg Ala Ala Glu Tyr Ala

210 215 220

Ser Leu Ile Met Glu Glu Leu Asp Pro Glu Cys Leu Gly Tyr Ile Glu

225 230 235 240

Leu Trp Gln Leu Glu Thr Leu Leu Leu Gln Arg Asp Asn Tyr Met Thr

245 250 255

Tyr Ser Arg Pro Leu Ser Thr Thr Ser Val Gly Trp Gly Gln Asn Leu

260 265 270

Gly Thr Leu Asn Lys Thr Lys Asn Leu Val Lys Arg Ala Ser Tyr Ala

275 280 285

Phe Lys Cys Leu Val Leu Asp Asn Trp Gln Arg Gly Trp Ile Leu Leu

290 295 300

Leu Trp Val Met Val Met Ala Val Leu Phe Thr Trp Lys Phe Leu Gln

305 310 315 320

Tyr Arg Gln Arg Ala Ala Phe Gln Val Met Gly Tyr Cys Leu Ala Thr

325 330 335

Ala Lys Gly Ala Ala Glu Thr Leu Lys Leu Asn Met Ala Leu Ile Leu

340 345 350

Leu Pro Val Cys Arg Asn Ile Leu Thr Trp Leu Arg Ser Thr Arg Ala

355 360 365

Lys Leu Leu Leu Pro Phe Asp Asp Asn Ile Asn Phe His Lys Val Lys

370 375 380

Cys Thr Gln Thr Glu Tyr Ile Val Gln His Pro Arg Ile Phe Leu Thr

385 390 395 400

Met Leu Phe Gln Leu Gln Ile Ile Ala Tyr Ala Ile Gly Val Gly Ile

405 410 415

Leu Leu His Ala Gly Asn His Leu Ala Cys Asp Phe Pro Arg Leu Ile

420 425 430

Asn Ser Ser Pro Glu Lys Phe Ala Leu Ile Ala Ser Asp Phe Asp Asn

435 440 445

Val Lys Pro Thr Tyr Lys Ser Leu Leu Thr Gly Ile Glu Gly Val Thr

450 455 460

Gly Ile Ala Met Val Ile Leu Met Ala Ile Val Phe Thr Leu Ala Thr

465 470 475 480

Arg Thr Phe Arg Arg Asn Val Leu Lys Leu Pro Pro Pro Phe Ser Arg

485 490 495

Leu Thr Gly Phe Asn Ala Phe Trp Tyr Ser His His Leu Leu Ala Val

500 505 510

Val Tyr Val Leu Leu Leu Val His Gly Thr Phe Leu Phe Leu Val His

515 520 525

Gln Trp Trp Gln Lys Thr Thr Trp Met Tyr Ile Ser Met Pro Leu Leu

530 535 540

Leu Tyr Val Ala Glu Arg Ser Leu Arg Thr Cys Arg Ser Glu His Tyr

545 550 555 560

Ala Ala Lys Ile Leu Lys Val Ser Val Leu Pro Gly Asp Val Phe Ser

565 570 575

Leu Thr Met Ser Lys Pro Asn Ser Phe Lys Tyr Lys Ser Gly Gln Tyr

580 585 590

Ile Phe Leu Gln Cys Pro Thr Ile Ser Ser Phe Glu Trp His Pro Phe

595 600 605

Ser Ile Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His Ile Arg

610 615 620

Met Val Gly Asp Trp Thr Asn Glu Leu Lys Arg Val Phe Thr Glu Asp

625 630 635 640

Asp Ser Ser Ala Cys Glu Ile Gly Arg Ala Lys Phe Arg Glu Arg Gly

645 650 655

Asn Val Asp Gln Arg Gly Leu Pro Arg Leu Leu Val Asp Gly Pro Tyr

660 665 670

Gly Ala Pro Ala Gln Asp Tyr Gln Asn Tyr Asp Val Leu Leu Leu Val

675 680 685

Gly Leu Gly Ile Gly Ala Thr Pro Phe Ile Ser Ile Leu Lys Asp Leu

690 695 700

Leu Asn Asn Ser Arg Ser Glu Glu Leu Asp Ser Thr Thr Glu Thr Ser

705 710 715 720

Ala Ser Asp Asp Ser Trp Thr Ser Leu Ala Ser Ser Ser Met Ala Ser

725 730 735

Thr Gly Lys Lys Lys Ser Leu Arg Thr Lys Ser Ala His Phe Tyr Trp

740 745 750

Val Thr Arg Glu Pro Gly Ser Phe Glu Trp Phe Lys Gly Val Met Asn

755 760 765

Glu Met Ala Glu Ile Asp His Lys Gly Leu Ile Glu Met His Asn Tyr

770 775 780

Leu Thr Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Thr Leu Ile Thr

785 790 795 800

Met Val Gln Ala Leu Asn His Ala Lys His Gly Val Asp Ile Leu Ser

805 810 815

Gly Thr Gln Val Arg Thr His Phe Ala Arg Pro Asn Trp Lys Glu Val

820 825 830

Phe Asn Lys Ile Ala Ser Lys His Pro Tyr Ser Thr Val Gly Val Phe

835 840 845

Tyr Cys Gly Leu Pro Ala Leu Ala Lys Glu Leu Lys Lys Leu Ser Gln

850 855 860

Glu Leu Thr Tyr Lys Thr Ser Thr Arg Phe Glu Phe His Lys Glu Tyr

865 870 875 880

Phe

<210> 7

<211> 865

<212> PRT

<213> tomato (Lycopersicon esculentum)

<400> 7

Met Glu Ile Glu Asn Thr Thr Asp Ser Val Arg Gly Ser Arg Val Gly

1 5 10 15

Phe Ser Gly Ser Leu Val Ser Gly Lys Lys Ser Ala Arg Phe Lys Asp

20 25 30

Asp Glu Ser Tyr Val Glu Ile Thr Leu Asp Val Arg Asp Asp Ser Val

35 40 45

Leu Val Gln Asn Ile Lys Gly Ala Asp His Glu Ala Ala Leu Leu Ala

50 55 60

Ser Lys Leu Glu Lys Arg Pro Asn His Thr Leu Gly Ser Gln Leu Ser

65 70 75 80

Phe His Leu Lys Gln Val Ser Lys Glu Leu Lys Arg Met Thr Ser Ser

85 90 95

Asn Lys Phe Gln Lys Ile Asp Arg Ser Lys Ser Gly Ala Ala Arg Ala

100 105 110

Leu Arg Gly Leu Gln Phe Met Asn Arg Asn Val Gly Thr Glu Gly Trp

115 120 125

Ser Glu Val Glu Ser Arg Phe Asp Gln Leu Ala Val Asp Gly Met Leu

130 135 140

Ala Lys Thr Leu Phe Gly Gln Cys Ile Gly Met Lys Glu Ser Ser Glu

145 150 155 160

Phe Ala Glu Glu Leu Phe Asp Ala Leu Ala Arg Lys Arg Cys Ile Thr

165 170 175

Ser Pro Ala Val Thr Lys Asp Glu Leu His Glu Phe Trp Glu Gln Ile

180 185 190

Thr Asp Thr Ser Phe Asp Ala Arg Leu Gln Thr Phe Phe Asp Met Val

195 200 205

Asp Lys Asp Ala Asp Gly Arg Ile Thr Glu Glu Glu Val Lys Glu Ile

210 215 220

Ile Ser Leu Ser Ala Ser Ala Asn Lys Leu Ser Lys Ile Glu Asp Asn

225 230 235 240

Ser Asp Glu Tyr Ala Ala Leu Ile Met Glu Glu Leu Asp Pro Gly Asn

245 250 255

Val Gly Tyr Ile Glu Leu Tyr Asn Leu Glu Thr Leu Leu Leu Gln Ala

260 265 270

Pro Ser His Ser Met Asn Leu Ser Thr Asn Ser Arg Val Leu Ser Gln

275 280 285

Met Leu Ser Gln Lys Leu Lys Pro Thr Lys Glu Arg Asn Pro Phe Lys

290 295 300

Arg Cys Lys Arg Arg Leu Asp Tyr Phe Ile Glu Asp Asn Trp Lys Arg

305 310 315 320

Ile Trp Val Met Val Leu Trp Leu Ser Ile Cys Ala Gly Leu Phe Thr

325 330 335

Trp Lys Phe Ile Gln Tyr Lys Arg Arg Ala Val Phe Asp Val Met Gly

340 345 350

Tyr Cys Val Ser Val Ala Lys Gly Gly Ala Glu Thr Thr Lys Phe Asn

355 360 365

Met Ala Leu Val Leu Leu Pro Val Cys Arg Asn Thr Ile Thr Trp Leu

370 375 380

Arg Ser Arg Thr Lys Leu Gly Lys Ile Ile Pro Phe Asp Asp Asn Ile

385 390 395 400

Asn Phe His Lys Val Ile Ala Phe Gly Val Ala Val Gly Val Gly Leu

405 410 415

His Ala Ile Ser His Leu Thr Cys Asp Phe Pro Arg Leu Leu His Ala

420 425 430

Thr Asp Glu Glu Tyr Glu Pro Met Lys Pro Phe Phe Gly Asp Glu Arg

435 440 445

Pro Asn Asn Tyr Trp Trp Phe Val Lys Gly Thr Glu Gly Trp Thr Gly

450 455 460

Val Val Met Val Val Leu Met Ile Ile Ala Tyr Val Leu Ala Gln Pro

465 470 475 480

Trp Phe Arg Arg Asn Arg Leu Asn Leu Pro Ser Thr Ile Lys Lys Leu

485 490 495

Thr Gly Phe Asn Ala Phe Trp Tyr Ser His His Leu Phe Val Ile Val

500 505 510

Tyr Val Leu Phe Ile Ile His Gly Tyr Phe Leu Tyr Leu Ser Lys Lys

515 520 525

Trp Tyr Lys Lys Thr Thr Trp Met Tyr Ile Ala Val Pro Met Ile Leu

530 535 540

Tyr Ala Cys Glu Arg Leu Leu Arg Ala Phe Arg Ser Gly Tyr Lys Ala

545 550 555 560

Val Arg Ile Leu Lys Val Ala Val Tyr Pro Gly Asn Val Met Ala Val

565 570 575

His Met Ser Lys Pro Gln Gly Phe Lys Tyr Thr Ser Gly Gln Tyr Ile

580 585 590

Phe Val Asn Cys Ser Asp Val Ser Ser Phe Gln Trp His Pro Phe Thr

595 600 605

Ile Ser Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His Ile Arg Thr

610 615 620

Leu Gly Asp Trp Thr Ser Gln Leu Lys Thr Leu Phe Ser Lys Val Cys

625 630 635 640

Glu Pro Pro Thr Gly Asp Gln Ser Gly Leu Leu Arg Ala Asp Ile Gly

645 650 655

Lys Ala Asp Tyr Lys Pro Arg Leu Pro Lys Leu Leu Ile Asp Gly Pro

660 665 670

Tyr Gly Ala Pro Ala Gln Asp Tyr Lys Lys Tyr Asp Val Val Leu Leu

675 680 685

Val Gly Leu Gly Ile Gly Ala Thr Pro Leu Ile Ser Ile Val Lys Asp

690 695 700

Val Leu Asn Asn Ile Asn Gln Gln Lys Asp Ile Glu Asp Gly Thr Lys

705 710 715 720

Gly Ser Lys Lys Ser Pro Phe Ala Thr Lys Arg Ala Tyr Phe Tyr Trp

725 730 735

Val Thr Arg Glu Gln Gly Ser Phe Glu Trp Phe Lys Gly Val Met Asp

740 745 750

Glu Val Ser Glu Asn Asp Gln Glu Gly Leu Ile Glu Leu His Asn Tyr

755 760 765

Cys Thr Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala Leu Ile Thr

770 775 780

Met Leu Gln Ser Ile His Gln Ala Lys Ser Gly Val Asp Ile Val Ser

785 790 795 800

Gly Thr Arg Val Lys Thr His Phe Ala Arg Pro Asn Trp Arg Gln Val

805 810 815

Phe Lys Arg Val Thr Ile Asn His Pro Asp Gln Lys Ile Gly Val Phe

820 825 830

Tyr Cys Gly Pro Gln Gly Leu Val Gly Glu Leu Arg His Leu Ser Gln

835 840 845

Asp Phe Ser His Lys Thr Asp Thr Lys Phe Glu Phe His Lys Glu Asn

850 855 860

Phe

865

<210> 8

<211> 12536

<212> DNA

<213> Rice (Oryza sativa)

