CN109679965B - Gene for regulating and controlling leaf type development of poplar and application thereof - Google Patents

Abstract

The invention relates to a gene for regulating poplar leaf shape development and an application thereof, belonging to the fields of plant genetic engineering and biotechnology; the nucleotide sequence for regulating and controlling poplar leaf shape development gene PagKNAT2/6b is shown in sequence 1 in the sequence table; PagKNAT2/6b The amino acid sequence of the expressed protein of 6b is shown in sequence 2 in the sequence listing. In the present invention, the PagKNAT2/6b gene is transferred into the 84k poplar, and the transgenic poplar overexpressing PagKNAT2/6b is compared with the wild type, and obvious curled and wrinkled leaf types appear, indicating that the PagKNAT2/6b gene is the key to regulating the development of poplar leaves It has important application value in the field of forest genetic engineering and clone forestry.

Description

A gene that regulates the development of poplar leaf shape and its application

technical field

The invention relates to a transcription factor for regulating the development of poplar leaves and its application, in particular to a gene for regulating the development of poplar leaves and its application, belonging to the field of plant genetic engineering and biotechnology.

Background technique

As an economic tree species widely distributed in the world, poplar has become the most famous fast-growing timber forest and greening tree species in my country due to its rapid growth, easy reproduction, wide use of wood and strong adaptability. Its cultivation and breeding work has always been paid attention to. .

In recent years, with the completion of poplar genome sequencing, molecular biology techniques have been gradually applied to the genetic improvement of poplar. The variety of poplars, luxuriant foliage, and late leaves in autumn make it one of the important street trees. Ornamental characteristics such as canopy shape, flower color, leaf shape, leaf color and seasonal changes in leaf color have become important indicators for the selection of urban greening tree species.

With its bright purple-red leaves, Populus chinensis has become a new variety for greening ornamental and timber use. With the intensification of urban, garden and road greening construction, the selection of new varieties of fast-growing poplar with excellent leaf type has become an urban garden. Greening, urban and rural greening, and channel greening are the top priorities for tree species cultivation.

The development of plant leaves is the key to the phylogeny of plant branches. The leaf type, leaf color and seasonal changes of leaf color have different visual effects, which can form different visual landscapes, which are different in garden viewing, horticultural configuration, and roadway greening. aesthetic value.

In the past ten years, great progress has been made in the study of the mechanism of plant leaf development using the model plant Arabidopsis thaliana, which provides many useful inspirations for the breeding and improvement of forest trees. Leaf development starts from the apical meristem (SAM), and the Arabidopsis class I KNOX gene affects the formation and development of lateral organs, especially leaf primordia, by regulating the activity of the shoot apical meristem. The leaf morphology of transgenic plants was significantly changed. Therefore, it is of great value and significance to study the relationship between class I KNOX gene and leaf development for the cultivation and production of new poplar varieties with ornamental value and landscape ecological value.

Studies have shown that abnormal expression of KNOX gene during the formation of Arabidopsis, maize and tobacco leaves can regulate the division and differentiation of leaf primordial cells, resulting in leaf phenotype variation, indicating that KNOX gene is related to the production of complex leaf shapes. The plant KNOX gene family is divided into two subclasses: class I KNOX genes and class II KNOX genes. The class I KNOX genes in Arabidopsis thaliana contain 4 members, namely: SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), KNOTTED- like from Arabidopsis thaliana 2 (KNAT2) and KNAT6. Class I KNOX genes play a key role in the establishment and functional maintenance of plant meristems, and are important regulators of plant meristem initiation and organogenesis. However, at present, only preliminary studies on the functions of STM and BP homologous genes ARBORKNOX1 (ARK1) and ARBORKNOX2 (ARK2) have been completed in poplar, and it is believed that they are involved in regulating the differentiation of woody plant cambium, while the other members of class I KNOX genes in poplar Function is unclear. Poplar class I KNOX gene (the homologous gene of KNOX1 in poplar) was genetically transformed into tobacco, petunia, carnation, celosia and other flower plants, and all showed a series of novel and peculiar ornamental leaf types in leaf shape and other forms.

At present, in woody plants, there are few reports on the cultivation of new varieties of leaf type development. Therefore, it is of great significance to use woody plants as the research object and combine molecular biology and genetic engineering technology to analyze the regulation of leaf type development by PagKNAT2/6b in poplar, increase the diversity of poplar leaf type through molecular breeding, and cultivate new poplar green varieties. .

SUMMARY OF THE INVENTION

Aiming at the deficiencies in the prior art, the main purpose of the present invention is to provide a gene PagKNAT2/6b that regulates the development of poplar leaves.

A gene that regulates the development of poplar leaf shape is named PagKNAT2/6b gene, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

Another object of the present invention is to provide an expression protein that regulates the developmental gene of poplar leaf shape.

An expression protein that regulates poplar leaf morphogenesis genes, the amino acid sequence of which is shown in sequence 2 in the sequence table.

Another object of the present invention is to provide a cloning method for regulating the developmental gene of poplar leaf shape.

