CN111662368A

CN111662368A - Rubber grass drought-enduring gene TkMYC2, protein, primer, vector, host bacterium and application thereof

Info

Publication number: CN111662368A
Application number: CN202010725600.XA
Authority: CN
Inventors: 闫洁; 黄�俊; 张云川; 孙军亭; 孙辉
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-09-15
Anticipated expiration: 2040-07-24
Also published as: CN111662368B

Abstract

The invention relates to the technical field of biology, and in particular relates to a hevea brasiliensis drought-enduring gene TkMYC2, a protein, a primer, a vector, a host bacterium and application thereof. The nucleotide sequence of TkMYC2 gene cDNA is shown as SEQ ID NO: 1 is shown. The amino acid sequence of the encoded protein is shown as SEQ ID NO: 2, respectively. Under natural drought stress, the transgenic hevea brasiliensis leaves overexpressing TkMYC2 grew well compared to the wild type. The over-expression of TkMYC2 gene can obviously improve POD, SOD and CAT enzyme activities to respond to drought stress. The TkMYC2 gene of the hevea brasiliensis provides a new solution for improving the drought resistance of the hevea brasiliensis.

Description

Rubber grass drought-enduring gene TkMYC2, protein, primer, vector, host bacterium and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a hevea brasiliensis drought-enduring gene TkMYC2, protein, a primer, a vector, host bacteria and application thereof.

Background

Taraxacum kok-saghyz Rodin (also called Taraxacum russiamensis) is a perennial diploid gum-producing plant. It has been found that the physical and chemical properties of the rubber produced by kochia is similar to the rubber produced by hevea trees and that the root tissue of kochia has up to 20% dry gum. However, the annual drought causes serious yield reduction of rubber, and huge economic losses are caused.

The TkMYC2 gene is a hevea brasiliensis drought-enduring gene, the drought tolerance of the hevea brasiliensis with the TkMYC2 gene is greatly improved, and the drought-enduring function of the gene is not reported at present.

Disclosure of Invention

One purpose of the invention is to provide a gene TkMYC2 related to drought tolerance of hevea brasiliensis, wherein the nucleotide sequence of the gene TkMYC2 is shown as SEQ ID NO: 1 is shown.

Another object of the present invention is to provide a protein encoded by the gene TkMYC2, the amino acid sequence of the protein is as set forth in SEQ ID NO: 2, respectively.

The third purpose of the invention is to provide a primer for amplifying the gene TkMYC2, wherein an upstream primer is shown as SEQ ID NO: 3, the downstream primer is shown as SEQ ID NO: 4, respectively.

The fourth purpose of the invention is to provide an expression vector containing the gene TkMYC 2.

The fifth purpose of the invention is to provide a host bacterium containing the gene TkMYC 2.

The invention further aims to provide application of the gene TkMYC2 in drought-enduring genetic improvement of the hevea brasiliensis.

The seventh purpose of the invention is to provide the application of the gene TkMYC2 in transformation of dicotyledonous plants to generate drought-tolerant transgenic dicotyledonous plants.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention finds that the protein encoded by the TkMYC2 gene of the kochia scoparia belongs to a bHLH family transcription factor, and contains a Pfam bHLH-MYC _ N and a helix-loop-helix conserved domain. The expression quantity of the gene is improved by PEG induction, which indicates that the TkMYC2 gene is a drought-resistant related transcription factor.

2. The invention reports that the TkMYC2 transcription factor can improve the drought resistance of the hevea brasiliensis for the first time. Firstly, analyzing transcriptome data of prophase methyl jasmonate treated hevea brasiliensis, and screening out a target segment. PEG induction treatment was performed for the TkMYC2 gene. The full length of TkMYC2 cDNA of the rubber grass is cloned by a PCR technology, and the cDNA and a 35S plant expression vector construct a recombinant plasmid which is transformed into GV3101 Agrobacterium tumefaciens. Agrobacterium-mediated TkMYC2 is introduced into the rubber grass to obtain the overexpression transgenic rubber grass. And then performing drought phenotype analysis and various physiological indexes determination on the over-expressed TkMYC2 hevea brasiliensis. The result shows that the drought resistance of the rubber grass plant with the over-expression TkMYC2 is obviously improved, and a new solution is provided for improving the drought resistance of the rubber grass.

