CN103626859B - Suaeda salsa protein as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses a suaeda salsa protein as well as a coding gene and application thereof. An amino acid sequence of the suaeda salsa protein is as shown in SEQ ID No.1, and a base sequence of the coding agene of the suaeda salsa protein is as shown in SEQ ID No.2. The application comprises the following steps: extracting total RNA (Ribonucleic Acid) of the suaeda salsa and reversely transcribing the total RNA into cDNA (Complementary Deoxyribonucleic Acid), and amplifying the suaeda salsa gene by taking the cDNA as a template and utilizing a primer; constructing a recombinant vector by utilizing the suaeda salsa gene, and transforming the recombinant vector into target plants by an agrobacterium-mediated method to obtain transformed plants; screening a stress-tolerance plant from the transferred plants. The suaeda salsa protein is highly expressed in the target plants, so that stress tolerance of the targeted plants can be obviously improved.

Description

A kind of Suaeda salsa albumen and encoding gene thereof and application

Technical field

The invention belongs to functional genomics field, be specifically related to a kind of Suaeda salsa albumen and encoding gene thereof and application.

Background technology

The threat of the soil salinization to agricultural is a global problem.The whole world has the saltings of 1,000,000,000 hectares, accounts for world land area 7.6%, nearly more than 100,000,000 hectares of China saltings, the underproduction that agriculture plantation causes because of salinification, abandoned land 333.5 ten thousand hectares nearby.In recent years, the fast development of China's industrialized agriculture, particularly vegetables and flowers booth are produced area and are constantly expanded, and according to statistics, the crop facility cultivation areas such as national vegetables, flowers, melons and fruits in 2005 reach 2,100,000 hectares.Developing into structure of agricultural production adjustment and improving agriculture production benefit of industrialized agriculture has played vital role, but along with facility cultivation time lengthening, the problem of soil secondary salinization is increasingly sharpened, have a strong impact on the yield and quality of facility cultivation crop, benefit also declines thereupon, thus affects the sound development of industrialized agriculture.

Solve facility operation salinification and generally take following two kinds of measures: one is improve soil by the chemical process such as gypsum and sulphur or with physical methods such as draining and the irrigation desalinization of soil by flooding or leaching; Two is cultivate salt tolerant crop kind by conventional breeding or animal nutrition, and the former input cost is high.Effectively can not only solve facility operation salinification problem by cultivating the anti-salt new variety of farm crop be suitable at varieties in saline-alkali areas cultivation and facility cultivation, but also alleviate the problem of China's land resources scarcity by effectively utilizing part salinization land widely.

In recent years, along with model plant Arabidopis thaliana and Sequencing of Rice Genome complete, Research of Plant Genomics research is transferred to functional genomics.The novel method of more current research functional genomicses and experimental technique system are as cDNA microarray, gene chip, gene expression system analysis (serial analysis of gene expression, SAGE), gene knockout (gene knock out) and RNAi analyze expression and the functional mode that all effectively can analyze lots of genes, and achieve certain progress on salt tolerance correlation function genetic resources are excavated.Some and osmoregulation genes involved from different floristics by successful clone and Transformation Application, as proline synthesis genes involved P5CS(KishorPBK, Hong Z, Miao G H, Hu CAA, Verma DPS.Overexpression of P5CSincreases proline production and confers osmotolerance in transgenic plants.Plant Physiol, 1995, 108:1387-1394) with betaine aldehyde dehydrogenase BADH gene (Xiao Gang, Zhang Gengyun, Liu Fenghua etc., prunella asiatica betaine aldehyde dehydrogenase gene is studied, Science Bulletin, 1995, 40 (8): 741-745).