A cloning method for regulating and controlling poplar leaf morphogenesis gene, the steps are as follows:

(1) Using 84K (P.alba X P.glandulosa) poplar as material, using RNeasy Plant Mini kit and RNase-free DNase I kit (Qiagen, Hilden, Germany) to extract RNA;

(2) By using the SuperScript III first-strand synthesis system (LifeTechnologies, Carlsbad, CA, USA) to synthesize the first strand of cDNA, use Primer 5 software to design primers (the amplicons include start codons and stop codons), perform gene Full-length amplification (GATEWAY linker was introduced into the primer);

Among them, the PagKNAT2/6b ORF forward primer is shown in sequence 3 in the sequence listing;

PagKNAT2/6b ORF reverse primer is shown in sequence 4 in the sequence listing;

(3) The high-fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR, forward primer, reverse primer, template is 84K poplar cDNA, supplemented by sterile ddH ₂ O; ℃, 20s; 56℃, 20s; 68℃, 60s, 28 cycles; 68℃, 10min;

Finally, the full-length cDNA sequence of the obtained gene was 942bp, which was named PagKNAT2/6b gene.

Another object of the present invention is to provide a construction method of the gene PagKNAT2/6b that regulates the development of poplar leaf shape.

A kind of construction method of the plant expression vector regulating and controlling poplar leaf shape development gene, its steps are as follows:

(1) utilize cloning technology to construct the overexpression vector of PagKNAT2/6b gene, use specific PCR primer (PagKNAT2/6b ORF primer), take 84K cDNA as template, carry out PCR amplification, construct PagKNAT2/6b gene ORF into entry vector, The entry vector is PDNOR207, and the sequence is shown in sequence 5 in the sequence listing;

The reaction system was fresh PCR product; PDNOR207 vector; BP Clonase II mixed enzyme; sterile ddH ₂ O supplemented; the reaction program was: 25°C for more than 5h;

(2) pick the positive clone from the screening culture plate to carry out PCR detection and sequencing verification, after the entry vector with the PagKNAT2/6b gene is linearized by Mlu I restriction endonuclease, the Gateway system is constructed to the plant expression vector PMDC32, The sequence is shown in sequence 6 in the sequence listing;

LR reaction was carried out, and the reaction system was: linearized entry cloning; purified target vector; LR Clonase II mixed enzyme; sterile ddH ₂ O supplementation; reaction conditions: 25 °C reaction for more than 5 h; after LR reaction, PagKNAT2/6b gene was introduced into plant expression vector In PMDC32, the strong expression promoter 35S is assembled at the 5' end of the PagKNAT2/6b gene, which enables the efficient expression of the PagKNAT2/6b gene in poplar; the 3' end of the PagKNAT2/6b gene is assembled into a strong terminator NOS, which is effective Terminate the transcription of PagKNAT2/6b gene;

(3) Assemble the hygromycin phosphotransferase HPT on the vector, as a selection marker for transgenic poplar, hygromycin can be used to screen the transgenic poplar; Assemble the LB and RB sequences on the vector to promote PagKNAT2 assembled in the middle The /6b gene expression framework and the selection marker gene HPT were integrated into the poplar receptor chromosome, and the overexpression vector was successfully constructed by PCR detection and sequencing verification, which was named PMDC32-PagKNAT2/6b.

Another object of the present invention is to provide a genetic transformation that regulates the gene for the development of poplar leaf shape.

A kind of genetic transformation regulating poplar leaf shape development gene, its steps are as follows:

The constructed PMDC32-PagKNAT2/6b overexpression vector was transferred into Agrobacterium GV3101 by electric shock method, and the PagKNAT2/6b gene was transferred into poplar mediated by Agrobacterium.

Another object of the present invention is to provide an application of the gene PagKNAT2/6b that regulates the development of poplar leaf shape.

The application of the gene PagKNAT2/6b regulating the leaf shape development of poplar in regulating the development of poplar leaves.

Beneficial effects:

In the present invention, the PagKNAT2/6b gene is cloned by using 84k poplar as material; at the same time, the overexpression vector PMDC32 is constructed, and the gene is located after the promoter P35S. Overexpressed, thereby regulating the development of poplar leaf shape. Among them, the PagKNAT2/6b gene is a key gene regulating the development of poplar leaf shape.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it does not mean to limit the scope of protection of the present invention.

Description of drawings

Figure 1 is a schematic structural diagram of the plant expression vector PMDC32-PagKNAT2/6b of the present invention.

Fig. 2-1 is a semi-quantitative detection chart of the transcription level of the transgenic poplar overexpressing PagKNAT2/6b of the present invention.

Figure 2-2 is a real-time quantitative detection chart of the transcription level of the transgenic poplar overexpressing PagKNAT2/6b according to the present invention.

Figure 3-1 is a graph comparing the phenotypes of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention in the first month of growth.

Figure 3-2 is a graph comparing the phenotypes of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention in the third month of growth.

Figure 4-1 is the leaf map of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention of one-month soil-cultivated seedlings.

Figure 4-2 is a statistical diagram of leaf size of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention for one-month soil-cultivated seedlings.

Figure 5-1 is a transparent view of the fourth expanded leaf of the transgenic poplar and non-transgenic poplar overexpressing PagKNAT2/6b according to the present invention, one-month soil-cultivated seedlings.