Drawings

Fig. 1 is an electrophoretic detection map of the TkMYC2 gene clone, in which M: DNA Marker III; 1: the TkMYC2 gene.

FIG. 2 is a TkMYC2 conserved domain analysis.

FIG. 3 shows the restriction enzyme identification of recombinant plasmid pMD-19T-TkMYC2, wherein M: DNA Marker III; 1 and 2: enzyme digestion of pMD-19T-TkMYC2 by BamH I and Pst I; 3: pMD-19T-TkMYC2 recombinant plasmid.

FIG. 4 shows the restriction enzyme identification of recombinant plasmid pCAMBIA2300-TkMYC2, wherein M: DNA Marker III; 1: enzyme digestion of pCAMBIA2300-TkMYC2 by BamH I and Pst I; 2: pCAMBIA2300-TkMYC2 recombinant plasmid.

FIG. 5 is a PEG-induced TkMYC2 gene expression analysis.

FIG. 6 is an identification of TkMYC2 rubber grass transgenic seedlings, where M: DNA Marker III; the bands 1-3 are transgenic plants overexpressing TkMYC2, the band 4, the band 6 and the band 7 are positive controls of an overexpression vector, and the band 5 and the band 8 are negative controls of the overexpression vector.

FIG. 7 is the expression analysis of transgenic Hevea brasiliensis TkMYC2, wherein WT represents the wild type; OE- #1 represents transgenic line 1 overexpressing TkMYC 2; OE- #15 represents the transgenic line 15 overexpressing TkMYC2, OE- #45 represents the transgenic line 45 overexpressing TkMYC 2; indicates significant difference between transgene and wild type (P < 0.01).

FIG. 8 is a comparison of TkMYC2 transgenic and wild-type rubber grass phenotypes under drought stress, where WT: wild type kokstroemia indica; #1, #15, # 45: three different TkMYC2 transgenic hevea strains; control: untreated group; drought: drought stress treatment group.

FIG. 9 is the physiological index assay for TkMYC2 transgenic hevea brasiliensis drought resistance, wherein A: POD enzyme activity determination; b: measuring SOD enzyme activity; c: measuring the enzyme activity of CAT; d: measuring the content of MDA; e: measuring the conductivity; indicates significant difference between the transgene and wild type (P < 0.05); indicates significant difference between transgene and wild type (P < 0.01).

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

The nucleotide sequence of the TkMYC2 gene cDNA of the kochia scoparia provided by the invention is shown as SEQ ID NO: 1 is shown. The amino acid sequence of the encoded protein is shown as SEQ ID NO: 2, respectively.

Example 1

Obtaining of TkMYC2 transgenic hevea brasiliensis

(1) Total RNA extraction from Hedychium kolomiktum

Total RNA was extracted using an RNA extraction Kit (EasyPure Plant RNA Kit) purchased from Beijing Quanjin biology, Inc.

1) 0.5g of different tissue samples were rapidly and thoroughly ground into powder in liquid nitrogen, 1ml of BB6 and 10. mu.L of beta-mercaptoethanol (ready-to-use) which were previously mixed, were added, and mixed by vortexing vigorously and shaking. Standing at room temperature for 3 min.

2) Centrifuging at 12000rpm for 4min, carefully sucking supernatant from centrifuge tube into RNase-free centrifuge tube

3) Adding 0.5 times volume of anhydrous ethanol into the supernatant (precooling at 4 ℃ in advance), and mixing uniformly.

4) Vortex and mix evenly, disperse and precipitate.

5) Adding the precipitate mixed solution into a centrifuge tube, centrifuging at 12000rpm for 30s, and discarding the effluent.