<400> 8

tttgtgcttt tggtcgacct gtctgtcacc catatattgc tctcccgctg ctcttcctct 60

ttcctcggct cggctcgtct cctctcccgc tccgacgcgt cgcgagggcg ccttttccca 120

gagattccac atccatccag cccgcggccg gcgagcattt ggcccatcac gcgagctgga 180

gaggttggcc cggcggcggc ggcggtgatg aggggcggcg cctcctcggg accccagcga 240

tggggctcgg cggggacgac accgcggtcg ctgagcacgg gctcgtcgcc gcgcgggtcc 300

gacgaccgga gctccgacga cggggaggag ctggtcgagg tcacgctcga cctgcaggac 360

gacgacacca ttgtgcttcg gagcgtcgag cccgcggcgg cggcggcggc gggggtgggg 420

gcgggggcgg gggcggcgtc ggcgcggggg gagctcacgg gtggcccgtc gtcgtcgtcg 480

tcgcggtcga ggtcgccgtc gatccggagg agctcgtcgc accggctgct gcagttctcg 540

caggagctca aggcggaggc catggcccgg gcgcggcagt tctcgcagga cctgaccaag 600

cggttcggcc gcagccacag ccgcagcgaa gcgcaggcgc cgtcgggcct cgagtccgcg 660

ctcgccgccc gcgccgcgcg gcggcagcgc gcgcagctcg accgcacacg ctccggcgcc 720

cacaaggcgc tccgcggcct ccgcttcatc agcagcaaca aggccaacaa cgcctggatg 780

gaggtgcagg ccaacttcga ccgcctcgcc cgcgacggct acctctcccg ctccgacttc 840

gccgaatgca tcggtatccc tacccaccga tcgcccacca ccaccatttt tccccccacc 900

gaattgcttt ctcacgcagc atccgcgaca ttgtcgtcgt gcagggatga cggaatcgaa 960

ggagttcgcg ctcgagctgt tcgacacgct gagccggcga cgacagatga aggtggacac 1020

gattaacaag gatgaactcc gcgagatctg gcagcagatc accgataaca gcttcgactc 1080

ccgtctccaa atcttcttcg aaatgtaatt agctacccaa tccactatct ctgcaatttt 1140

gccattatta ccagtactac tagataaaca gagtatcaac tctgctaaat tttgtggcga 1200

ttttcgtctg catttttacc agggtggata agaacgcgga cggccggatt acggaggcgg 1260

aggtgaaaga ggtgagcaaa acataatcca cgcccatgtc tatgctgttc ctacgcttgg 1320

tggtccagca gtacgagttc ttctgtgctt tttttctcca ttctttttaa atttgctatt 1380

tcaggatatc aaatcgaata atttagaccg ttttgacaac cgtagttgta tatttggtcg 1440

attgagctgt cctcgccttg cttgggccac accaatattt gttttctccc tccaaaatat 1500

gcacacatct aagtcgcgat tcgcaacctt accggatagg attacttctt aattctgatt 1560

agagggaaga acagttctta attaacacgt aacaatccga taagagagga cattttagat 1620

cagaagcgta ctacgcaggg aaaggagaga ggcatgcagt acaaaggtac gagaaaagaa 1680

gaaaaaatgc ttggcttcca atcggagtcg gtgagcgaaa ccggcgggga aagcatcacg 1740

gcgacaccaa ctcctacctg ccagttttgg gacactcatt ttctttaact tttaggaaaa 1800

gccgaaaagg ggtacccttt ctagaacgtg tgagggcagt ttcggtaatc cggtgggctt 1860

cgtggcttac agaagccctg tgaagcggca gtaggtgagg ggcatttctg tcaaacccac 1920

taagggcaag ttttatagta gaggtggcca ccacatccaa tttcatgcat gacatatata 1980

attaaattaa gaggtgatat gtataataga ttacctctac tacacaatcc ccaatatctc 2040

taatactctc acatgtttat tgaagtttat ccccaatatc cccaatatcc cttgtatgag 2100

aggtgactat ttctctctca tccttgttct ctcctccacc tcactattta gtcccatgtg 2160

gcatcttagg gcttgtgctt ggatgcccat agtacttgcc ctaatgctct ctgatcagct 2220

caaacgatgc gttgacaaga tgagttataa agtagccttt taaaaaaaaa acgattcgct 2280

aattttttgc ctctcactga tgtcactaac agtgctgcgg agcaaattga gttggtaggg 2340

gtttttttgg ctgctctggg ttgctttctt gaggtttgga agatgttgga tcatgttgac 2400

tcagctcgtg tttctccttt cacctacgcg cagattatta tgttgagcgc gtctgccaat 2460

aaactgtcga ggcttaagga gcaagcagaa gagtacgccg ctttgatcat ggaggagctt 2520

gatcctgaag ggctcggcta cattgaggta agctcgatgt tcttaagcag ttaagctggt 2580

gtaaaccctc ataatgcgct tatatctgta atagttgtac tcatgcactg taaacccaca 2640

ctatttactt ccaccaaacc tttaggaata gttgactgaa gctgtctgtt ttgggatggt 2700

agtgctgtag tggggtagta ccagtagagc ctaattgatg acttcgtgca gttctatttt 2760

atctgtcaca ttgctccggt gatgggtgtc ccccaaccgg ccaagcgcgc ttgttatgat 2820

aattaaacaa aaatttgtcc gtcacagcgt caaggccaac tacttgcaaa gggacgcatc 2880

attcataaca aaaagatgat gctggtaccc cacaaacttc ttgcccaccc atcatcattt 2940

atatcgaagt tgttgagcag acgtagacca gtgtagctgt gtagcatcca tttcagtttg 3000

ggaacagttt cgcttcgctc ccctgcctaa ttcaatgcca tccttgctgc ccacagcttg 3060

ttgccgcacc atttacaaag gaatcattta caatgccaga aatgttccat cacggttcca 3120

tggctcttgg tcgtgcattg ctctgccttg cccgggtgca agggtaggga agctgatgta 3180

gtaactaata agcagggtag caagtactag ggatttagga accaggatta acccatgcgc 3240

tcatgtacta gtaggaatgt cgatcaaggg tgtttatctg gaccatgcag ccagtggctt 3300

tggtgattgc catccatgat ccatgttagg cctcatttgt tatggtttta gttcaccagg 3360

tgattcaagt cccacctttt ctagcagtgg gatagctaca acttcagttt ggtggggatt 3420

catatttttg tgcaaccgta ccaattagat cgccgtgtag tgttaacttg tctcgtactt 3480

tttttaccct catagtattt gaagtaaatt aactttacat gttcgataag ttgacgcttc 3540

tttttcaaaa gatagcaaga gataactagt tgacacaatt gtttcatttg agtcaacact 3600

gatttaatat ttgtttgtcc aaaatggtta gttaagcttg agaaagttca tgtacatatt 3660

tcatagggta atttcaggac acatttgcca atattagtaa agcagcaaac tgtaaattcg 3720

agtttctctt aaataaatta ttgaactatg aattatatac atctgaagaa aatgtttgat 3780

atcttcttga tgagaagcgt tgcatctatt cacaaatcac aagtaccatg tactgcatct 3840

ggtatgactg cacaactttt tacatgaaag tcctttcata tataaaaaag gaggtgtttt 3900

acaatgatct ctactagtag tgcaacattt tattttttgc cattgcagca taattctgtt 3960

gttatataat cttgatatta ttatgtattg gatggtgaaa attgatgtgc tgttgttgac 4020

atgctcatgt gtttcttgca aacaatttgt tgagctaatg gaagtgtgag gacgtcccaa 4080

agcctaggtg gtgcaacgct ccgaggcatg tcagcgcgaa atatctggat ggatgggttc 4140

tcaaataaag cccaaataaa tacggtaacc atccaacctc atcgcagacc ccgacatgtc 4200

ttgaagagtg cactacatag gctctaagcc atcggatatg aaatagaaga cacaactgtt 4260

ggaatagcca caccagtcat ctacgtcata tgcccgtcaa ccactcgatg gtggcgacaa 4320

aaaacatacc ccagagactc gatctcaaca gaggcatatt ccccggttat ctcaaccatt 4380

tcaaacccat cctcatgtgg gactagtcgc ccaggaatgc aacatagtct cacataaatc 4440

atctcaagca tgatatccaa ataataattg gttctaagca ataagcataa atagtcttaa 4500

taaggttcta agtagtactt ggcgacaagc cataaatatt ggttcatgcg gtaagcgtgc 4560

tacccaaaag cccataggca aaccgactag ggtaaatccc tagttagaac catcctggaa 4620

atctgcaaca tcacaagctg catcaaactc ctcatccaaa cagtcattac ctgcagcgcg 4680

gtctgagcat aaacaagtaa aaaaaagcaa gaaatcagta cattaatcaa attaatgtat 4740

tggcaagtcg gaaacaatcc tagcatacta catgttggcc atattcaagg ataagctgtg 4800

tataggttaa tttgcataaa tgccaatttt agttcacaac atgtttcgca acggaatttt 4860

ataacaatag tatagagtaa gtaaaacttg taatgtgaat gaataaataa tacacatcta 4920

cccctcaagt catcacatac cacatatcat taatcaattt catcatatca cgaaccatag 4980

gaagttccga taagttacca aaacccggta acccataccc ttcggcctac caagatgcaa 5040

ggctaaactc taaacaactt aataggttga ggccagccca tacttagcac atgtggtttg 5100

tactgttttc attagttggt gacatagagt ggggtcctta atcgacccgg gcgaacgctc 5160

cccctatcgg tgtacaacta ccaccctatg tacactcgcc gcccaagtct aaaaacaagt 5220

tatttccatt ttcagttcac aatactcaca cacttaaatt tctgtccagc aacatcaaaa 5280

taaccatttt cacatataac atttatcaaa acgagtaatt tgcaaggcgg taaccaaata 5340

taagcatata gcagcaatat aagcatagta taatcccaaa gaacacaata attgtattca 5400

aaagcactct atggttacaa ccagcaaggg ttcaatattt caaggtggat tatgcagcaa 5460

ttaggtcata tctaccattc ccacatttat ataatatttt ttttaccaaa attaatacta 5520

gctaacgtat gcatgtttgc aatagaatca tttagcccaa tagaacatag gatcaatgtg 5580

gtcaaaggag gggcaaactt tccttcgtct gcaaactcct gagcctggtc gtcaacaaac 5640

tcaccctctt cttcttcgac gtattcttcc tctatacaaa atacgtgtat acgataaata 5700

caaaaatgca taaaaaccaa aatgcatgaa aatgcatgaa atttatgcag tgagtgtgtg 5760

ctggtctcag gtggtctctg gtgaaggaat ttttattttg tcacttttta tttacagaga 5820

tatgcatttt acaattaaaa aggtttaaaa gggctaagtt ttattaagca acatttttgt 5880

acagaagtga acaaaattcc ttttacaaag ttatagagca tgattttaga aagttaacag 5940

aagtggtttc acaattttta gagctatatt caattcgtta tggatttaac aagttctagg 6000

caacattatg gcaaaacaaa aataaaggaa aagtttgtac agaaagtacc catttttata 6060

tttttgtaaa atatttcaca gctttatgga tctaacaaaa ctggtttcac aatttttgaa 6120

gttaaaatga attttctaca aattttagaa gttctgctca ttctctgctt taaaccaaaa 6180

tagaaatcaa aatttgaaat tgcattccag ccgggtccac aggtcagtga aagaagattt 6240

gcagagaggc cctcggaagt tgggggaatc atcccgcact tcttctgcca cctggacagc 6300