Figure 5-2 is a graph comparing the cell size of the dorsal-ventral surface of the fourth leaf of the transgenic poplar overexpressing PagKNAT2/6b of the present invention and the non-transgenic poplar, one-month soil-cultivated seedlings.

Fig. 6 is a graph comparing the tissue sections of the first month and the 4th leaf of non-transgenic poplar (84k) and the transgenic poplar of the present invention (OE5, OE7).

Figure 7 is a graph comparing the phenotype of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention after the annual soil cultured seedlings are flattened.

Detailed ways

The present invention will be further described below with reference to specific examples. For operations not described in detail in the following examples, reference may be made to molecular cloning and the operations described in the relevant kits for use.

Example 1 Cloning of PagKNAT2/6b gene

Using 84K (P.alba X P.glandulosa) poplar as material, using RNeasy Plant Mini kit and RNase-free DNase I kit (Qiagen, Hilden, Germany) to extract total RNA from soil cultured seedlings for one month, each sample About 1.0 μg of RNA was taken, and the first-strand cDNA was synthesized by using the SuperScript III first-strand synthesis system (LifeTechnologies, Carlsbad, CA, USA). With reference to the published genome sequence of P. trichocarpa, primers were designed using Primer 5 software (amplicons containing start codon and stop codon), perform full-length gene amplification (GATEWAY linker is introduced into the primer);

Among them, the PagKNAT2/6b ORF forward primer is shown in Sequence 3 in the sequence listing, see Table 1 below;

Table 1

The sequence of the PagKNAT2/6b ORF reverse primer is shown in Sequence 4 in the sequence listing, and is shown in Table 2 below;

Table 2

The high-fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR 12.5μl, forward primer (10μM) 1μl, reverse primer (10μM) 1μl, template (84K poplar cDNA) 100ng, sterile ddH ₂ O to make up to 25μl; Reaction program: pre-denaturation at 95°C, 5min; 95°C, 20s; 56°C, 20s; 68°C, 60s, 28 cycles; 68°C, 10min;

The full-length cDNA sequence of the finally obtained gene is 942bp, named PagKNAT2/6b gene, and the sequence is shown in sequence 1 in the sequence table, and is shown in Table 3 below;

table 3

The compiled and expressed protein sequence is shown in Sequence 2 in the sequence listing, and is shown in Table 4 below;

Table 4

Example 2 Construction of PagKNAT2/6b gene plant expression vector

Utilize cloning technology to construct the overexpression vector of PagKNAT2/6b gene, use specific PCR primer (PagKNAT2/6b ORF primer of Example 1), take 84K cDNA as template, carry out PCR amplification, construct PagKNAT2/6b gene ORF into entry vector , the entry vector is PDNOR207, the sequence is shown in sequence 5 in the sequence listing, see Table 5 below;

table 5

The reaction system is Fresh PCR product (fresh PCR product) 150ng; PDNOR207 vector (vector) 75ng; BP Clonase (clonase) II enzyme mix (mixed enzyme) 0.8 μl; sterile ddH ₂ O supplemented to 4 μl; reaction program: 25°C The reaction is more than 5h.

The positive clones were picked from the screening plate for PCR detection and sequencing verification. After the entry vector with the PagKNAT2/6b gene was linearized by Mlu I restriction enzyme, the Gateway system was constructed to the plant expression vector PMDC32, and the sequence was as follows Sequence 6 in the list is shown, see Table 6 below;

Table 6

LR reaction was carried out, and the reaction system was: Linearized entry clone (linearized entry clone) 75ng; purified destination vector (purified destination vector) 150ng; LR Clonase (clonase) II enzyme mix (mixed enzyme) 0.8 μl; sterile ddH ₂ O supplemented 4μl; reaction conditions: 25°C for more than 5h; after the LR reaction, the PagKNAT2/6b gene is introduced into the plant expression vector PMDC32, and the 5' end of the PagKNAT2/6b gene is assembled into a strong expression promoter 35S, which can make the PagKNAT2/6b gene in the plant expression vector PMDC32. It is highly expressed in poplar; it is assembled into a strong terminator NOS at the 3' end of the PagKNAT2/6b gene, which can effectively terminate the transcription of the PagKNAT2/6b gene. As shown in Figure 1, it is the plant expression vector PMDC32-PagKNAT2/6b of the present invention. The structure of , among which, from the top left-handed, are the strong terminator NOS, resistance to hygromycin, resistance to kanamycin, PBR332 terminal, 2*35S strong expression promoter.

Assemble hygromycin phosphotransferase HPT on the vector, as a selection marker for transgenic poplar, hygromycin can be used to screen transgenic poplar; Assemble LB and RB sequences on the vector to promote the PagKNAT2/6b gene assembled in between The expression framework and screening marker gene HPT were integrated into the poplar receptor chromosome, and the overexpression vector was successfully constructed by PCR detection and sequencing verification, which was named PMDC32-PagKNAT2/6b. The gene is located behind the promoter 35S. Driven by the promoter 35S, the PagKNAT2/6b gene can be highly expressed in poplar.