Na in plant materials ⁺ionic equilibrium is the important mechanisms that plant self salt tolerant regulates.Zhu's health research group finds that the adjustment signal of Arabidopis thaliana SOS gene series is plant self-regulation Na ⁺one of important channel of ionic equilibrium.2005, Lin Hong declared research group to cooperate with U.S. professor Luan Sheng, the Quantitative Trait Genes SKC1 that successful clone Rice Salt is correlated with.This gene can control the content of rice plant overground part sodium ion and potassium ion, maintains sodium and potassium ion balance, makes excess sodium ion not in position accumulation such as cauline leafs, and make sodium ion be back to root, alleviate sodium ion to poison, have additional nutrients Element Potassium ion simultaneously, thus increase Salt Resistance of Rice.

Suaeda salsa (Suaeda salsa (L.) Pall.) is annual Chenopodiaceae Suaeda herbaceous plant, is a kind of euhalophyte being grown on saltings and sandy beach, beach.At present, Suaeda salsa genome is not sequenced, though have some genes such as SsAPX, SsGST, SsNPS, SsPSI etc. to be cloned, but the sequence of most gene and Unknown Function.

Summary of the invention

The invention provides a kind of Suaeda salsa albumen, in target plant, this Suaeda salsa albumen of high expression level, can significantly improve the resistance of reverse of target plant.

A kind of Suaeda salsa albumen, aminoacid sequence is as shown in SEQ ID No.1.

This Suaeda salsa albumen can raise the activities of antioxidant enzymes such as hemocuprein, catalase, ascorbate peroxidase enzyme; remove because high salt or low temperature stress produce too much active oxygen (superoxide ion or hydrogen peroxide); maintain cell activity in vivo oxygen level in normal range; protection seedling, plant exempt from the oxidative damage because high salt or low temperature cause, and improve plant stress-resistance ability.

Present invention also offers the encoding gene of described Suaeda salsa albumen, and expression unit, recombinant plasmid or the transformant containing described encoding gene; The base sequence of described encoding gene is as shown in SEQ ID No.2.

Present invention also offers described Suaeda salsa gene and improve the application in plant stress tolerance.

Based on the performance of this Suaeda salsa albumen, plant stress tolerance described in the present invention is at least one in resistance to high salt and drought tolerance; Described plant is dicotyledons, as Arabidopis thaliana, strawberry, capsicum, eggplant, tomato etc.The present invention checks the effect of this Suaeda salsa gene in raising plant stress tolerance for dicotyledonous model plant Arabidopis thaliana, shows that this Suaeda salsa albumen can significantly improve plant resistance to high salt and drought tolerance really.

Particularly, described Suaeda salsa gene is improving the application in plant stress tolerance, comprising:

(1) extract Suaeda salsa total serum IgE reverse transcription is cDNA, be template with cDNA, utilize Suaeda salsa gene described in primer amplification;

The base sequence of described primer is:

Upstream primer: 5 '-TCTAGAATGGTGAGAGAAAAGATTAA-3 ';

Downstream primer: 5 '-GGTACCTTAATATATCCCCCTGTTTC-3 ';

(2) utilize the gene constructed recombinant vectors of this Suaeda salsa, and this recombinant vectors is imported in target plant by agrobacterium-mediated transformation, obtain conversion of plant;

The initial carrier of described recombinant vectors can select Super1300 or pCAMBIA1300;

(3) from conversion of plant, screening obtains resistance of reverse plant;

The T1 of conversion of plant is dull and stereotyped in hygromycin B screening for planting seed, get the many generation breedings of positive plant, obtain the resistance of reverse plant of genetic stability; Preferably, in described screening flat board, the concentration of hygromycin B is 80 ~ 100 μ g/mL.

Compared with wild Arabidopis thaliana, the T3 that the present invention obtains for stable strain Arabidopis thaliana can in containing the MS solid medium of 200mM sodium-chlor normal growth, then all albefaction is dead for wild Arabidopis thaliana; When carrying out dehydration-rehydration test to two plants, the T3 that the present invention obtains can restore normal growth for stable strain rehydration, and cannot restore normal growth after wild Arabidopis thaliana rehydration, and namely dead in the near future.