6) Add 500. mu.L of CB6, centrifuge at 12000rpm for 30s and discard the retentate.

7) Add 80. mu.L of DNase1 working solution to the center of the column, let stand at room temperature for 15min, and repeat the experimental step 6 once.

8) Add 500. mu.L of WB6, centrifuge at 12000rpm for 30s and discard the effluent.

9) The experimental step 7 was repeated once.

10) Centrifugation was carried out at 12000rpm for 2min at room temperature to completely remove the residual ethanol.

11) Adding 50 μ L of RNAase-free H2O into the centrifugal column, and standing for 1 min.

12) The RNA was eluted by centrifugation at 12000rpm for 2min at room temperature.

13) The RNA was stored at-80 ℃.

(2) Reverse transcription into cDNA

RNA was reverse-transcribed into cDNA using a reverse transcription Kit (EasyScript One-Step gDNA Removal Kit and cDNAsynthesis Supermix) purchased from Beijing Quanyu gold organism, Inc.

1) Total RNA 2.5. mu.L, Anchored Oligo (dT)₁₈Primer 0.5. mu.L, 2 × ES Reaction Mix 5. mu. L, RT/RI Enzyme Mix 0.5. mu.L, gDNA Remover 0.5. mu. L, Rnase-Free Water 1. mu.L, in total, 10. mu.L.

2) Incubate at 42 ℃ for 30 min.

3) Then heated at 85 ℃ for 5 s.

(3) Cloning of the TkMYC2 Gene

Primer5.0 was used to design specific primers (Table 1).

TABLE 1 primer sequence information I

The Megasphaea root cDNA is used as a template for amplification, and the PCR reaction system is a system with 20 mul of upstream and downstream primers of 0.5 mul, 1 mul of root cDNA template and 10 mul L, ddH2O 8 mul of PCR Mix. The PCR reaction program is set to be the pre-denaturation temperature of 95 ℃ for 5 min; the denaturation temperature is 95 ℃ for 20s, the annealing temperature is 58 ℃ for 20s, the extension temperature is 72 ℃ for 2min for 30s, and 35 cycles are performed; further extension was carried out at 72 ℃ for 8 min. The PCR product was detected by 1% agarose gel electrophoresis and then recovered using a general agarose DNA recovery kit, as shown in FIG. 1, and the TkMYC2 conserved domain was analyzed using the Normal SMART MODE and NCBI database CD-search on-line tool, as shown in FIG. 2.

The target product is connected with a pMD-19T vector to construct a pMD-19T-TkMYC2 recombinant plasmid, the connection system is shown in Table 2, and the connection is carried out overnight at 16 ℃.

TABLE 2 connection System

(4) Transformation of E.coli

Slowly adding 10. mu.L of the recombinant ligation product into 100. mu.L of the E.coli competence; gently blowing and beating by using a pipettor to mix the components evenly; immediately carrying out ice bath treatment for 30 min;

1) performing heat shock treatment for 90s at 42 ℃ in a constant-temperature water bath kettle, and immediately and rapidly performing ice bath treatment for 2-3 min;

2) then 450 mul LB culture medium liquid is added; incubating the cells on a shaker at 37 ℃ and 220rpm for 1 h;

3) centrifuging at 5000rpm for 5min, and discarding 300 μ L of supernatant; uniformly coating the residual 200 mu L of bacterial liquid to an LB solid culture medium carrying antibiotics (resistance corresponding to the carrier), and culturing at the constant temperature of 37 ℃ overnight;

4) the positive single colony is selected for colony PCR detection, a plasmid recombinant vector pMD-19T-TkMYC2 is extracted, the recombinant vector is sent to Huada GenBank for detection, and the result is shown in figure 3.