aaactttatt aagagtctct attcttttgt ttatttccat caaggtcctg ctatagtaat 6360

atgagtcatg gcagttttca gattggaccc tagagtcgat caaattcaag aaatgaagtc 6420

cccagctatc tcacatcggg agcagccatg gctgagctcg gtctacggct caccggcggc 6480

gatcagtgga tcgaatcggt tcgagcacaa aagagaaaat gtagagaacg ttcttgcgca 6540

tccgtgagtg gtgctggtga gggaaatggt ggtctgtagc ccatagaaca tgagctacag 6600

cggtggtgtg tctcggtatg cccatgtatg tgcatgcaga ggcttggtta gtgcaaaggg 6660

ttagtgcatg tgctctcata tggtaccagg gtgctcacca agcagagaaa cggaggcagc 6720

gaggtccagg gagcggcgga gacggtggtt gatggccaac ggcggcaaaa ctccggcgtc 6780

gacgtcgccg ggtcgactca aggcgcgaac tgtaacttcg agagtacggg ggtgttagag 6840

atgagccaca gagggtgcat gcgcgaggaa tccggcggaa actcaccgga gcttggtgtt 6900

gcttcggtgg tgaggaacgg cggccggagc ggcgtcggcg gttcgggtgg ccggcggcga 6960

ggacggtgac gcccaccacg tgctcgacag attggtccta gggcaaaagg ggtacgggtg 7020

agctcggcgt gatgcgtagg tcacgttcca taggaggaaa gagagaggga ctcaccgggg 7080

atgcacggcg acgacggcta agtcgccgga gccggggaag atggccgagc tctgggcatt 7140

ctgtgcgtgg ggtgtgtttt cttggacttg gaacgcgatg tggggagcga gctgttgctt 7200

ctggtgcttt ggcttgggaa ataaggaaga gtggagtggg ggagtgagag agagagaggt 7260

ggctggtgca ctcattgtgt tccttgggca tttattgggc atgaggaacg gtggatgctg 7320

tgaggggaga gtgggaggct gtcatgtggg tcccgggggt tgtggggccc actgtcagtg 7380

aaagaaattc gaaaatagcc ttggaggcta agtcaatgag ggttacctgc caaggatctt 7440

tggaggggat tcaaggtgga ttttgagcag caagaaatct ggctagatat cctcttggtc 7500

aggggtgatg tggagagtaa atgtgctgaa gtgggttggg ttagaagtct ctggtttagg 7560

tttgatggag tgatgaacat gtgttgacag tgttggttag cagcagttga cagtgcagtt 7620

agcagctgat tggatttttg gagggagaga agaaagggtt agagagtgat ccagggtatg 7680

ccactcctca gggtgttaga agaaagttag tagaagactt tggctgaaac caaacttcaa 7740

aaagaggttt tgaaataaat tttacagaga gagcaagttt gaatctgcta agcagttttt 7800

tttttcagga ttttggagag attaaagtag atcaacaagt tagcaaacct tactgaaaaa 7860

ctctgaaaat ttgtgggttg atttctgaca tatcaaggta tttttgagaa tttttgtgga 7920

atttttggat gcatataatt accagattaa ttgtttttgt gactctaggc ctatcaagat 7980

gcatgtgatg atgagatgat taagatccaa aaacaattaa aatcactcca caaccacatg 8040

atgcatttaa attaaattta gttctcaggt ttataggctt gggtatgtta caggaaggga 8100

ccaactttca tttttaatag ggagtatcta aaaaactgcc gcaagttttt ttgagtgaaa 8160

aactttcaca tgtaactata gtataatcga agtgtaacta cattgtaact atactataac 8220

tgcattgtaa ctatactgta actatgatat aacttatata aaacttgtat ttaactatgg 8280

tttgattggt tagcaccgga atattacacg tgacatgtgc gcaaatttct ttctagcaat 8340

tttttccctt catgcacaaa tctcgaggca atccgatggc tacaaatccg aaagttttgg 8400

aggcaaaaga acttgtggaa gttttctagc aattccattt taatatcaca gaacactata 8460

tatattaatt taggcggtct tgtgctatgt attcagtatc ctccatgtgt agtacatgta 8520

tagtggccca gtggttgagc ttatggacct tcatagtaca atttctttct ttaggaacca 8580

gttttgagag agtagcttat gttttgttca tagaatatta tcctcaaatc aactaaatgt 8640

gcttatagtt ttgcatcaat gaaatgtgaa tgacatgact tctgaaaaat acactgtttt 8700

tttttttgtt tgtgttacag ctatggcaat tggagacact tctgttgcag aaagatacct 8760

atatgaacta tagtcaggcc cttagttaca caagccaagc actgagccag aatcttgcag 8820

ggctaaggaa gaagagttca atccgcaaaa taagcacctc tttaagctac tatttcgagg 8880

acaactggaa acgtttatgg gtgcttgcat tgtggattgg gataatggct ggactgttca 8940

cctggaaatt catgcagtat cgtaaccgat atgtctttga tgtgatgggc tactgtgtca 9000

caacagcaaa aggagctgct gaaaccctaa agctgaatat ggcaattatc ctcctgccag 9060

tatgccgtaa caccattact tggttgcgaa gtacaagggc tgcacgggca ctaccttttg 9120

atgacaacat caacttccac aaggttagca aatagtctgt cattgcctct gttatcccct 9180

tgatacaatc tagcacagtg atctgtattg aaatcaatga atttagacct tgacccagta 9240

ctctagaatg tagatgctgc atttgagata caagcacaag taatagggtc atgacacaac 9300

tgaaaaattt ataatataag tagatatatt gaacattctt ctgctgacaa actcgaaaaa 9360

gaaactaaca acttgtgttt ttgttgtaac ggcagactat tgcagcagca attgtggttg 9420

gtataatcct ccatgcaggg aaccaccttg tatgcgattt tccacggtta ataaaatcat 9480

cagatgagaa gtatgctcct ttgggccagt attttgggga aataaagcca acatatttta 9540

cattggtcaa aggagtggag ggcatcactg gggtaatcat ggttgtatgc atgataattg 9600

cttttactct agcaacccgg tggttccgcc gtagcttggt taagcttcca aggccatttg 9660

acaaactgac tggcttcaat gccttttggt attctcatca tctgttcatc attgtgtata 9720

tcgcgctcat tgttcatgga gagtgtctat accttattca tgtctggtac agaagaacgg 9780

taagatattt ttgaatttat cttcattcat tggtctaaga taattcgaat ttctaatgtc 9840

ctcatgcttt tatttcaatg cagacatgga tgtatctttc agtgcctgtt tgcttgtatg 9900

taggggagag gattctaagg ttcttcaggt ctggcagtta ttctgtccgg ctattgaagg 9960

tcaatccaaa gacattacaa aattgagcat tgatactttg tgccttgtac taattcagta 10020

actttcttag caggtggcca tatatccagg taatgttttg acactgcaaa tgtccaagcc 10080

tcccacgttc cgttacaaga gtggacaata tatgtttgtt caatgtccag cagtgtctcc 10140

ctttgaatgg tacttggtgc ttttcagctg aaatttaatt gaattatgta cactggataa 10200

cattgctaag atagattact tctaattttc atgtgtagtg catatgtacg aaaggcaaac 10260

tcagggcaat taatctaaca ttaacattta ttgtctcttg taggcatccc ttctcaatta 10320

cttcagcacc tggggatgac tacctcagca ttcatgttcg acaacttggt gattggacac 10380

gagaactcaa gagagtattt gctgcagctt gtgagccccc agcgggtggt aaaagcggcc 10440

ttcttagggc agatgagaca actaagaaaa tgtatgaaca ctccctatgt ttaccacaac 10500

aaaattttgt actttgtgtg cactaacatt tataattttg tccttttggc tatgcagctt 10560

acccaagctt ctgattgatg gaccgtatgg ttctcctgct caggattaca gcaagtatga 10620

tgttttatta cttgttggat taggaattgg tgcgacaccc tttattagca tattaaaaga 10680

tcttctgaat aacatcatca aaatggagga agaggaggta tgttttctgc agttgtcttc 10740

ctagtattgc aagtaaataa aatattactc ttttccttat catgcttatc ttctttgtgt 10800

agtatagtaa ccatttacgt ttttagctgt gccttatgtt ttcggtgtcc tctgtttctc 10860

cgtatttgca ggatgcttct actgatcttt atccaccaat gggtcggaat aagccacatg 10920

ttgatctggg cacacttatg acgattacct caagaccaaa gaagatcttg aagaccacaa 10980

atgcttactt ttactgggtg acacgtgagc aaggctcttt tgattggttc aaaggagtca 11040

tgaatgaaat tgctgacttg gatcaaaggg taaatagacc aattgttttt tttccagtgc 11100

agtttgtttg tcttcagaaa tttgccatgc atttccttgt gaacttgtag ttacggttgc 11160

ctttgattca tgctagaata tcattgagat gcacaactac ctaacaagcg tctatgagga 11220

gggggatgcc aggtcagcac tcatcaccat gctccaagct ctgaaccatg ccaagaatgg 11280

agttgatatt gtctctggga caaaagtaag tattggcagg acgtttaaca tctccacacg 11340

gatgctgtaa agctgttttg tttgatatta agtttttcac ctcactggtt attatcgtaa 11400

agaactcaaa gttaatagaa atcacatttg ccttgtccat ttaattgtca ctgcacttgt 11460

ttttcaggtc cggacacatt ttgcacgacc aaattggaga aaggtccttt ctaaaatttc 11520

ctccaagcat ccatatgcca aaataggtta gtttacatgc ttcaagtcta actaatcttt 11580

atcttgattt cctttggtca ttcatgccta ctacctaact ctgcatatat aacttattag 11640

atgtactgag tactacataa aagaaacgca aaacttccaa tgatatataa taaaaaatgg 11700

gaaaggggaa aacttgaaat tccacgcctt ctacactttc agctctgttg taaatgagca 11760

gatctgtaaa caagttacca cctcgggctg agttactcat caacatttga tctatatagt 11820

taatatagtg atatcgtaga cacattatat atggtttgag tacttatttg tgttgcatac 11880

aggtgtattc tactgtggag ctccagtcct ggcacaagaa ctaagcaaac tttgccatga 11940

attcaacggg aaatgcacaa cgaagttcga attccataag gagcatttct gaaatcaagt 12000

aaatacttga ggggaaacaa ggatagatta tgttatgtgt atagcatttt tgtcttcgta 12060

tattcttcat tataatggac aggagatagc aaggtgagga agcaagcata tagttagccc 12120

aacagttctt gctaccaatc aataaaagga gcaacaacaa gagtgatttg ggataatcat 12180

ataaatttgt gcgatgtcca tcatggcttt tgcagcatgt cttatgcagt catgaatgta 12240

cagagctcac ttgaagacct tgagcttctt ctgattttag ttgagattat aagtaggatg 12300

gtatagcaca taggaaggtc acaaggctaa ttaactgtat attattcatg cagctgagtg 12360

tctggctaag gccagaagca cgttttactt ttgtatttat acataacaga aaacgagaaa 12420

aaaaaagtga aaagggaaag gtatactcga attgagtgtt tgtaatctgt aatttataca 12480

gtgcattggc agttttattg gctccaggtt gggcgcaccg cgcaccaacc acatca 12536

<210> 9

<211> 6629

<212> DNA

<213> Rice (Oryza sativa)