Example 3 Genetic transformation of PagKNAT2/6b gene

The constructed PMDC32-PagKNAT2/6b overexpression vector was transferred into Agrobacterium GV3101 by electroshock method, and the PagKNAT2/6b gene was transferred into poplar (P. silver glandularis, 84K poplar, the same below) mediated by Agrobacterium tumefaciens, The transformation steps were as follows: the hybrid poplar clone 84K tissue culture seedlings used for genetic transformation were cultured under the conditions that the culture temperature was 23-25°C, the light was 16/8h (day/night), and the light intensity was 50 μM m ^-2 s ^-1 , Agrobacterium containing PMDC32-PagKNAT2/6b expression vector infects 84K leaf discs at OD600=0.6～0.8, and the infected leaf discs are in adventitious bud induction medium (SIM, Murashige-Skoog (MS) basic medium) Add 0.5mg/l 6-benzyl aminopurine (6-BA, 6-benzylaminopurine) and 0.05mg/l naphthaleneacetic acid (NAA, naphthalene acetic acid), and co-culture at 23±2°C in the dark for 3 -4 days; the leaf discs after co-cultivation were transferred to SIM containing 3mg/L hygromycin B (hygromycin B) and 200mg/LTimentin (Timentin), and the culture temperature was 23-25°C and the light was 16/L. The resistant adventitious buds were induced and screened under the conditions of 8h (day/night) and a light intensity of 50 μM m ^-2 s ^-1 ; 200mg/LTimentin rooting medium (RIM, 1/2MS basic medium supplemented with 0.05mg/L IBA (indole butyric acid) and 0.02mg/L NAA) until adventitious roots were induced; DNA was extracted from the leaves of rooted plants, PCR verify.

The application of embodiment 4PagKNAT2/6b gene

The 84K poplar was genetically transformed by the Agrobacterium-mediated leaf disc method (Liu et al., 2014a). The 84K tissue culture seedlings used for genetic transformation were grown at a temperature of 23-26°C, and the light conditions were 16h light/8h dark, The light intensity was 50 μM·m ^-2 ·s ^-1 in a culture room, and 84K poplar leaf discs were infected with Agrobacterium containing 35S:PtGRF1/2d and OD ₆₀₀ =0.6-0.8, and the infected leaf discs were indeterminate. Bud induction medium SIM, namely Murashige-Skoog (MS) minimal medium supplemented with 0.5mg/L 6-BA (6-benzylaminopurine) and 0.05mg/L NAA (naphthalene acetic acid), at 24±2℃ Cultured in the dark for 3 days at the same temperature, the leaf discs after dark culture were transferred to SIM medium containing 200 mg/L Timentin and 3 mg/L hygromycin B, and the culture temperature was 23 Resistant adventitious buds were induced and screened under the conditions of -26 ℃, light conditions of 16h light/8h dark and light intensity of 50μM·m ^-2 ·s ^-1 . Adventitious shoots were transferred to RIM rooting medium containing 200mg/L Timentin and 3mg/L hygromycin B (1/2MS minimal medium supplemented with 0.05mg/L IBA and 0.02mg/LNAA) until adventitious roots were induced, and transgenic poplars overexpressing PagKNAT2/6b were obtained, and transgenic poplar (OE5, OE7) plants were selected respectively.

Compared with non-transgenic poplar (CK), transgenic poplar (OE5, OE7) plants obtained by transgenic technology showed leaf phenotypes of smaller leaves, shriveled leaves, disorganized leaf veins, curled leaves, and wrinkled leaves. With the increase of planting time, the leaves of OE5 and OE7 showed a phenotype that was not easy to senesce and fall off (Figure 3-2). Figure 7. From the comparison of the phenotypes of the biennial transgenic poplar (OE5, OE7) plants and the non-transgenic poplar (CK) after stubble, it can be seen that the leaf type of the transgenic poplar (OE5, OE7) plants is relatively stable.