Compared with prior art, beneficial effect of the present invention is embodied in:

The present invention finds that the Suaeda salsa albumen shown in SEQ ID No.1 is the functional protein making Suaeda salsa have resistance to high salt, and by gene (the SEQ ID No.2) arabidopsis thaliana transformation of this Suaeda salsa albumen of coding, significantly improve resistance to high salt and the drought tolerance of Arabidopis thaliana; By this Suaeda salsa gene transformation as in other dicotyledonss, the output of crop on saltings can be increased on the one hand, realize the effective utilization to China's coastal region saltings; The growing barrier that the plant such as vegetables, flowers under facility condition causes because of the soil accumulation of salt in the surface soil and seasonal variation cooling (winter and early spring) can be overcome on the other hand, improve its yield and quality, increase farmers' income.

Embodiment

Suaeda salsa gene is improving the application in plant stress tolerance, comprising:

1 target gene obtains

(1) preparation of samples

With the Suaeda salsa seedling of 3 weeks for material, water planting 1 week at 25 DEG C, then containing 400mMNaCl water planting liquid process 24h, collects the blade of Suaeda salsa seedling.

(2) protein extraction

With cold acetone/trichloroacetic acid precipitation (see Salekdeh G H, Siopongco J, Wade L J, Ghareyazie B, Bennett J.A proteomic approach to analyzing drought-and salt-responsiveness in rice.Field Crop Res, 2002,76(2-3): 199-219) rapid extraction leaf total protein, concrete operations are as follows:

The Leaves of Suaeda Salsa liquid nitrogen grinding of 1. getting 1g cleaning becomes fine powder, be distributed in 1.5mL centrifuge tube, add the acetone soln that 1mL protein extract I(contains 10% trichoroacetic acid(TCA) and 0.07% beta-mercaptoethanol) at-20 DEG C of precipitation crude protein 1h, at 4 DEG C, centrifugal 20min under 13000r/min, get precipitation, abandon supernatant;

2. in precipitation, 1mL protein extract II (acetone soln containing 0.07% beta-mercaptoethanol) is added, at-20 DEG C of suspension crude protein 1h, at 4 DEG C, centrifugal 20min under 13000r/min, get precipitation, abandon supernatant, repeat with protein extract II again, after suspension extraction under the same conditions 3 times, vacuum drains precipitation;

3. use lysate (containing 7mol/L urea, 2mol/L thiocarbamide, 4%Chaps(Ameresco company, the U.S.), 50mmol/L DTT(Promega company, the U.S.) and 40% amphotericeledrolyte of 0.5%pH3-10) dissolution precipitation, lysate consumption is 25 μ L lysates/mg precipitation, then at room temperature 1h is placed, continuous vortex 5-6 time between burst times;

4. according to Bradford method (see Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem, 1976,72:248-54) measure the protein content in above-mentioned lysate with Coomassie brilliant blue G-250 (Sigma company), the albumen shotgun proteomics method qualification in above-mentioned lysate;

(3) analysis of protein

1. enzymolysis and peptide section are extracted: carry out enzymolysis with 1 μ g/ μ L pancreatin (Trypsin) to the leaf total protein extracted, enzymolysis time 20h, at 40 DEG C, extract above-mentioned enzyme with 50%ACN cut peptide section 1 hour (for the first time), use 50%ACN and the 2.5%TFA(Merk company of same volume again, Germany) solution in 30 DEG C extract 1 hour (for the second time), finally use 25 μ l ACN(Fischer companies, the U.S.) supersound extraction 5min(third time), after merging No. 3 extracting solution freeze-drying, add 0.1% formic acid solution 10 μ L, mixing is dissolved, and is transferred in automatic sampling bottle, for subsequent use; 2. stratographic analysis: efficient liquid phase chromatographic analysis is carried out to the sample that 1. step is prepared, wherein, A liquid is for containing 0.1% first aqueous acid, and B liquid is the acetonitrile solution (acetonitrile is 80%) containing 0.1% formic acid; Chromatographic column is with after the A liquid balance of 95%, and sample is by automatic sampler loading (applied sample amount 5 μ L); Elutriant is: 0 ~ 15 minute, 95%A liquid; 15 ~ 45 minutes, B linear gradient was from 5% to 60%; 45 minutes ~ 50 minutes, B linear gradient was from 60% to 100%; 50 ~ 55 minutes, B liquid maintained 100%; 55 ~ 60 minutes, B linear gradient was from 100% to 5%;