<400> 9

cctcctgctg ccgccactac tactaccatt gccaagccag gccaggccag accggccatc 60

tgcattgcat ttgctctctc tctctctctc tctctctcgg caccacggac actcgtacag 120

ctgccgcctg ccggagggtt ggtggtgtgt ttcaggttcc agaaaaatcc tccagctttt 180

tgtggtcgct tgaggagctg agctgtgtga gccttgcaag gtttgcatca acaagcttgg 240

gggtttttcc tatcttgttg ttctgaggga attcaagaaa tgctccaaat ggtctgaatt 300

cgtaggttta tttgggctgg aatctctcag agggtttaag gtaccgttcc tttcgtcact 360

tcccccaaag ttttagcctg caaagtttgt tcttcttctt cttcttcttc ttcttcttct 420

tcttcttctt ttttccctct tgctctgtgt ctgattcttc ctttcccttt tcgttttgtt 480

tctccacttg tgaataagct tttgtttttt ttacattcag tagtacccac tcactagtag 540

aaatgttcct ctgtttcttc tccactactg ttggacatgt ttgcttgctg tttgtgggtt 600

gtggcaccaa gaaacggatg gttgattgag ggggtgggtg ttggtgtgtt cctgcatttt 660

cttggaagca gcagcttcca gatctgagat ggcagaggta ttttgcggct catggtttga 720

gttccatttc gagtgaattc gttcattcct tccttcagtt tcttggttta tttttgaggg 780

gtagtttctt ggtttatctt ggggcagttt taaagttttc ccctctttta ttagctaatg 840

ctgctgaagc tgtacatccc tggaggctat cttctccata tttaatggat tggtagtatc 900

ttagattcct agtttattgg attttctctc tcccgaattt tgggttttgg aatagatgtt 960

ctggatctca tagacttcaa gagaggcagt tctgctattt aaattgctat ttctttcttc 1020

agttatatat ttttttctgt acctagactg cagataattt tggtctgagt gtctcttaaa 1080

aaaaaggaga taattttggt ctgaatgaac agtgaagtgt tttatttcac tgatttttgt 1140

ccgtatcact atattagtag ttattttcat ttttgcccag acttcacctt tttctttttt 1200

gttgttgtgc taaaagacca aattatcact ttcaatcact gaagttcatc tttccttggt 1260

tgctaactaa attatccgtt gatcagagga aaaaggaaag aggagaaatt gtaaggacaa 1320

gtagagaaga acatggctga cctggaagca ggcatggttg ctgctgccac agaccagggc 1380

aattcaacaa ggtcacaaga tgacgcagcc acactgatcc cgaacagtgg caatctgggc 1440

tcgagcaaca ggagcaccaa gacggccagg ttcaaggacg acgacgagct ggtcgagatc 1500

accctcgacg tgcagcgcga ttcggtggca atccaagaag tgagaggggt ggatgagggt 1560

ggctccgggc acggtaccgg gttcgacggc ctgccactgg tgtcaccctc gtcgaagagc 1620

ggaaagctga cgtcaaagct caggcaggtg accaatgggc tcaagatgaa gagctccagc 1680

aggaaggcgc catccccgca ggcgcagcag tctgcgaaga gggtgaggaa gaggctggac 1740

aggaccaaga gcagcgccgc cgtggcgctc aaaggattgc agtttgtgac tgcaaaggtt 1800

ggcaatgacg gctgggccgc ggtggagaag cggttcaatc agctgcaggt ggatggtgtg 1860

ctgctccgtt caagatttgg gaaatgcatt ggtaaatttt ttttttcaga agttgagctg 1920

aatttgaatt gttcatacat ctgttaggag aaatggtgtt tattccatta tttggattga 1980

ttgtgcagga atggatgggt ccgacgagtt tgcggtgcaa atgttcgatt ctctggcgag 2040

gaagagaggg atagtgaagc aggtgctcac taaggacgag ctcaaagatt tctatgagca 2100

attgactgat caggggtttg acaatcgtct tcggacattc tttgacatgt tagtactctc 2160

tgaaaacact tctagtgttg tcatgtttcc tttctgtact attataggca taaatatatt 2220

tcatgtggtt aatttgccaa atcaaatttc caccacagag tagctcaata tcttgcatag 2280

aattcaagca agtgagcgac aagcttttca tgcttctgtt ttgctaactg ctaagctatt 2340

cattgtagca cttaacaatg ttcattcttg ttgtgcaggg ttgacaagaa cgctgatgga 2400

aggctcacag cagaagaggt taaggaggta agtcagaaaa cacaaaaccg aaaaaaaata 2460

taacaatttc tatatgttct gtgtttaaat ggtattaaat ggatgattat gcttttgcag 2520

attattgccc ttagtgcatc agcaaacaaa ctttccaaga tcaaggagcg agctgatgag 2580

tacacagcac tcattatgga agagcttgac cctacaaact tgggatacat cgaggtttgt 2640

ccaattccaa gcatcaaagt aatattgtgc acaacctatg acctaaccgc attaataatt 2700

ttgacagatg gaggacttgg aagcactatt gcttcagtca ccatctgaag ctgctgcaag 2760

atcaacaacg acgcacagct ccaaacttag caaagctctt agcatgaagc ttgcgtctaa 2820

caaagaaatg agcccagttc gtcattactg gcagcagttc atgtacttcc ttgaagagaa 2880

ttggaagcgc agttgggtta tgactctgtg gatctcaatc tgcattgccc ttttcatttg 2940

gaagttcatt cagtaccgta atcgagccgt attcggcatc atgggttatt gtgtgaccac 3000

tgcaaagggt gctgcagaga ccctcaaatt caacatggct ttggtcctac ttcctgtctg 3060

cagaaataca atcacatgga ttcggtcaaa gacacaggtt ggagctgttg tacccttcaa 3120

cgacaatata aactttcata aggtaaaatt atgttgattc aatcaatatt gaactgatgg 3180

attttaccgt gttgtgaata tcatccagtc cattttgcgt agtgctttcc ataatcaagt 3240

aaagtcaata tttttttaag ataatggaag ctttattgaa ctcagccaat gacatcaaga 3300

tgatagaact gtactaagaa cactctcgac ctctgcataa ctaagatgca cacagtcaaa 3360

acacaccaac actagaattt tttttaaaga aaaaactaaa caaaagatag gagggattaa 3420

gggccatcaa tccatagact agatcgccac ccacgctcct aagtaaaaaa accccctcgc 3480

tacctgctcc aattgtgaca tgccacagca ataaaattat gcagacccga cttccgaaga 3540

atatcccaag aacggagcca atgagtaatc aaagagataa cctggaaagg agagtcaatc 3600

tttatgttat caaaaaccac atcattactg cctagccaca atgaccagca tatagcagcc 3660

gctcctaata ggactaaagg tctcaaatat ttagataaac acgaaagcaa actcgcaaac 3720

atatgtgaaa cactatgggg gcagacatag gtttgaaact atttggatta aagaccaaat 3780

agaatgagca agacgacatt ggaagagtaa atgtttaatc atctcttctt tatgacaaaa 3840

gcagcatatc ttgctacctt gctaattaca cttggcaaga ttatcctttg ttagtactac 3900

acgtctacac cacggcgtaa gcaccaaaga aagatcttga cttttagagg accctttaaa 3960

ttctaaagtc gtttatttag taacatatga gtaacacatt aaacctgttc atctggtaca 4020

ttttttcaat aatggaaatt agcatatcct ctacatcaaa aaatgtgtcc ggccatatag 4080

ttgagaacta ggcaaaaaaa tatgagttgt gagactaata tcgactttac ataatctatg 4140

atttactact gtgcaggtca tagccgcagg tgttgcagtt ggtgttgctt tgcatgcagg 4200

tgctcatctg acatgtgatt ttccccggct gctccatgcg agtgatgcac aatatgaact 4260

aatgaagccc ttctttgggg agaagaggcc accaaattac tggtggtttg taaagggaac 4320

tgaaggctgg acaggtgtgg tcatggtggt gctcatggca atagcattta cattagccca 4380

accatggttc cgacgtaaca agctcaagga ctccaatccc ctcaaaaaaa tgactggctt 4440

caatgccttc tggtttaccc accacctgtt tgtcattgtg tacactttgc tctttgtcca 4500

tggaacgtgc ttgtatctaa gcaggaaatg gtacaagaag acggtaaaag tctataccct 4560

gaacacacaa gaaatcccaa tgatcatagt tggtaccttc tttgtgagga gatcataact 4620

gatacttatg gttcattttt atcattttca gacatggatg tacctcgctg ttcctgttgt 4680

cctgtatgta agtgagcgta ttcttcggtt gtttaggagc catgatgcag ttgggattca 4740

gaaggtacca tcaatcaaat ctctttcctc aacttacata ttactactat ccatcgccat 4800

ccacatcttt ccttttttta tgttgtattt tatttaaagg gtttgaactg aaactagcct 4860

tggaatgtct ggaagctctg aatccgtctt ttttttttgg gtttaacatc tctgctttca 4920

acaggttgca gtgtatcccg ggaatgtatt ggctctttat atgtcgaagc cacctggttt 4980

cagataccgt agtgggcagt acatcttcat aaaatgcact gctgtgtctc catatgaatg 5040

gtgctggtct tacacatttt atggaaagaa aatgtattat tttgaaataa agtccgcaga 5100

taactaaaag agcaatactt tgtctgttac aggcatccat tttccataac atcagcacct 5160

ggagatgatt atcttagtgt tcatattcgc acaaggggtg attggacttc acggcttaga 5220

actgttttct ctgaggtaat tgaaccaact gccttttatt ttttttgctt tgttctatag 5280

ataaactcct aagaaacatg acttacgaac tttgtaagca cctgaattgc attgtctttc 5340

atttgtcata tccaatttat ctgcttgatg gattcaatcg acaacaagta tgataacaaa 5400

atctttgaac caggcatgcc gaccccccac tgagggagaa agtggactac ttagagctga 5460

cctttccaag ggaataacgg acgaaaaagc aaggtgcaaa ggacttcatt tgatttgttt 5520

gcgaaaagca gaaatgcatt atatatcttg cttcatctca tggtaggact ttccgttatc 5580

taaagaaaaa tcttccgtct aacgataatt ctgctttaca tgcactagat tcccaaaact 5640

tttggtcgat ggaccgtatg gtgcaccggc acaagattac cgtgaatacg atgtgctact 5700

tctcatcggg ctgggcatcg gagccacccc tttgattagc attgtgaagg acgtgcttaa 5760

ccacattcaa ggtgagggat cagttggaac cacggagccg gagagcagca gcaaggcgaa 5820

gaagaaacct ttcatgacga agagagccta cttctactgg gtgacgagag aggagggctc 5880

gtttgagtgg ttcagaggcg tcatgaacga ggtgtctgag aaggacaagg atggagtcat 5940

tgagctccat aaccactgct caagcgtgta ccaggaaggc gatgctcgtt ctgctctcat 6000

tgtcatgctc caagaacttc agcatgcgaa gaagggcgtc gatatcttgt cgggaactag 6060

tgtgaagacc catttcgcac gacctaattg gcgaagcgtc ttcaagaagg ttgcggtcag 6120

ccatgagaac cagcgcgtcg gtatgttctt cttcctgtcc tttcaaatcc agtagtagga 6180

atgtccatga tccattgcat attaaccctg tatatttctt caggtgtgtt ctactgtggt 6240

gagcctgtgc tggttcccca actaaggcag ttgtcagcag atttcaccca caagacaaac 6300

acaagatttg atttccacaa ggagaacttc taatggtaca aattgagaaa tacccgtgta 6360

tggttttgta tgtagttctt tatcatgtga attatatggt ttctaatata tataaagttg 6420

gacaaaataa atgaaatgat ggaagctatt ttgtttttta agatgtcaaa agtctgcaat 6480

atctttttac aagagtgctg tctattcatg tatacaccta gtggaagaag ctgtgcttca 6540

tgttgtagct tacatagatg aagggaagtt ctctttgttg tgaccaaagg atgcctagaa 6600

tcatgtaaca ttgtgatgtt ccctttcat 6629

<210> 10

<211> 4543

<212> DNA

<213> Rice (Oryza sativa)