The experimental results are shown in Figure 2-1 and Figure 2-2, Figure 2-1 is a semi-quantitative detection chart of the transcription level of the transgenic poplar overexpressing PagKNAT2/6b of the present invention (actin is actin in the figure), Figure 2 -2 is the real-time quantitative detection chart of the transcription level of the transgenic poplar overexpressing PagKNAT2/6b according to the present invention, using tissue culture seedlings about 1 month old as the material, extracting RNA and performing reverse transcription, using SYBR Premix Ex TaqTM Kit (TaKaRa ) kit, with ACTIN gene as the internal reference, completed with LightCycler 480 (Roche) real-time quantitative instrument). (for 2 lines of transgenic poplar overexpressing PagKNAT2/6b). As can be seen from Figure 2-2, compared with the non-transgenic poplar (84K), the mRNA level of the transgenic poplar (OE5, OE7) was significantly higher; as shown in Figure 3-1, the non-transgenic poplar ( 84k) The phenotype comparison diagram of the soil culture seedlings of the present invention (OE5, OE7) in the first month of growth; Fig. 3-2 is the non-transgenic poplar (84k) and the present invention transgenic poplar (OE5, OE7) Phenotypic comparison chart of soil culture seedlings in the third month of growth; photographs were taken with an ordinary camera. As can be seen from Figure 3-1 and Figure 3-2, compared with non-transgenic Populus alba (84K), transgenic poplars (OE5, OE7) have smaller leaves, shriveled leaves, and disordered leaf veins, indicating that PagKNAT2/6b gene It is a key regulatory gene that regulates the development of poplar leaves; as shown in Figure 4-1, it is the leaf map of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention (from left to The right is from the top to the bottom), as shown in Figure 4-2, it is a statistical diagram of the leaf size of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention for a month of soil culture seedlings, from Figure 4-1 As can be seen from Figure 4-2, the leaves of the transgenic poplars (OE5, OE7) become smaller and the wrinkle increases; as shown in Figure 5-1, the transgenic poplars and non-transgenic poplars that overexpress PagKNAT2/6b of the present invention , the transparent diagram of the 4th unfolded leaf blade of soil culture seedlings in one month (the white bar in the figure is 3 cm), as shown in Figure 5-2, is the transgenic poplar and the non-transgenic poplar that the present invention overexpresses PagKNAT2/6b , a comparison chart of the cell size of the dorsal-ventral surface of the fourth leaf of one-month soil cultured seedlings, it can be seen from Figure 5-1 and Figure 5-2 that the leaf cells of transgenic poplars (OE5, OE7) became smaller and increased; as shown in Figure 6 , is the comparison chart of the tissue sections of the first month and the fourth leaf of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention; Note: xy, xylem (xylem); ph, phloem ( phloem), it can be seen from Figure 6 that the leaves of the transgenic poplars (OE5, OE7) become smaller; as shown in Figure 7, it is the biennial soil of the non-transgenic poplar (84k) and the transgenic poplar (OE5, OE7) of the present invention The phenotype comparison diagram after planting and flat stubble, it can be seen from Figure 7 that the leaf shape of transgenic poplar (OE5, OE7) plants is relatively stable.

In the present invention, the PagKNAT2/6b gene is transferred into 84k poplar, and the transgenic poplar overexpressing PagKNAT2/6b is compared with the wild type, and obvious leaf types with small leaves, shriveled leaves, disordered leaf veins, curled leaves, and folds appear. PagKNAT2/6b gene is a key regulatory gene regulating poplar leaf development, and has important application value in the field of forest genetic engineering and clonal forestry.

sequence listing

<110> Forestry Research Institute, Chinese Academy of Forestry

<120> A gene regulating the development of poplar leaf shape and its application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 930

<212> DNA

<213> PagKNAT2/6b

<400> 1

atggacggaa cgtacggtct gcactcaacg gtggcggact actcagacaa ggcgttgatg 60

tcgccggagg attttatttt acaatcggag taccagagct tgctgtcttc agaaactctt 120

cggctgcgga ttccgattct tggatccgaa gagttgcttt cagaggcggc ttcgatcaga 180

acagaagaag atatgtccgc tttgatcaaa gccaaaattg cctcgcatcc ttgctatcct 240

cgtttactcg aggcttatat cgattgccag aaggtaggggg cgcctccggg gatagcgtgt 300

ttcttggatg aaatccggcg agaaaacgac cttttcaagc aaggcgctgt ctccacgtac 360

tggggagctg atcccgagct agacgagttc atggaaacct actgtgattt gttggtgaag 420

tataaatctg atcttgaaag gcctttagat gaagcaacaa ccttcttgaa caagattgaa 480

atgcagcttc gaaatctctg caccggtgcc tccatcagaa gcctctctga tgaaggtgcg 540

gcatcttcag atgaggagtt aagtgtaggg gagttggata tgcatgaagc tcaaccaagc 600

ggtgaagacc gagagcttaa agataaactt ctccgcaggt ttggaggtca tattggtacg 660

ctgaagctgg agttctcaaa gaaaaaaaag aaaggaaagc taccgaagga agctaggcaa 720

accctacttg gatggtggga tgctcactat aaatggccat atccaactga agctgataag 780

atagcattgg ccgaatcgac gggactagat cagaagcaaa ttaataattg gtttataaat 840

caacgtaagc gccactggaa accatctgag aacctgcaat ttgctgttat ggataatctt 900

tctgggcagt tctttacaga agacgactga 930

<210> 2

<211> 238

<212> PRT

<213> Expressed protein of PagKNAT2/6b (unknown)

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Met Asp Gly Thr Tyr Gly Leu His Ser Thr Val Ala Asp Tyr Ser Asp

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Lys Ala Leu Met Ser Pro Glu Asp Phe Ile Leu Gln Ser Glu Tyr Gln

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Ser Leu Leu Ser Ser Glu Thr Leu Arg Leu Arg Ile Pro Ile Leu Gly

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

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Met Ser Ala Leu Ile Lys Ala Lys Ile Ala Ser His Pro Cys Tyr Pro