(5) Plant overexpression vector construction

Primers were designed with sites for BamH I and Pst I using primer5.0 software (Table 3). Carrying out double enzyme digestion on the empty vectors of the recombinant plasmids pMD-19T-TkMYC2 and pCAMBIA2300 (containing 35S promoter) carrying the two enzyme digestion sites by using BamHI and Pst I to respectively obtain a target fragment and a vector fragment, wherein the enzyme digestion systems are 0.5 mu L BamHI, 1 mu L Pst I and 2 mu L TangoBuffer, 16.5. mu.L vector, cut at 37 ℃ for 1 h. Recombinant ligation of the fragment of interest with the vector fragment (Table 4) and transformation (same step (4)) 50mg.L^-1Positive single colonies were picked from the kan-resistant plates, subjected to colony PCR (same step (4)) and double-enzyme digestion, plasmids were extracted to obtain recombinant vector pCAMBIA2300-TkMYC2, which was sent to Huada GenBank for sequencing, the results are shown in FIG. 4.

TABLE 3 primer sequence information II

Note: the underlined parts are the cleavage sites, BamHI (GGATCC) and Pst I (CTGCAG)

TABLE 4 connection System

(6) Plant over-expression recombinant vector transformation agrobacterium tumefaciens

The pCAMBIA2300-TkMYC2 recombinant plasmid was transformed into Agrobacterium tumefaciens (GV3101) by Agrobacterium freeze-thaw transformation.

The freeze-thaw transformation method of agrobacterium comprises the following steps:

1) 2 mu L of recombinant plasmid is absorbed by a liquid transfer gun, added into 100 mu L of agrobacterium tumefaciens competence, and slowly blown and uniformly mixed;

2) standing the mixed solution on ice for 10min, immediately and rapidly freezing in liquid nitrogen for 5min, and thermally shocking in a 37 deg.C water bath for 5 min;

3) adding the mixture into 1mL LB liquid culture medium without any antibiotics, and respectively setting the temperature and the rotating speed of a shaking table to be 28 ℃ and 220rpm to ensure that the thalli are recovered and cultured for about 3 hours;

4) centrifuging the bacterial liquid at the rotating speed of 5000rpm for 2min, discarding the thallus supernatant, reserving 100 μ L of the supernatant to blow out thallus, and mixing completely;

5) coating the bacterial liquid on a solid LB plate containing three antibiotics (Kan, Rif, Gen), wherein the bacterial liquid is required to be fully coated;

6) the plate was inverted and placed in a 28 ℃ incubator and allowed to incubate for about two to three days until positive colonies appeared.

(7) Agrobacterium-mediated genetic transformation of kochia

Shearing a sterile seedling of the rubber grass into small pieces of about 1cm × 1cm, flatly paving the small pieces with the front facing upwards on an MS solid culture medium, culturing in dark for 2 days, carrying out propagation on agrobacterium tumefaciens carrying pCAMBIA2300-TkMYC2 recombinant plasmids in 50mL of LB liquid culture medium, performing shake culture at 220rpm and 28 ℃ for 8 hours, performing thallus centrifugation at 5000rpm for 5min to collect thallus precipitates, sucking 1mL of MS liquid culture medium, blowing the precipitates into a bacterial suspension, adding the bacterial suspension into 50mL of MS liquid culture medium, and simultaneously adding 10mmol^-1MES、10mmol.L^-1MgCl₂And 200. mu. mol.L^-1And (3) an AS solution. The entire experiment was run under sterile conditions. Co-culturing the rubber grass leaf disc and the agrobacterium tumefaciens suspension by using an agrobacterium infection method, and performing shake culture at 220rpm and 28 ℃ for 10-15 min (the co-culture time is determined according to the concentration of the agrobacterium liquid). Placing the upper epidermis of the leaf disc on sterile filter paper, sucking the redundant bacterial suspension on the epidermal cells of the rubber grass by the filter paper, placing the rubber grass on the filter paper of the MS solid culture medium with the front face upward, and performing dark culture for 2 d. The leaves were transferred to a rubber grass callus induction medium. And the culture medium is prevented from being polluted through multiple plate turnover. When the bud is bigger, the bud is transferred to a rooting culture medium to continue to root and grow seedlings. The whole tissue culture process uses 50mg.L^-1Kan antibiotics were used for transgenic shoot screening.