3. mass spectroscopy: carry out mass spectroscopy to the sample that 1. step is prepared, the mass-charge ratio of the fragment of polypeptide and polypeptide gathers according to following method: each full scan (full scan) gathers ten fragment patterns stored (MS afterwards ²scan), source document (raw file) the corresponding storehouse of BIOWORKERS software search obtained, filtration parameter is: Xcorr(1+>=1.9,2+>=2.2,3+>=3.75) and DelCn(>=0.1);

According to mass-spectrometric data comparison to Suaeda salsa SsMBTF1 albumen, matching sequence accounts for overall amino acid sequence 30%.According to SsMBTF1 protein amino acid sequence (SEQ ID No.1), obtain encoding gene (refer to step (3) " target gene acquisition ", base sequence is as SEQ ID No.2) by gene clone.The open reading frame (ORF) of this gene for 399bp, mRNA length be that 1192bp(base sequence is as SEQ ID No.3).

(3) target gene obtains

With Suaeda salsa seedling (after broadcasting 3 weeks) total serum IgE (extracting with Trizol reagent) for template, utilize RT-PCR method amplification SsMBTF1 gene, concrete operations are as follows:

1. mRNA reverse transcription is become the first chain cDNA, Reverse Transcription box used is the High Fidelity PrimerScript of TaKaRa company ^tMrT-PCR Kit; Reaction system is 20 μ L: add 1 μ L20M random primer (Random6mers), 1 μ L10mM dNTP, 2 μ L total serum IgE and DEPC water to 10 μ L successively; 65 DEG C of sex change 5 minutes, rapidly cooled on ice 2 minutes, centrifugal a little, then add 4 μ L5 × PrimerScript RT buffer, 0.5 μ L RNase inhibitor, 0.5 μ L PrimerScript RTase and 5 μ L DEPC water successively; Slightly mix, 30 DEG C are reacted 10 minutes, and 42 DEG C are reacted 30 minutes, and 95 DEG C make enzyme deactivation in 5 minutes;

2. in order to remove the RNA chain with cDNA complementation, 1 μ L RNase H is added at 37 DEG C of incubation 20min ,-20 DEG C of preservations;

3. then with the first chain cDNA for touching plate amplifying target genes SsMBTF1, the primer is:

SsMBTF1-F：5’-TCTAGAATGGTGAGAGAAAAGATTAA-3’；

SsMBTF1-R：5’-GGTACCTTAATATATCCCCCTGTTTC-3’；

PCR reaction system is 50 μ L: add 2 × PCR buffer25 μ L, 2.5mM dNTPs4 μ L, reverse transcription product 2 μ L, 20 μMs of forward primer (SsMBTF1-F) 1 μ L, 20 μMs of reverse primer (SsMBTF1-R) 1 μ L, 2.5U/ μ L Tag archaeal dna polymerase 0.5 μ L successively, finally add water to 50 μ L;

PCR reaction conditions: denaturation 94 DEG C of 3min, sex change 98 DEG C of 10s, anneal 55 DEG C of 15s, extends 72 DEG C of 30s, 30 circulations, finally extends 72 DEG C of 10min, 4 DEG C of preservations;

4. amplified production carries out 1% agarose gel electrophoresis, and carries out recovery purifying to target fragment;

5. the DNA fragmentation and pMD19-T carrier (purchased from TakaRa company) that reclaim purifying are carried out ligation, linked system is 10 μ L, comprising: 0.5 μ L pMD19-T carrier, 4.5 μ L DNA fragmentations, 5 μ L Solution I; At 14 DEG C-16 DEG C, connect 8-12 hour, then will connect product conversion in bacillus coli DH 5 alpha competent cell, and send order-checking;