<400> 10

ttacccgcca tatctgtctc cctctcccct tctcatctcc cgagaagtag tagtagtagt 60

cgtctagtag tagtagccta ccaccacctc cctcggcgag cgggtcccac cggccaccgt 120

ccgtactccg gtgagccggc attgcgtgtt ccgggatggc gtcgccgtac gaccaccagt 180

cgccgcatgc gcagcacccg tcggggttgc cgaggccgcc gggggcgggg gcgggtgcgg 240

cggcgggcgg gttcgcgcgg gggctgatga agcagccgtc gcggctggcg tccggggtga 300

ggcagttcgc gtcgagggtg tcgatgaagg tgccggaggg ggtggggggg atgcggcccg 360

gtggcgggag gatgacgcgg atgcagtcca gcgcgcaggt ggggctccgg gggctccgct 420

tcctcgacaa gacgtccggc gggaaggagg ggtggaagtc cgtcgagcgc cgcttcgacg 480

agatgaaccg caacggccgc ctccccaagg agagcttcgg caagtgcatc ggtgagtccc 540

cggcccgcag cttccatcga tggtgcgatc cagaaacaga tagccgtaaa ttcagttcca 600

aaagggcgat tagttgttgc taacaagaac acagcagaaa tatctctgat tgaaggaata 660

ttgtgttggc gccatctttc ttgttttctg tcaatcattt ttatgctccg caacagttgt 720

gtgatgatgc cgtgaactgt tcatctttgc ccaaagagtt ggcagggaca gctctgccta 780

actgagatcg gctctttgct tctcaactaa tcccctcgaa ttaatgcttt tgctttgcga 840

aatggtccat tattaagcag taaatcggta cagcaaaaga aacgagtact ctgtacttct 900

cttttgttat tgcacatgct tacggagaaa atggagttat agcgaccggt tcatgctgga 960

ggcagttagg cgatcgatgg aataataatc gtgtagcgct aagcgaatca tttctctttt 1020

tagatagttt ggatgtctaa caatgcccat gattcatgat cttttaattg aactaagaaa 1080

aattaatcca tggtggatct gcttggcatc aaaaggcatg ggggactcca aggagttcgc 1140

cggcgagctg ttcgtggcgc tggcgcggcg gaggaacctg gagccggagg acggcatcac 1200

caaggagcag ctcaaggagt tctgggagga gatgaccgac cagaacttcg actcgcggct 1260

tcgcattttc tttgacatgt aatggttcac cgtcccagtt ataatttcca agatgaacac 1320

gataaatcga tcgccaactg acgaaatgga tttttttttt ccttccccct ctctctgatg 1380

catccaggtg cgacaagaat ggcgatggga tgctcacgga agatgaggtc aaggaggtga 1440

ggacgaaaga attcttctgg attggatctc atctgccatt ctggaacatg gagagagagg 1500

attaaatatc gttttcttgt ttggctggaa tggatttgca ggttattata ctgagtgcgt 1560

cggcgaacaa gctggcgaag ctgaagggac acgcggcgac gtacgcgtcg ctgatcatgg 1620

aggagctgga cccggacgac cgcgggtaca tcgagatctg gcagctggag acgctgctgc 1680

gcggcatggt gagcgcgcag gcggcgccgg agaagatgaa gcggacgacg tcgagcctcg 1740

cgaggacgat gatcccgtcg cggtaccgga gcccgctgaa gcggcacgtg tccaggacgg 1800

tggacttcgt gcacgagaac tggaagcgga tctggctcgt cgcgctgtgg ctcgccgtca 1860

acgtcggcct cttcgcctac aagttcgagc agtacgagcg gcgcgccgcg ttccaggtga 1920

tgggccactg cgtgtgcgtg gccaagggcg ccgccgaggt gctcaagctc aacatggcgc 1980

tcatcctcct ccccgtgtgc cggaacacgc tcaccacgct caggtccacg gcgctcagcc 2040

acgtcatccc cttcgacgac aacatcaact tccacaaggt gatcgcggcg accatcgccg 2100

ccgccaccgc cgtccacacg ctggcgcacg tcacctgcga cttcccgagg ctgatcaact 2160

gccccagcga caagttcatg gcgacgctgg ggccgaactt cgggtacagg cagccgacgt 2220

acgccgacct gctggagagc gcccccggcg tcaccggcat cctcatgatc atcatcatgt 2280

ccttctcctt cacgctggcc acgcactcct tccgccggag cgtcgtcaag ctgccgtcgc 2340

cgctgcacca cctcgccggc ttcaacgcct tctggtacgc gcaccacctc ctggtgctcg 2400

cctacgtcct cctcgtcgtg cactcctact tcatattcct caccagggag tggtacaaga 2460

aaacggtaaa attctcatca ggcgctcgat cttcctgtga ctgcaatgaa attaagtgac 2520

atcatctcaa aatgtcaatc ttgttcctgc agacatggat gtacctgata gtcccagtgc 2580

tcttctatgc atgcgagaga acgatcagaa aagttcgaga gaacaactac cgcgtgagca 2640

tcgtcaaggt aagcagctga ctcatgtaac tgggatccat tctttcttag ctgtttgcct 2700

gttactcata aaattggtga tgtgggtgtg gcacatgcag gcagcgattt acccaggaaa 2760

tgtgctctct cttcacatga agaagccgcc gggtttcaag tacaagagcg ggatgtacct 2820

gtttgtgaag tgccctgatg tctctccttt cgaatggtaa ccaattcacc cttgtcgcat 2880

gaatgcatgt ccaaaatgtg gtttatctga gactatttct aactgttttt cctgcctttg 2940

caggcatccc ttctccatca cttctgcacc tggagatgac tacctgagtg tgcatatccg 3000

tacactaggt gactggacga ctgaactcag aaacctgttt gggaaggtca gttgaggcag 3060

cacagctgca aagagttgca agaacaatta gtttccctcg ttaaacaatt ctaggagaat 3120

aatgcagatt gccaaaagat ggagaactaa atcttagtat tcatgtaggc ttgcgaggca 3180

caggttactt ctaagaaggc taccctttca agacttgaaa ctacagttgt ggcggacgct 3240

cagacagagg atactaggtg actagagaag ctcttctatt tcacctacca aatgcatttg 3300

gtctacaatc attttctaac cctggttatc ttcacaatgt tgcttcaggt ttcctaaggt 3360

ccttattgat gggccctatg gtgcaccggc gcaaaactac aagaagtatg acattctttt 3420

gcttattggt cttggaattg gtgctactcc tttcatcagc attctgaagg atctgttgaa 3480

caacattaaa tccaacgaag taaaattctt ccctgataaa tagtgtttcc ttatttccta 3540

caatgttgta taactggagg aaatagataa ctaaaatgag ttgatggcaa caggaggtgg 3600

aaagcataca tggttctgag ataggcagct tcaagaacaa tgggccagga agagcttact 3660

tctactgggt gaccagagag caagggtcct tcgagtggtt taaaggagtc atgaacgatg 3720

tcgctgaaag tgatcacaat gtactgtctc actctcaatt cttcgctgta ttttcaaatt 3780

tccatagttc cttcagtcat agatagaaca taaactaata tatgtgggaa ttgcagaata 3840

ttatagagat gcacaattac ctgaccagcg tgtatgaaga aggcgacgca aggtcagctt 3900

tgattgccat ggttcagtca cttcaacatg ccaaaaatgg tgtggatatc gtctccggca 3960

gcagggtatt cgacatcttc cccctcttct atctgaaata cttatttatt tccagtgtat 4020

tcctcttatt tctgattgat ctgttgcaga ttcgcacaca ttttgcgagg cctaactgga 4080

gaaaggtgtt ctctgacttg gcgaatgccc acaaaaactc acgcataggt gagcctcttt 4140

tttcagcgaa catcgtcgat gcaacaattt tgagctttgc agtaacggtt gttaacaaat 4200

ccataaatat gcaggtgttt tctattgtgg atcccctaca ctcacgaaac aactcaagga 4260

tctttcaaaa gaattcagcc agacaaccac aactagattc cacttccaca aggaaaactt 4320

ttaagaccgt accaaaggac gtctaaacag actgcatata cttgtatagg aaagaaatac 4380

gatagcattg ggatagcaaa tatagtctta caagttttgc tctacgatgg attgtacaaa 4440

atatatgttg aaagctagtg tcaccatcgt gcatagattc tggaatgttt acagatatat 4500

gtattggaac agtgggaatg catgttagaa aaattgagtt cca 4543

<210> 11

<211> 2748

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 11

atggtgagtg cttcagcaaa taagctatcc aaattgaagg aaaatgcagg agagtacgct 60

tgtttgataa tggaggagtt agatccagaa aatgttggtt acattgaggt atgttcctct 120

gtcttgctaa gtgtgttcaa aactagcaac actaatatgt tccttttcct tcagatatgg 180

cagttggaaa cacttcttct acaaagggac aattacatga gccacagtac gcctttaagc 240

acaaccactg tcgactggag tccaaacata ggaaccagca atgccaacaa catagtaaag 300

aaagcaagtc gtaccgtcaa atgccttgtc ttagaaaact ggcatagagg cttgattatc 360

ttgctgtggt tgcttgctat gattggactt ttcatctgga aattcatgca gtataggaaa 420

atggaagcat ttcaagtaat gggttactgt ttggctacag ctaaaggagc tgctgagaca 480

ctaaagttta acatggcact aatccttcta ccagtttgtc gaaacatgtt aactaggtta 540

cgttcaacaa gagccaggat acttattcca tttgatgaca acatcaattt tcataaggta 600

aacattccat acctcaattt atcactagta tcatgtattc cttaactaca cattgacttg 660

cagattattg cacatgccat agctgttgcc ataatacttc atgcagccaa ccatttagcg 720

tgtgactttc cccatttggt taactcatcg ccagaaaaat ttgcactcat agcttctgat 780

tttgacaagt tgaagccaag ctacataagt cttttgactg gtgttgaggg catcaccggt 840

atttctatgg taattttgat gactatagcc tttatactgg caacacgacg cttcaggagg 900

agcgtaccca agttgccgac ccctctgaac cgattaacag gatttaatgc attttggtat 960

tctcatcacc ttttggcatt tgtctatata ttgctactac tacatggaac gttcttgttc 1020

tttgtccaca aatggtatca gaagacggta aggactatga atataattag tacaaaaaaa 1080

gcaagcatta aattgaaaaa acatttatcc aagattatat gctaacaatg tatcaccaat 1140

aactattgca gacgtggatg tatatctcca ttccgttaat cctgtacatt gcagagagga 1200

ctttgagaac atggcgatca gaagactatg cagttaagat cttaaaggta tgtaagggca 1260

tgaaactgat acatgtggct aaatagatgg cattttcatc aattgcactt atattctcgg 1320

tacagtagaa gaaactatct cctaaaatga aactgtaaaa ccacttaaga atataaggag 1380

aggaagatca agaacaagat aggggaaaac gaataaagaa ttaatttttg gaccttacag 1440

gtttctgtac ttccaggaga tgtcttgagc ttgatcatgt caaaaccagc tggcttcaaa 1500

tacaagagcg ggcagtatat attcctgcag tgcccaacaa tatcctcatt tgaatggtag 1560

aaattgttat ttcacataca agccgattat gttccctaca ggcattgaac atatatttca 1620

atatttctcc aggcatccat tttctataac atcagcacca ggagatgact atatcagtgt 1680

ttatatccgg atcgtaggtg actggacaaa agaacttaag agggtattca cagagattcc 1740

agcatgttta gttggtcgag caaagtttga agaacaagaa aatgttgatc aaagagggta 1800

actttactat ttctgtccag tgatcaaacc tacattgtga tttcctttac aagctctaaa 1860

tatgctatcg accatgcagt ttacctcgat tgctagttga tggaccatat ggagctccag 1920

cacaggacta tcaaaattat gatgtcttac ttcttgtagg acttggtatt ggagctacac 1980

cttttataag tatcctaaag gatctactga acaatacaag agcagatgaa aatgtggtaa 2040