65 70 75 80

Arg Leu Leu Glu Ala Tyr Ile Asp Cys Gln Lys Val Gly Ala Pro Pro

85 90 95

Gly Ile Ala Cys Phe Leu Asp Glu Ile Arg Arg Glu Asn Asp Leu Phe

100 105 110

Lys Gln Gly Ala Val Ser Thr Tyr Trp Gly Ala Asp Pro Glu Leu Asp

115 120 125

Glu Phe Met Glu Thr Tyr Cys Asp Leu Leu Val Lys Tyr Lys Ser Asp

130 135 140

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

145 150 155 160

Met Gln Leu Arg Asn Leu Cys Thr Gly Ala Ser Ile Arg Ser Leu Ser

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Asp Glu Gly Ala Ala Ser Ser Asp Glu Glu Leu Ser Val Gly Glu Leu

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Asp Met His Glu Ala Gln Pro Ser Gly Glu Asp Arg Glu Leu Lys Asp

195 200 205

Lys Leu Leu Arg Arg Phe Gly Gly His Ile Gly Thr Leu Lys Leu Glu

210 215 220

Phe Ser Lys Lys Lys Lys Lys Lys Gly Lys Leu Pro Lys Gly Ser

225 230 235

<210> 3

<211> 56

<212> DNA

<213> PagKNAT2/6b ORF forward primer (artificial sequence)

<400> 3

ggggacaagt ttgtacaaaa aagcaggctg catggacgga acgtacggtc tgcact 56

<210> 4

<211> 57

<212> DNA

<213> PagKNAT2/6b ORF reverse primer (artificial sequence)

<400> 4

ggggaccact ttgtacaaga aagctgggta tcagtcgtct tctgtaaaga actgccc 57

<210> 5

<211> 5585

<212> DNA

<213> Entry vector PDNOR207 (artificial sequence)

<400> 5

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagcc aggaagagtt 60

tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca 120

gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc 180

gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga 240

aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct 300

cgcgttaacg ctagcatgga tctcgggccc caaataatga ttttattttg actgatagtg 360

acctgttcgt tgcaacaaat tgatgagcaa tgctttttta taatgccaag tttgtacaaa 420

aaagcagaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat 480

aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg aatcaactac 540

ttagatggta ttagtgacct gtagtcgacc gacagccttc caaatgttct tcgggtgatg 600

ctgccaactt agtcgaccga cagccttcca aatgttcttc tcaaacggaa tcgtcgtatc 660

cagcctactc gctattgtcc tcaatgccgt attaaatcat aaaaagaaat aagaaaaaga 720

ggtgcgagcc tctttttttgt gtgacaaaat aaaaacatct acctattcat atacgctagt 780

gtcatagtcc tgaaaatcat ctgcatcaag aacaatttca caactcttat acttttctct 840

tacaagtcgt tcggcttcat ctggattttc agcctctata cttactaaac gtgataaagt 900

ttctgtaatt tctactgtat cgacctgcag actggctgtg tataagggag cctgacattt 960

atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg tggctgagat 1020

cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg gtcatcatgc 1080

gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact ttatctgaca 1140

gcagacgtgc actggccagg gggatcacca tccgtcgccc gggcgtgtca ataatatcac 1200

tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa accttaaact 1260

gcatttcacc agtccctgtt ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg 1320

acctcagcca tcccttcctg attttccgct ttccagcgtt cggcacgcag acgacgggct 1380

tcattctgca tggttgtgct taccagaccg gagatattga catcatatat gccttgagca 1440

actgatagct gtcgctgtca actgtcactg taatacgctg cttcatagca cacctctttt 1500

tgacatactt cgggtataca tatcagtata tattcttata ccgcaaaaat cagcgcgcaa 1560

atacgcatac tgttatctgg cttttagtaa gccggatcca cgcgattacg ccccgccctg 1620

ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 1680

gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 1740

tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 1800

actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 1860

agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 1920

ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 1980

gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 2040

catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 2100

aaacttgtgc ttattttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 2160

ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 2220

ttgggatata tcaacggtgg tatatccagt gattttttttc tccattttag cttccttagc 2280

tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg 2340

aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg 2400

cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac 2460

aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc 2520

actaatacca tctaagtagt tgattcatag tgactggata tgttgtgttt tacagtatta 2580

tgtagtctgt ttttttatgca aaatctaatt taatatattg atatttatat cattttacgt 2640

ttctcgttca gctttcttgt acaaagtggg cattataaga aagcattgct tatcaatttg 2700

ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgcagc 2760

tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat 2820

gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 2880

aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata 2940

aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc 3000

ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag 3060

atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt 3120

ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat 3180

tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt 3240

tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat 3300

ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg 3360

atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc 3420

cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg 3480

acgagggggaa attaggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta 3540

cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc 3600

actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg 3660

gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg 3720

gagactgcga gatcatagat atagatctca ctacgcggct gctcaaacct gggcagaacg 3780

taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc 3840

ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct 3900

acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca 3960

gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg 4020

cgactgccct gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc tccataacat 4080

caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac 4140

aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc 4200

ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca gtttacgaac 4260

cgaacaggct tatgtcaact gggttcgtgc cttcatccgt ttccacggtg tgcgtcaccc 4320

ggcaaccttg ggcagcagcg aagtcgaggc atttctgtcc tggctggcga acgagcgcaa 4380

ggtttcggtc tccacgcatc gtcaggcatt ggcggccttg ctgttcttct acggcaaggt 4440

gctgtgcacg gatctgccct ggcttcagga gatcggaaga cctcggccgt cgcggcgctt 4500

gccggtggtg ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg aaggcgagca 4560

tcgtttgttc gcccaggact ctagctatag ttctagtggt tggctactaa taggttgtat 4620

tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg 4680

cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa 4740

tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt 4800

ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc 4860

atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 4920

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 4980

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5040

aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5100

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5160

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5220

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5280

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5340

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5400

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 5460

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 5520

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 5580

atgtt 5585

<210> 6

<211> 11672

<212> DNA

<213> Plant expression vector PMDC32 (artificial sequence)