Example 2

PEG induced expression assay

Three months old wild type RUBENCAO aseptic seedlings were acclimatized in water for three days, and the acclimatization method was described in the reference (De novo transformation Sequencing of MeJA-Induced Taraxacum koksaghyz rotation identification Genes Related to Rubber Formation). Wild type rubber grass was treated with 20% PEG to completely submerge the roots. The PEG induction time gradient is set to be 0, 6, 12 and 24 hours, the root tissues (1 cm under the leaf base) of the rubber grass are respectively collected, and the same parts are kept to be obtained as much as possible; repeating the three biology procedures, immediately quick freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C. Total root RNA extraction of various samples Using RNA extraction Kit (Easypure Plant RNA Kit) and reverse transcription Kit (EasyScript One-Step gDNA R)emusal Kit and cDNA Synthesis Supermix) with immobilized Oligo (dT)18 primer to synthesize single-stranded cDNA, experimental methods were as in example 1, step 1) and step 2), in preparation for subsequent quantification experiments. Quantitative primers were designed using Primer5.0 (table 5); real-time fluorescent quantitative PCR reaction was performed on a Light Cycler480System instrument using GAPDH as an internal reference gene. The reaction program is that the pre-denaturation temperature is 95 ℃ for 30s, the denaturation temperature is 95 ℃ for 10s, the annealing temperature is 58 ℃ for 15s, the extension temperature is 72 ℃ for 20s, and the total time is 45 cycles. The reaction system consisted of PCR Mix 5. mu.L, ddH2O 4. mu.L, upstream and downstream primers 0.25. mu.L each, and cDNA template amount 0.5. mu.L, totaling to 10. mu.L. Three biological replicates per tissue and three experimental replicates per sample. By using 2^–ΔΔCtThe method calculates the expression quantity of the TkMYC2 gene at different time points of PEG induction treatment. Data processing and analysis were performed using GraphPad Prism 8 software. The results are shown in FIG. 3.

TABLE 5 primer sequence information III

Example 3

Identification of TkMYC2 transgenic rubber grass plant

Indoor hardening of TkMYC2 transgenic rubber grass grown in example 1 for 2d, cleaning excess culture medium at root, and transplanting into nutrient soil. The nutrient soil consists of vermiculite and humus in a ratio of 1: 2. The transgenic rubber grass candidate was cultured in a cultivation room under normal conditions for about 20 days, and DNA extraction was carried out according to the Kit (Easypure Plant Genomic DNA Kit) protocol.

1) Taking 0.5g of the tissue of the rubber grass leaf, adding liquid nitrogen and fully grinding.

2) 250 μ L of RB1 and 15 μ L of RNase A were added to each centrifuge tube.

3) And (3) adding the sample into the centrifuge tube in the step (2), uniformly mixing, and carrying out water bath at 55 ℃ for 15 min.

4) Centrifuge at 12000rpm for 5min and aspirate the supernatant.

5) Add 100. mu.L PB1, mix well, ice-wash for 5min, centrifuge at 12000rpm for 5 min.

6) The supernatant was aspirated into a fresh centrifuge tube, 375. mu.L of BB1 was added, and the mixture was mixed well.

7) The mixture was aspirated into a spin column, centrifuged at 12000rpm for 30s, and the effluent was discarded.

8) Add 500. mu.L of CB1, centrifuge at 12000rpm for 30s and discard the effluent.

9) Add 500. mu.L of WB1, centrifuge at 12000rpm for 30s and discard the effluent.

10) Repeat step 9).

11) The WB1 solution was removed completely by centrifugation at 12000rpm for 2 min.

12) Add 100 μ L EB preheated at 65 ℃ to the center of the centrifugal column, let stand for 1min, centrifuge at 12000rpm for 1min, elute DNA.

13) The second elution was performed and the DNA sample was stored in a freezer at-20 ℃.