aatcttactt catcataatt ttaaagcaac aacgttgtaa ttatgctcca agtaaatatt 2100

tttcggccat gcatgattct gtttcaggat tcgaatactg agaccagcgc atcagatgat 2160

agctggacaa gttttggttc ttcaagctca gcatcaagtg taaaaaagaa atcacaaagg 2220

gcaagaagtg cacattttta ttgggttacc agagaacctg gatccttaga gtggttcaaa 2280

ggagtgatga atgaagttgc agagatggat cacaaagtag tgtacacata cttctcgcta 2340

catgtcctta atttgaattc taagttggtc gatttctgtt ttaataatat tgccactcaa 2400

ctcagttatg ttcctgtatt ctggtgatgg aacagggtca gatagagatg cataattatc 2460

taactagtgt atatgaagaa ggtgatgcca gatcaactct tatcaccatg ctccaggcac 2520

ttaatcatgc aaaacatggt gttgacatct tgtctggcac taaggtatga tccatcacat 2580

aaaccttctc aaagtgaaat atttttaatt ctactgacat tgaaactaat tcttctgcag 2640

gtgaggacac attttgcaag gcccaagtgg actgaagtgt tcaagagaat agctttgaaa 2700

catccctatt ccacagtagg taaagaattt ctacaatctc aagtatga 2748

<210> 12

<211> 6105

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 12

atgaatgaag gtagtgaaag aggagaagag gaagcaccgc ccaatggatt tctggctcgt 60

agtgcatcgg cggcctccaa actccgccgg aaattctcgt ggatccgatc gccgtcggtg 120

atgtcacgga catcggcagc agcatcggaa gtgtcggatg ataattatca gcttcaccat 180

acatcaaaca ccttatctgc acgagaggaa atgaaatcga aattgaaact agtaaggagt 240

aaatcaacgg ctcaacgtgc gcttggtgga ttgagattta ttagcaaaac cactggggaa 300

tctgacacaa atgtgctgtg gaaaaaagtg gaggcgcggt ttgatgcttt agccaaagac 360

ggattacttg ctagagaaga ttttggtgaa tgcattggta cattcaaagt tctcaattca 420

tgtaaaacca catgaatgcc atctattttt actagtaata aaatcatgca tgaaattagt 480

tttaactgag atgcaataat actttatatt gaacgggtgc atgtgcagga atggaggatt 540

cgaaggaatt tgcagttggg gttttcgacg cattgatacg gcggcggcgt caaaaagcag 600

caaagataac caaaattgag cttcatgatt tttggttaca gatttctgac caaagttttg 660

atgctaggct tcagattttc tttgacatgt gggtttctct ttttcttttt gtccactttg 720

ttcgaaagca tgcattatta ctaatctcaa ttctcaacac ggaaaagcaa ctcttttttg 780

tcctatatct aaaataagaa ttgaagttac aaacgtacac cataaagaca tatcagtgtg 840

cttaaactat tataccatgt taatcagtta tgattggtcc acttctttgt tttacctaaa 900

ataaaataaa aaaatacatt ctgctgaaat tcagctcctc ctcctatatg attttggagt 960

gggctaaact agaattttga cctccttaca gattacaaca catcaaatcc aatatatttt 1020

gattttttag atcttaaatc gctaatcata tattgaaaaa taaaaaaaat actcatatta 1080

ctctatataa gttgttagta ttttatatat atattcgtct tactatttta cttgataatg 1140

atattacatg tagggcggat agcaatggag atggaaaaat cacgagagat gaagttcaag 1200

aggtaagatt atcgtgggtt aactactaaa acaataaaac ttagcattta aagtcaaacc 1260

atattctatt agtgcaaaat ccattagggg catcttcaat aattcgaaaa agcattagtt 1320

atgtgtttag ttattaataa taatatggaa cagaataaaa tacgtaaatg gcggaggaat 1380

attttattta atagtaataa taataataat aatatatagt tttccctcaa agctttgtta 1440

tggtatttat attttgttca tccaacagct aatcatgctg agtgcttcgg caaataagtt 1500

gtccaagttg aaggaacgtg cagccgagta tgcttcttta attatggagg aattagaccc 1560

tgaatgtctt ggttacattg aggtaacatt atcttctctt ttacttagtg tctaacaaac 1620

atttttatgt ataagtatct tcttaaacca caatttataa aataaaaatg aatatctgat 1680

tattgacaat tccaagattt gattaattaa tacgtatttc tgttagtgtt agtaggtgtc 1740

ttgtctatac cacgacttgt acatatccgg acccacaaac ttcgtttttc ttttgaataa 1800

gtgtttagca cgaacttctt ttagtcgacc acgtctaagt caaaacatag ggaatcctcg 1860

cactgtcttt ggactttggg ggaccacaag aacgttgttg aagaaaaaaa attctctaaa 1920

tattatctta atacatagat aattcctctt aattattcgt aaaaggaaat ctaattatat 1980

tttataacta atgaaatatc atgtcatgtt tttttaaaaa gaaaaaaaat cgcaaacttg 2040

taatatttgt caaagtcatt tcaatcgtgt ccgctaagaa gtacttttat tggtttggca 2100

aaatgacttt ttgatatatt atctttttct ttacagttat ggcagttgga aacgcttctt 2160

ctgcaaaggg acaattacat gacctacagc agacccttaa gcacaacaag tgttggatgg 2220

ggtcaaaact taggaactct taataagacc aagaacctag taaagagagc aagttatgca 2280

tttaaatgcc ttgtattaga taactggcaa agaggctgga ttcttttgct atgggtgatg 2340

gttatggctg tacttttcac ctggaaattc ttgcagtata ggcaaagagc agcatttcaa 2400

gtcatgggtt actgcttggc aactgctaaa ggagctgcag agactctcaa gctcaacatg 2460

gcactaattc ttctcccagt ttgtcgaaac attttaactt ggttacggtc tactagggcc 2520

aagctcctcc ttccttttga tgataatatc aattttcaca aggtaaaatg cacccaaaca 2580

gagtatattg ttcaacatcc aagaattttc ttaacgatgt tgtttcaact gcagattatt 2640

gcatatgcta ttggagttgg aatcttgctt cacgcaggga accatttagc ctgtgatttt 2700

cctcgtctca ttaactcatc tcctgaaaag tttgcactga tagcttctga ttttgacaac 2760

gtgaagccca cttacaaaag tctattgact ggtattgaag gcgtcactgg tattgctatg 2820

gtgattttga tggcaattgt cttcacacta gcaacacgca ccttcaggag aaatgtattg 2880

aagctaccac ctcctttcag tcgattaaca ggcttcaatg cattttggta ctcccatcac 2940

cttctagcag tggtctatgt actactactg gtacatggaa catttttatt cttggttcac 3000

caatggtggc aaaaaacagt aaggccatag ataaaacacc aaatttgctt tatataaact 3060

aaacatgggg tgtgctgaca atttatttcc catattgcag acatggatgt acatttccat 3120

gccgttgctc ctctatgtag cagagagaag tttgagaaca tgccgatcag aacactatgc 3180

agcaaaaatt ttgaaggtaa cttgctttat ctatcaactt ttccatctat acagaaatta 3240

gactcctttt ccgcatacca ataagctgta atcaacacct gcttctagcc tccctctaca 3300

ccccttaata attcaactct ccaacgaaat ctcacaacac cacaaaccat ttattcaatc 3360

agttacttcc ctaagcagca gagtttctct tattccatgg caaacgtcca ccgtgtaaca 3420

tcgtaaacat gaccatcgag caccaacata aatcaatcaa atactggcac tccaaccacc 3480

agcgccccaa caattttcaa aaccttccaa tatagaacta agtcaagtgt gaaaatttac 3540

cgttagcaaa ctcaaagtcg agagataaga gggggggaaa gggtagcctg gtgcactaag 3600

ctaccactat gccagggtcc tgggaagtgc cggaccacaa gggtctactg tacgtatccc 3660

taccttgcag ttctgcaaga agctgtttcc acagctcaaa tttgtgaact cctggtcacg 3720

tggaggcaac tttactagtt ctgcaaggct ccccttgtta taaataaagt cattcactaa 3780

tgatagtctt ttctctaagt atttaatagc attcctaaat tactagagat gatataagca 3840

tctggttcct acagcctcca cccacaagac agtagtaaag aaacaaataa aacatattga 3900

gataaacatg gtagcaggga gagaaggaaa ggaacaaaac acgaaaccaa tgaagatggt 3960

tttttctttg atccttacag gtttctgtac ttccaggaga tgtatttagc cttaccatgt 4020

caaagcctaa tagtttcaaa tacaagagtg ggcaatacat attcctgcag tgcccaacaa 4080

tctcctcatt tgaatggtaa gtgcagtatt tctggattta actcaatcat gcctcaacca 4140

acagatattt aacatatatc ttgatgcttc tccaggcatc cattttctat aacatcagcc 4200

ccaggagatg actacctcag tgttcacatt cgcatggtgg gtgattggac caatgaactt 4260

aagcgggtat tcacagagga tgatagttca gcatgtgaaa tagggcgggc taagtttaga 4320

gaacgtggga atgttgatca aagagggtaa cttcctttgt gtgatttttt aacagaaaca 4380

tatcaagatt tccttattga gaagtctaaa gggcttgtaa tgacgcagtt tacctcgatt 4440

gcttgttgat ggaccatatg gtgccccagc acaggactat caaaattatg atgtcttgct 4500

cctcgtggga ctaggtattg gagctacgcc ttttataagt atcctaaaag atctattgaa 4560

caattcaaga tcagaagaac tggtacaaat cttacttcaa gttaatttgg aactaatcag 4620

gtacaatatt aaaagcccca acttacagtc tctttgcctg tgattctacc ttaggattca 4680

actaccgaaa ccagcgcttc ggatgacagc tggacaagtc ttgcctcttc aagcatggca 4740

tcaactggaa aaaaaaaatc actcaggaca aaaagtgcac acttttattg ggttaccaga 4800

gaaccaggat cctttgagtg gtttaaaggg gtgatgaatg aaatggccga aatagatcac 4860

aaagtaatgt acagagattt gttacaggat atctttaagt gccccccccc ccccctcttt 4920

catcttaatt cagctatgat atattagtaa ttgactcacc tatatctttt ctgatgattt 4980

aaatgaaaca gggtctgata gagatgcaca attatcttac aagtgtttat gaagagggtg 5040

atgccaggtc aactctcatc accatggtcc aggcactcaa ccatgcaaaa cacggtgttg 5100

acatcctgtc tggcactcag gtattataat ccatcaatat tctcaaccaa gccttcttga 5160

agagaaatgg aaaagaaata taatgtagta aggaatttta gcttagctca acgagcgcca 5220

tgttctagct aaaaaaatgt tttattattc aaaagctact taatttcatg aaaagttttc 5280

aatcacaaca cagattagag agaagcaaac tagaggagaa atatactcca cccccaggac 5340

taattaaact aattctactg caggtaagga cacattttgc aaggcccaac tggaaagaag 5400

tattcaacaa aattgcttca aagcatccat attccacagt aggtaagaaa tttctacaac 5460

ctaaactcca ataaactgta agcagaagtg gttctaagaa gtgttgtgtc atacctatta 5520

gttaacctct cacctacaat atgtccaaac ctcctgccag cggaaattat cacataatag 5580

aggcgaagga agaaaacaac tatgtataat caaacacatg aaacatgaag cagaacataa 5640

tgatgcagca gcattaccaa ttataagaat ggcatccttt ttgtctagat gaagatagaa 5700

aatatagctc taaaatgcat tgtcatgatg aagtagcaag gctttataaa tcttaatttg 5760

tctttcgaag gaacctaaag aatgtaaatg agattgaaat gtgttcttta aacttcagaa 5820