<400> 6

atgtataatt gcgggactct aatcataaaa acccatctca taaataacgt catgcattac 60

atgttaatta ttacatgctt aacgtaattc aacagaaatt atatgataat catcgcaaga 120

ccggcaacag gattcaatct taagaaactt tattgccaaa tgtttgaacg atcggggaaa 180

ttcgagctcc accgcggtgg gcggccgctc tagaactagt taattaagga attatcgaac 240

cactttgtac aagaaagctg aacgagaaac gtaaaatgat ataaatatca atatattaaa 300

ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact 360

atggtcgacc tgcagactgg ctgtgtataa gggagcctga catttatatt ccccagaaca 420

tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc acttcttccc 480

cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag ctttcatccc 540

cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga cgtgcactgg 600

ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt acatccacaa 660

acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt tcaccagtcc 720

ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc gggcgacctc agccatccct 780

tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt ctgcatggtt 840

gtgcttacca gaccggagat attgacatca tatatgcctt gagcaactga tagctgtcgc 900

tgtcaactgt cactgtaata cgctgcttca tagcacacct ctttttgaca tacttcgggt 960

atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg catactgtta 1020

tctggctttt agtaagccgg atcctctaga ttacgccccg ccctgccact catcgcagta 1080

ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacagacgg catgatgaac 1140

ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa 1200

aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 1260

ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 1320

gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 1380

gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 1440

agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaattccgg 1500

atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 1560

tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 1620

ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 1680

ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcgc 1740

cggatcctaa ctcaaaatcc acacattata cgagccggaa gcataaagtg taaagcctgg 1800

ggtgcctaat gcggccgcca tagtgactgg atatgttgtg ttttacagta ttatgtagtc 1860

tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt 1920

tcagcttttt tgtacaaact tgtttgatag cttggcgcgc ctcgagggggg ggcccggtac 1980

ccggggatcc tctagagtcg aggtcctctc caaatgaaat gaacttcctt atatagagga 2040

agggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg agatatcaca 2100

tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 2160

gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttcaacga tggcctttcc 2220

tttatcgcaa tgatggcatt tgtaggagcc accttccttt tccactatct tcacaataaa 2280

gtgacagata gctgggcaat ggaatccgag gaggtttccg gatattaccc tttgttgaaa 2340

agtctcaatt gccctttggt cttctgagac tgtatctttg atatttttgg agtagacaag 2400

tgtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg tggttggaac 2460

gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 2520

agaggcatct tcaacgatgg cctttccttt atcgcaatga tggcatttgt aggagccacc 2580

ttccttttcc actatcttca caataaagtg acagatagct gggcaatgga atccgaggag 2640

gtttccggat attacccttt gttgaaaagt ctcaattgcc ctttggtctt ctgagactgt 2700

atctttgata tttttggagt agacaagtgt gtcgtgctcc accatgttga cctgcaggca 2760

cgccaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 2820

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 2880

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct 2940

tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga 3000

tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag 3060

ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc 3120

tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg 3180

ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc 3240

tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa 3300

tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct 3360

gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca 3420

cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc 3480

ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct 3540

agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc 3600

cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg 3660

catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg 3720

cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac 3780

cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt 3840

gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg 3900

cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt 3960

gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa 4020

ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg 4080

atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa 4140

atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa 4200

gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat 4260

gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga 4320

aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc 4380

tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg 4440

gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg 4500

accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg 4560

acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc 4620

tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg 4680

ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg 4740

cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc 4800

ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca 4860

caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg 4920

ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac 4980

aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag 5040

cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac 5100

caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata 5160

catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg 5220

ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca 5280

tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca 5340

atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg 5400

gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc 5460

gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct 5520

gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag 5580

ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc 5640

cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga 5700

gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg 5760

gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc 5820

gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca 5880

cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac 5940

ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag 6000

gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag 6060

atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg 6120

taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt 6180

ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag 6240

gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc 6300

aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat 6360

ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc 6420

gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta 6480

gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca 6540

aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac 6600

cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac 6660

tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag 6720

cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc 6780

gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca 6840

ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat 6900

caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 6960

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 7020

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 7080

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 7140

atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 7200

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7260

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7320

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 7380

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 7440

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 7500

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 7560

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 7620

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7680

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7740

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7800