In the TkMYC2 overexpression rubber grass body, a 35S primer is used as an upstream primer and a TkMYC2 primer is used as a downstream primer (Table 6) to verify a transgenic rubber grass plant, the procedure is the same as that in example 2, the screened transgenic rubber grass plant successfully overexpressing TkMYC2 is marked as T0 generation, and the result is shown in FIG. 6.

TABLE 6 primer sequence information IV

Example 4

TkMYC2 transgenic rubber grass TkMYC2 transcription level, drought phenotype and physiological index analysis

(1) TkMYC2 transcript levels

Collecting the T0-generation over-expression TkMYC2 transgenic hevea brasiliensis seeds, transplanting the seeds into flower pots after the seeds germinate into small seedlings, recording as T1 generation, and planting four small seedlings in each pot. Under normal growth conditions, seedlings were cultured for three months. Transgenic hevea brasiliensis overexpressing TkMYC2 was identified at the RNA level and primers used in table 5. Total RNA extraction as in example 1 step 1), reverse transcription process as in example 1 step 2). The quantitative test, data analysis, and plotting methods were the same as in example 2, and the results are shown in FIG. 7.

(2) Drought phenotype

T1 generation plants with the size of three months are selected, natural drought treatment is carried out on the transgenic rubber grass and wild type plants for 22d under the same culture condition, the experiment results are recorded by photographing, and the results are shown in figure 8.

(3) Physiological index

Carrying out drought stress treatment on transgenic hevea brasiliensis and wild plants, sampling and collecting wild type and overexpressed TkMYC2 transgenic hevea brasiliensis leaf tissues before drought treatment, immediately carrying out liquid nitrogen quick freezing, and storing in a refrigerator at-80 ℃; until drought stress is 22d, the phenotype of the wild type and the transgenic plant is obviously different, the leaf tissues of the wild type and the transgenic plant are collected again, quick frozen by liquid nitrogen, and stored in a refrigerator at the temperature of-80 ℃. The method for measuring various physiological indexes of the rubber grass adopts a kit provided by Nanjing institute of biological engineering for construction. The experimental method refers to the kit instructions. Conductivity measurements conductivity was measured using a conductivity meter (BANTE 540) and experimental procedures as follows: taking 0.5g of wild type and transgenic plant leaves, respectively washing with distilled water for three times, and absorbing water on the tissue surface by filter paper. The leaves are cut into small strips (15 sections each) of 1-2 cm, and the cut materials are put into a 50mL conical flask to immerse the leaves. Standing at room temperature for 2h, and gently stirring the blade with a glass rod to uniformly mix the solution; conductivity in the solution was measured with a conductivity meter. Then putting into boiling water and boiling for 30min to achieve the purpose of plant tissue death, cooling the tube wall for 10min by flowing water, and measuring the conductivity of the solution at room temperature. Each of the above physiological index determinations followed three biological replicates and three experimental replicates, and the results are shown in fig. 9.

It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> river university

<120> hevea brasiliensis drought-resistant gene TkMYC2, protein, primer, vector, host bacterium and application thereof

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gatccgcaca agatagtcaa cgatttcaat ccggatactt tacagcaacg cctccagggg 240

ttgattgata cggcacggga gtcgtggact tacgctatat tctggcagtc gtctgacgtc 300

gattacacgg atactccggt gttggggtgg agagatgggt attacaaggg ggaggtgaat 360

aaggtgaaaa cgaagccgtc ggcgacttct ttggcggagc aacagtaccg gaagaaagtg 420

ctccgggagc ttaattcatt gatctctggc tcacaggcgc cggagagcga tgccgtggat 480

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acaatcgtgt gtatcccttc agccgatggg gttattgaat taggttcaac ggagttgata 720