agaaaaaaaa aactctaact tcatgacaat gaccatttaa aaagaagcag cctccaacag 5880

ggcactttcc tgataacatt ttgcattttc aacagaggag gggagagaaa aaagaactaa 5940

aaggaagaag atatttaata gattagttaa caacacaatt ttatgtgatt cacaggagtc 6000

ttctactgcg ggttgcctgc cttggcaaag gaattgaaga agctatcaca agaattgact 6060

tataagacat ccacacgatt tgagttccac aaagagtact tttga 6105

<210> 13

<211> 4100

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 13

caataatcac aattattcaa aaacaataca aaatccgccc cctgatttcc ctcttgactt 60

ttcccttcta ccttaattcc ttttatatat atatacacat caccgttctt ctataatttc 120

tcaacatatt actatttttt caatctttct cagaatttat tttttttaca tttttgttac 180

ataggtcgat actagttata aatttcattt tgaaaaaaaa cagaggaaaa aagagcggga 240

aaatcaaatg gagatcgaaa acacgacaga tagtgtgaga ggatcgagag taggattcag 300

cggttcatta gttagtggga agaaaagtgc aaggttcaaa gatgatgaat cgtatgtaga 360

gatcactctt gatgttcgcg atgattctgt tttggttcaa aacattaaag gtgctgatca 420

tgaagctgca ttgttagcta gcaagctcga aaagaggcct aaccatacgc ttggctcaca 480

gctttcgttt catttgaaac aagtttcaaa ggaattgaaa agaatgactt cttctaataa 540

gtttcagaaa attgatagga gtaagtctgg tgctgctcgt gctctacgtg gacttcagtt 600

catgaacagg aatgtaggaa ctgaaggatg gtctgaagtt gaatcgcgat tcgatcaact 660

tgctgttgat ggaatgctag caaaaacatt gtttggtcaa tgcataggtt tgttcattac 720

acaaattctt ttggatttat acaataaaag tttctaataa ttagcatttt tgcaattgta 780

attaaggtat gaaggaatca agtgagtttg ctgaggaatt atttgatgct ctagctagga 840

agagatgcat cacatctcct gcggtgacta aggatgaact gcacgaattc tgggaacaaa 900

taactgatac tagctttgat gcccggcttc aaactttctt cgacatgtaa gattatgatc 960

actcaactaa atagttatat tgtggttttc tgagataata actatatgat gaatcaaccc 1020

tacaacataa tgaatgctaa attgctaatg atgtcttgaa ctgttacact agtctattac 1080

atgcaaccta tcgctcctac tttgaacatt ctcgattaag ttagaacttt gtcatcagat 1140

tctcaatagg atagattgtt gataaaatgg tataagatat tgttgatagc tcataatggt 1200

atgatgaaaa aaaaataggg tggataaaga tgcagatggg agaattactg aagaagaggt 1260

gaaagaggtg agagcttaag atgattattg aattaattca tgaaattata caaattcgaa 1320

gagctcaaaa ctcatacttc gttgatatgc ttatagatca tcagtcttag tgcttctgca 1380

aataagctgt caaaaatcga ggacaattca gatgaatatg cagctctcat catggaagaa 1440

ctagatccag gcaatgttgg atacattgag gttcgttttg aaacaagaat ttaagttata 1500

tacactgaca gtataaaaca aattcttaga caaggacatg agcttataaa tgattgtgtt 1560

tgatacagct gtacaacttg gagactttgc ttcttcaggc cccgagccac tcgatgaatc 1620

tttcaactaa cagcagagtt ctgagtcaga tgctaagtca gaagcttaag ccaacgaaag 1680

agcgaaatcc ctttaaaaga tgcaaaagaa ggctggacta tttcattgag gacaattgga 1740

agaggatttg ggtgatggtt ttgtggctat caatctgtgc aggactcttt acatggaaat 1800

ttattcaata taaacgtcgc gctgtctttg atgttatggg ctattgtgtc tctgtagcta 1860

aaggaggtgc tgaaacaaca aaatttaaca tggctctagt tcttttgcca gtatgcagaa 1920

acaccataac ttggctaaga agtaggacta agctcgggaa aataattcct tttgatgata 1980

acatcaattt ccacaaggta tatagcctga gaatataaat ggaaacacga aatcgttgaa 2040

cttaaatgtc tgattttttt cgtgttcttt gttgcaatct tttttaggta attgcatttg 2100

gagtagcagt tggtgtaggc ttacatgcaa tttcacactt aacatgtgat tttccccggc 2160

tgttacatgc cacggatgag gaatacgagc caatgaagcc attttttgga gatgaaagac 2220

caaacaacta ctggtggttt gtgaaaggca cggaaggatg gaccggtgtt gtgatggttg 2280

tccttatgat catagcgtat gtactagccc aaccatggtt tcgtagaaac cgactcaatc 2340

ttccatcaac aatcaagaaa ctcactggat ttaacgcttt ttggtactcc catcacttat 2400

tcgttatagt ctatgtcctc ttcatcattc acggatactt cctctacctc tcgaaaaaat 2460

ggtacaagaa gacagtaagt tcttaacaaa ataaattata tctaatgcta atcatgccct 2520

gccttgccat ataatggatc taatatcttt ctttttactc tgaaatattg agcagacatg 2580

gatgtatatc gcggtgccta tgatactata tgcttgtgaa cgtctgcttc gtgcatttag 2640

gtccgggtac aaggcagtga ggattttgaa ggtacataag agcttagacc attgcaagca 2700

ttaagattcc tagtttgatt acatgtgcat agaattttta tctaaaattc gagttttcag 2760

gtagctgtat atcctggaaa tgtaatggca gtgcacatgt ctaagcctca gggctttaag 2820

tacacaagtg gacagtacat ttttgtgaac tgttctgatg tttcttcatt tcaatggtaa 2880

atttgaaata aaccaagctc ctaatcaaac aaactgtata caaattatta ttattattat 2940

tactgctaaa atcatacata ttttgtactg taggcatcct tttaccatct cctcagctcc 3000

gggagatgat tacctaagtg tgcacattcg aacattgggc gattggacat ctcagcttaa 3060

aactctcttt tctaaggtat cgaattcatt aaatcagtta taacacagca aagacgaaat 3120

cttttgaaga aactttggaa gactaataat gcttgtactg atcatatttt aggtctgtga 3180

gcctccaact ggtgatcaaa gtggcctttt aagagcagat atagggaaag ctgactataa 3240

gcctaggtaa gggagtaaag tttgaaaatt tccaagtgct tttaacttat tttcccctga 3300

attttgattc atatcaatgt aaacagattg ccaaaactat tgattgatgg cccttatgga 3360

gcaccagcac aggattacaa gaaatacgac gtagtcctct tagtaggcct tggcattgga 3420

gcaacacctt tgataagcat agtaaaagat gtgctcaata acatcaacca acaaaaggac 3480

attgaagatg gtactaaggg tagtaaaaaa agtccttttg caactaaacg agcttacttc 3540

tattgggtaa cacgcgagca aggctcgttt gagtggttta agggtgtgat ggatgaggtc 3600

tcggagaacg atcaagaagg cctaattgag cttcacaact actgcaccag cgtttatgaa 3660

gaaggcgatg cccggtctgc actaatcaca atgcttcaat caatccacca agctaagagt 3720

ggcgtagaca ttgtctctgg gacaagagtc aagactcatt ttgctagacc aaattggcgc 3780

caagttttca aacgtgttac aattaatcat cctgatcaaa aaattggtta gtacgcatct 3840

ttgattacta ttcattaaaa ttaaaccgcc tctctacttc acaaactagg ggtaaggtcg 3900

atctttctgg actatattat atccagacct cacttatgaa attatacgtg ttaaattgac 3960

attcttattt tgaatttatg atattacagg tgtgttttat tgtggcccac aaggtctagt 4020

tggagaacta agacatctat ctcaagattt ctcacacaag acagacacaa agtttgaatt 4080

tcataaagaa aatttctaag 4100

<210> 14

<211> 5434

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 14

gttgaaaatt gtgtgtttgt tttttttgta tcaatcggtt gagaagtgcc tctcttgttt 60

ttttttcctc tctttcttct ttattcatat ttcactttct ttctttcttt caaaatttct 120

caccttcttt ctcttctctt ctttgcttct acagttttca tggccatcgg tgaaaaccgt 180

gtagagaaat attaaatttg atatatattg tgttaagtgt tggttggttg gttaatctct 240

ctcactgatg aagttatcgc ctctgagttt ctctacgagt tcgagtttca gtcacgccga 300

tggaatcgac gacggtgttg agttaatatc atctccattt gccggcggcg caatgctgcc 360

ggtttttctc aacgatttga gtcgtaacag tggagaatcc ggaagcggta gtagctggga 420

aagagagctc gtggaggtta cgttagagct cgacgtcggc gatgactcca tactcgtctg 480

cggaatgtct gaggcggcgt ctgttgattc tcgggcgaga tcggtggatt tggtaaccgc 540

gaggttatcg aggaatttat cgaatgcgtc tacgagaatc cgccagaaac tagggaagtt 600

actgaggtcg gaatcgtgga aaacgacgac gtcttctacc gccggcgaga gagatagaga 660

tcttgaacgt cagacggcgg tgactttggg gattctgacg gcgagggata aacggaaaga 720

agacgcgaaa ctacagcgat caacgtcgag tgcgcaaaga gcgttaaaag gattacagtt 780

tataaataaa accacgagag gaaatagctg cgtctgcgat tgggattgcg attgcgatca 840

gatgtggaaa aaagttgaaa agcgattcga atcgctttct aaaaatggat tactcgcccg 900

agacgatttt ggcgagtgcg ttggtaagtc aataaatatc tatatataca tacgaaaatg 960

atcatggacc aatccactgt tttgtaaatt taaagggatg gtggattcga aggactttgc 1020

ggtgagtgta ttcgacgcgt tggctaggag aagaagacag aaactggaga agataacgaa 1080

agatgaactt catgatttct ggttacagat ttctgaccaa agcttcgacg cacgtcttca 1140

aattttcttc gacatgtacg tgtctctttg attctctctt tctttttatt tttaagtcaa 1200

gtttttgggt ttgtctttac gtttataact gttacgggaa taacgcactg caattagcaa 1260

aagcataaga aaacaaatgt attgatccaa gtgcaacgaa taattcagtt gacgttaata 1320

attgaagaaa gttttatcta taattgatat tccaaattct gaataacgcc ggcgaggtct 1380

catcaacttg cattttaata agatttgagt tgatttttgt tttctggtgg tattttatga 1440

agtttagctg attaaattca cttattgagt aaaaatgttg ttgattaaag ataatgttag 1500

cttaccaatc attctttgat tgtaatttgt tatctcttta gtgagaacta ccgcaacttc 1560

acaataattt tctacaattt tgggaattat tagccttaaa taaaataaca tgtgttggtt 1620

aagaacttaa gatgaattgt gattgttttt aaaaccaaat atcatatcct tgtagagaga 1680

tatagataga tacatcattt aaatcacgtg gtgttcttag ccgttggatc tggtagttca 1740

cgcaatcaat ggcgaaaatg gtctagtgag tttaagtaat gggccgcggt ttacttggcc 1800

cagtaaaaaa taaactttta ccaaattttc aaaaagtttc ctctttcagc tttctttttc 1860

ccgcaataag tttcttgcaa gtataagata caatgactct tatatacacc aagctgccaa 1920

aatgagtttg caatttttta ctctttttat ggccaaacat ttaattcagt aacattatgg 1980

tttacactca actcgttttg ttattctgtc atttttatga cataactaaa tatattttgt 2040

tgtttaatga ttctacaatt ctgttataat ttgtagtgtt actgaaagaa aaaaaaagat 2100

tctaactggt accggttttt tgttacaggg ctgatagtaa cgaagacggg aagatcacta 2160