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7860

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7920

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 7980

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 8040

cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca 8100

ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc 8160

ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca 8220

agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg 8280

ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg 8340

cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg 8400

ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa 8460

tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt 8520

tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg 8580

agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt 8640

ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac 8700

cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta 8760

gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat 8820

tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa 8880

agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct 8940

aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca 9000

tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc 9060

gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt 9120

tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct 9180

cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag 9240

ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt 9300

gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga 9360

attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta 9420

ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca 9480

tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac acattattat 9540

ggagaaactc gagcttgtcg atcgacagat ccggtcggca tctactctat ttctttgccc 9600

tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg 9660

tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc 9720

ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca 9780

agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc 9840

ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc 9900

acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc 9960

tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat 10020

ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga 10080

gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat 10140

ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg 10200

gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag 10260

cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga 10320

caccctgtgc acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa 10380

gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt 10440

agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc 10500

tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact 10560

tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt 10620

gccccccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 10680

cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 10740

agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 10800

aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 10860

tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 10920

atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 10980

aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 11040

ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 11100

tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 11160

tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 11220

tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 11280

ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 11340

tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 11400

agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 11460

gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 11520

gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 11580

ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 11640

tcacacagga aacagctatg accatgatta cg 11672

Claims (7)

1. A gene for regulating poplar leaf development, the nucleotide sequence of which is shown in Sequence 1 in the Sequence Listing. 2. The expressed protein of claim 1 regulating and controlling poplar leaf morphogenesis gene, the amino acid sequence of which is shown in sequence 2 in the sequence table. 3. the cloning method of the described regulation and control poplar leaf shape development gene of claim 1, its steps are as follows: (1) Using 84K silver gland poplar as material, use RNeasy Plant Mini kit and RNase-free DNase I kit to extract RNA; (2) Synthesize the first strand of cDNA by using the SuperScript III first-strand synthesis system, and use Primer 5 software to design primers for full-length gene amplification; Among them, the PagKNAT2/6b ORF forward primer is shown in sequence 3 in the sequence listing; The PagKNAT2/6b ORF reverse primer is shown in sequence 4 in the sequence listing; (3) The high-fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR, forward primer, reverse primer, the template is 84K poplar cDNA, and sterile ddH ₂ O is supplemented; Finally, the full-length cDNA sequence of the obtained gene was 942bp, which was named PagKNAT2/6b gene. 4. the cloning method of regulating poplar leaf development gene according to claim 3, is characterized in that: the reaction program of reaction system described in described step (3) is as follows: pre-denaturation 95 ℃, 5min; 95 ℃, 20s ; 56℃, 20s; 68℃, 60s, 28 cycles; 68℃, 10min. 5. the construction method of the plant expression vector of the described control poplar leaf shape development gene of claim 1, its steps are as follows: (1) Use cloning technology to construct an overexpression vector that regulates poplar leaf development gene PagKNAT2/6b, uses the ORF primer that regulates poplar leaf development gene PagKNAT2/6b, and uses 84K cDNA as a template to carry out PCR amplification, and will regulate poplar leaf shape. The developmental gene PagKNAT2/6b ORF was constructed into the entry vector, the entry vector is PDNOR207, and the sequence is shown in sequence 5 in the sequence listing; The reaction system was fresh PCR product; PDNOR207 vector; BP Clonase II mixed enzyme; sterile ddH ₂ O supplemented; the reaction program was: 25°C for more than 5h; (2) pick positive clones from the screening culture plate and carry out PCR detection and sequencing verification, after the entry vector with the PagKNAT2/6b gene is linearized by Mlu I restriction endonuclease, the Gateway system is constructed to the plant expression vector PMDC32, The sequence is shown in sequence 6 in the sequence listing; LR reaction was carried out, and the reaction system was: linearized entry cloning; purified target vector; LR Clonase II mixed enzyme; sterile ddH ₂ O supplementation; reaction conditions: 25 °C reaction for more than 5 h; after LR reaction, PagKNAT2/6b gene was introduced into plant expression vector In PMDC32, the strong expression promoter 35S is assembled at the 5' end of the PagKNAT2/6b gene, which enables the efficient expression of the PagKNAT2/6b gene in poplar; the 3' end of the PagKNAT2/6b gene is assembled into a strong terminator NOS, which is effective Terminate the transcription of PagKNAT2/6b gene; (3) Assemble the hygromycin phosphotransferase HPT on the vector, as the selection marker of the transgenic poplar, and use hygromycin to screen the transgenic poplar; Assemble the LB and RB sequences on the vector, and promote the PagKNAT2/ The 6b gene expression framework and the screening marker gene HPT were integrated into the poplar receptor chromosome. PCR detection and sequencing verification confirmed that the overexpression vector was successfully constructed and named PMDC32-PagKNAT2/6b. 6. the genetic transformation method of the plant expression vector PMDC32-PagKNAT2/6b of the plant expression vector PMDC32-PagKNAT2/6b of the regulation and control poplar leaf shape development gene described in claim 5, its steps are as follows: (1) Transfer the constructed PMDC32-PagKNAT2/6b overexpression vector into Agrobacterium GV3101 by electric shock method; (2) The PagKNAT2/6b gene was transferred into poplar through the mediation of Agrobacterium. 7. the application of the control poplar leaf shape development gene of claim 1 in the control poplar leaf development process.

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