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gacagagcaa ctgctggcag aaatgggaac actttatacc ctaagccaga atccggcaga 1140

atcctggatt tcagcgagag taaagggagt tctacaaaca acgcagcact gttctccggt 1200

caatcccagt tcctcggagc agaggagaac aacaacatca acaataagaa caagagcaag 1260

aagaagagat cgccaggttc gtgcggcagc aacgaagacg ggatgctttc tttcgtctcc 1320

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gaaccggaga aaaaaccacg aaaacgagga cgaaaaccgg cgaacggcag ggaggagcca 1500

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385 390 395 400

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

405 410 415

Asn Lys Ser Lys Lys Lys Arg Ser Pro Gly Ser Cys Gly Ser Asn Glu

420 425 430

Asp Gly Met Leu Ser Phe Val Ser Gly Val Leu Pro Ser Ser Ser Met

435 440 445

Gly Lys Ser Asp Gly Gly Ala Phe Thr Gly Ala Asp Pro Asp His Ser

450 455 460

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

465 470 475 480

Glu Pro Glu Lys Lys Pro Arg Lys Arg Gly Arg Lys Pro Ala Asn Gly

485 490 495

Arg Glu Glu Pro Leu Asn His Val Glu Ala Glu Arg Gln Arg Arg Glu

500 505 510

Lys Leu Asn Gln Arg Phe Tyr Ala Leu Arg Ala Val Val Pro Asn Val

515 520 525

Ser Lys Met Asp Lys Ala Ser Leu Leu Gly Asp Ala Ile Leu Tyr Ile

530 535 540

Asn Glu Leu Lys Ser Lys Leu Asp Asn Thr Glu Cys Asp Lys Glu Glu

545 550 555 560

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

565 570 575

Ser Arg His Ser Ser Ser Ser Ala Ile Ser Leu Pro Glu Asp Met Lys

580 585 590

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

595 600 605

Lys Val Ile Gly Trp Asp Ala Met Ile Arg Val Gln Arg Asn Lys Lys

610 615 620

Asn His Pro Ala Ala Arg Leu Met Ala Ala Leu Lys Asp Leu Asp Phe

625 630 635 640

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

645 650 655

Gln Ala Thr Val Lys Met Gly Gly Arg Leu Tyr Thr Gln Asp Gln Leu

660 665 670

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

675 680

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<400>3

atgacggatt accggctaca g 21

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

ggagggacct tcacctgata at 22

<210>5

<211>27

<212>DNA

<213> Artificial sequence

<400>5

ggatccatga cggattaccg gctacag 27

<210>6

<211>32

<212>DNA

<213> Artificial sequence

<400>6

ctgcagcaat ggatctgaaa atctgtttgt aa 32

<210>7

<211>25

<212>DNA

<213> Artificial sequence

<400>7

gctctagaac caacccccaa tgaat 25

<210>8

<211>22

<212>DNA

<213> Artificial sequence

<400>8

cctcgagacc gcccattttc ac 22

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

cgcctcattt aacatcatcc 20

<210>10

<211>20

<212>DNA

<213> Artificial sequence

<400>10

gtcataccac gaaaccagct 20

<210>11

<211>23

<212>DNA

<213> Artificial sequence

<400>11

ccatcattgc gataaaggaa agg 23

Claims

1. A gene TkMYC2 related to drought-enduring traits of hevea brasiliensis is characterized in that the nucleotide sequence of the gene TkMYC2 is shown as SEQ ID NO: 1 is shown.

2. A protein encoded by the gene TkMYC2 of claim 1, wherein the amino acid sequence of the protein is as set forth in SEQ ID NO: 2, respectively.

3. The primer for amplifying the gene TkMYC2 of claim 1, wherein the upstream primer is shown as SEQ ID NO: 3, the downstream primer is shown as SEQ ID NO: 4, respectively.

4. An expression vector comprising the gene TkMYC2 of claim 1.

5. A host bacterium comprising the gene TkMYC2 of claim 1.

6. The use of the gene TkMYC2 in claim 1 for genetic improvement of drought tolerance of hevea brasiliensis.

7. Use of the gene TkMYC2 of claim 1 to transform a dicot to produce a drought tolerant transgenic dicot.