gagaagaaat caaagaagta actttcttta ttcttcaaca tagagattac gtttaaaaca 2220

atcaatctct ttaacaactc ttgtactttt gaaacgcagc ttctaatgct aagtgcatca 2280

gcaaacaagt tagcaaaact caaggaacaa gctgaagagt acgcatctct gatcatggaa 2340

gagcttgatc ctgagaattt tggatacatt gaggttcaaa aaaacataat atattcgaac 2400

acacaatcaa cgattctttt atgtttctta acttttttgt gttgtctcag ttatggcaac 2460

tcgagacact cttacttcag agagacgcat acatgaatta cagtagacca cttagcacaa 2520

caagcggtgg agtaagtaca ccaagacgga atctaataag acctcgtcat gtggttcaaa 2580

agtgtagaaa aaaacttcaa tgtctgattc tagataactg gcaaagaagc tgggttcttt 2640

tagtgtgggt catgcttatg gccatcctct ttgtctggaa gtttctagag tacagagaaa 2700

aagctgcgtt taaggtcatg gggtattgtt taaccactgc taaaggagct gcagagactc 2760

ttaagctcaa catggctctg gttttattac ctgtctgtag aaacacattg acttggctaa 2820

gatctacacg cgctcgagct tgtgttcctt ttgatgacaa catcaatttc cataaggtac 2880

cttccttcag tctttcgatt tttcagtttt agtcagagca caacatttga gataaactct 2940

ttctctctct cttgtcctgt ttgtgtgcca gattattgct tgtgccatag cgattgggat 3000

acttgttcat gctggtactc atttggcgtg tgatttcccc cggattataa actcgagtcc 3060

agaacaattt gtcttgatcg cttctgcctt caatggtact aagcctacat tcaaagacct 3120

gatgacaggt gcagaaggaa tcaccgggat ctcgatggtg atcttgacaa ccattgcatt 3180

cacattagca tcaactcatt tcaggagaaa ccgtgtgagg cttccggcac cacttgatcg 3240

gttgactggc tttaacgcat tctggtacac tcatcacctt ctagtggttg tctacatcat 3300

gctcattgtc cacgggacct tcctgttctt tgctgataag tggtatcaga aaacggtaag 3360

cagcagcaac cataatctct ctttacttta aaccgaaaaa ccaaatgtcc tgaaacctaa 3420

attattgttg attaccactg ttcccagact tggatgtaca tctcggttcc tttggtgctc 3480

tacgtggcag aacgaagtct gcgagcttgt aggtcaaagc attactctgt caaaatcctc 3540

aaggcaacca ccataaaccg agcaaattca ttcctgttaa aaggtgtttt aataacaaaa 3600

taaacccatg aaccttcaat ctgatctctg caggtttcca tgctacctgg agaagtactc 3660

agcttaatca tgtcaaagcc tcctggattc aagtacaaga gcggtcagta catattcttg 3720

cagtgtccaa ctatctctcg atttgaatgg tgagtactga taccgaattc ccatagtgtt 3780

aacaaacatc tcaggtttca ctcagaaaga tattccctaa acttttccag gcacccattt 3840

tctattacct ctgcaccagg agatgaccaa cttagtgttc acatccgaac actcggagac 3900

tggacagagg agctacgacg agttctaact gtgggcaaag atctttcaac atgcgtgatt 3960

gggcgttcaa aattctctgc ctattgcaat attgatatga taaagtaaat ttcaagacat 4020

ctttgctttc tccttactca cttggatacc tattattaac atggcttgtt gatcctgcac 4080

tgacagccga ccaaaattac tagtagacgg tccatatgga gctccagcac aggactacag 4140

aagctatgat gtcttgctcc tcattggact gggaatagga gctactcctt tcataagcat 4200

cctgaaggat ctgctgaaca attcaagaga cgaacaaaca gtaagtccaa ttcagtgcac 4260

cagaaaagtt agtcttttct agaaccttgc accaaataca tatctctaaa cttcatatgg 4320

cacaggacaa tgaattcagc agatcggatt tcagctggaa tagctgtaca tcttcatata 4380

ccacagcaac cccaacttca acacatggag ggaaaaagaa agcagtgaag gctcactttt 4440

actgggtcac aagggagcca ggatccgttg aatggtttag aggagtaatg gaggaaatat 4500

cagacatgga ctgcagagta atactaaaca ccagcatatc attcatttgc atcacctctt 4560

taccattgat cgatatacta acatattcct tcatcttctc gtaatacagg gccagattga 4620

gttgcacaac taccttacaa gcgtatatga tgaaggtgat gcaagatcaa ccctgataaa 4680

aatggtccag gctttgaacc atgccaaaca tggagttgat attctatccg gaacacgggt 4740

aagaattgat gcagggcata taaccagaaa ctatttgcca atattaccta taactgacaa 4800

aataacatca ttcaaacagg tgagaacaca ttttgcaagg cccaactgga aggaagtctt 4860

cagtagcata gcaagaaagc atccaaattc gacagtcggt aattattcta tacaatcagc 4920

tgcttaccat caagacatct tataaatctc tcttctagga ctaacgaatt aataccattg 4980

gaaaagaaag aaaacttgca ataggaatta tgacacatgc aaacacatga acatgtctgc 5040

taatatttag gccaggtatt acgaagcata tgtgaaaaca atttatataa ttgtgcagga 5100

gtgttttact gcggcataca aaccgtggca aaggagttga agaagcaagc acaagacatg 5160

agtcaaaaaa caaccacacg gttcgagttc cataaggagc atttctaatt caatgttaca 5220

agccagcttc tatcactttc cctaaagtga gattcaaatc cagcccgcca ttattttttt 5280

tcacactaac ttcacagtca cttagcaaag tagattatat acactatgtt aaaaaaggaa 5340

acattgtctt gcctgtttgt atagaccccg cctaaaggca tgaaaataac aaatctaagt 5400

tactatacga aataatatac gtgtctttcc caag 5434

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 15

tccgacttcg ccgaatgcat 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

gcaatctggg ctcgagcaac 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

ggtgaggcag ttcgcgtcga 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

tctagcaagt aatccgtctt 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 19

taagcacaac cactgtcgac 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

tagctagcaa gctcgaaaag 20

Claims (9)

1. A method of breeding a plant sterile line comprising the steps of: reducing expression or activity of a Rboh gene or a protein encoded by the Rboh gene in the plant, wherein the plant sterile line is a high temperature sensitive plant sterile line; the high temperature is more than or equal to 30 ℃;

the plant is rice, the Rboh gene comprises OsRBOHA, osRBOHB and OsRBOHE, the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 1, the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 2, and the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 3;

or the plant is tomato, the Rboh gene comprises LeRBOHB, leRBOHE and LeRBOH, the amino acid sequence of the LeRBOHB is shown as SEQ ID NO. 5, the amino acid sequence of the LeRBOHE is shown as SEQ ID NO. 6, and the amino acid sequence of the LeRBOH is shown as SEQ ID NO. 7.

2. The method of claim 1, wherein the method is performed at a maximum temperature of the growing environment of at least 30 ℃ during anther development.

3. The method according to claim 2, wherein the method is carried out under conditions such that the highest temperature of the growing environment is not less than 30 ℃ during the flowering period of the plant.

4. The method of claim 1, wherein the Rboh gene is a gene that is specifically expressed during plant anther development.

5. The application of the Rboh gene or the inhibitor of the coded protein thereof is characterized in that the Rboh gene or the inhibitor of the coded protein thereof is used for cultivating a plant sterile line, or is used for preparing a reagent or a kit for cultivating the plant sterile line, wherein the plant sterile line is a high-temperature sensitive plant sterile line, and the high temperature is more than or equal to 30 ℃; the inhibitor is a gene editing reagent;

the plant is rice, the Rboh gene comprises OsRBOHA, osRBOHB and OsRBOHE, the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 1, the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 2, and the amino acid sequence of the OsRBOHE is shown as SEQ ID NO. 3;

or the plant is tomato, the Rboh gene comprises LeRBOHB, leRBOHE and LeRBOH, the amino acid sequence of the LeRBOHB is shown as SEQ ID NO. 5, the amino acid sequence of the LeRBOHE is shown as SEQ ID NO. 6, and the amino acid sequence of the LeRBOH is shown as SEQ ID NO. 7.

6. A method for restoring fertility to a plant sterile line, comprising the steps of: cultivating the plant sterile line of claim 1 under a low temperature condition, wherein the low temperature condition is that the highest growth environment temperature is less than 25 ℃.

7. A method of converting a plant from sterile to fertile comprising the steps of: culturing a plant sterile line cultivated by the method of claim 1 under a low temperature condition that the highest growth environment temperature is less than 25 ℃.

8. A plant breeding method, characterized by comprising the following steps: crossing the plant sterile line of claim 1 with another parent plant to obtain a hybrid.

9. A plant breeding method comprising the step of maintaining sterility of a plant; a step of changing the plants from sterile to fertile; and/or maintaining the plant fertility and breeding steps;

in said step of maintaining sterility of the plant, comprising maintaining a sterile line of the plant grown according to the method of claim 1;

in the step of changing a plant from sterile to fertile, comprising changing the plant from sterile to fertile using the method of claim 7.