6. correct through checking order, with Xba I and Kpn I(purchased from TakaRa company) carry out double digestion, the enzyme system of cutting is 40 μ L, comprises 4 μ L10 × buffer, 8 μ L connect product, 1 μ L Xba I, 1 μ LKpn I) and 26 μ L water, incubation 6h in 37 DEG C of water-baths;

7. Agarose Gel Extraction Kit(is used purchased from TakaRa company) reclaim gene fragment, operate as follows: above-mentioned hybrid dna, after gel electrophoresis, cuts required DNA fragmentation from gel, be placed in the Eppendorf pipe of 1.5mL; Add the Buffer QG-A of 3 times of volumes, 55 DEG C of water-bath 5-10min, period jog Eppendorf pipe makes glue dissolve completely several times, adds the Buffer QG-B that 2/3 recovery colloid is long-pending; In the DNA purification column of sky, add 250 μ L Buffer BL, the centrifugal 1min of 10000g, outwells raffinate; Pour the DNA glue after dissolving into DNA purification column, the centrifugal 1min of 10000g, outwells raffinate; In purification column, add 500 μ L Buffer W2, the centrifugal 1min of 10000g, outwells raffinate; In purification column, add 700 μ L Buffer W2 again, the centrifugal 1min of 100g, outwells raffinate; The purification column of sky centrifugal 2min under 15000g is added the sterilized water dissolving DNA of 10-15 μ L70 DEG C of preheating, the centrifugal 1min of 10000g after making its drying, the solution of gained is the gene SsMBTF1 of purifying.

2 build recombinant vectors

Gene SsMBTF1 is connected in Super1300 carrier, linked system 10 μ L, comprise: gene SsMBTF, 1 μ L10 × T4 ligase enzyme buffer of 2 μ LSuper1300 carriers, 6 μ L purifying and 1 μ L T4 ligase enzyme, 12h is connected at 4-10 DEG C, then product conversion will be connected in bacillus coli DH 5 alpha competent cell, extract plasmid to identify, through being accredited as positive plasmid, for subsequent use.

3 transformation Agrobacterium

1. 200 μ l Agrobacterium competent cells are got, add 5-10 μ l restructuring positive plasmid, 30 DEG C of ice bath 30min, quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 5min, (1 liter of YEB substratum is containing 1g yeast extract, 5g beef extract, 5g peptone, 5g sucrose and 0.5g MgSO then to add 1mL YEB substratum ₄7H ₂o, pH7.0), cultivate 4h for 28 DEG C;

2. the centrifugal 30s of 10000g, abandon supernatant, add 0.1mL YEB substratum Eddy diffusion cell, (1 liter of YEB substratum is containing 1g yeast extract, 5g beef extract, 5g peptone, 5g sucrose, 0.5g MgSO for the YEB flat board coating containing 100 μ g/mL kantlex and 125 μ g/mL Rifampins ₄7H ₂o and 12g agar, pH7.0) on, cultivate about 48h for 28 DEG C;

3. picking positive colony is as template, identifies with colony polymerase chain reaction (PCR) method;

4. through identify correct after, by the Agrobacterium bacterium colony containing object plasmid in 10mL YEB substratum (containing 0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.05%MgSO47H ₂o, 1.2% agar, 100 μ g/mL kantlex and 125 μ g/mL Rifampins) in, 28 DEG C, 200r/min shakes overnight incubation;

5. transform and be inoculated in 200mL containing enlarged culturing in identical antibiotic YEB nutrient solution to OD by 1:50 the day before yesterday ₆₀₀for 0.6-0.8; Get bacterium liquid, in the ratio of 1% ~ 2%, proceed in the YEB liquid nutrient medium of the antibiotic-free of new preparation, after 6 hours, bacterium liquid OD ₆₀₀conversion of plant is can be used for when being 0.2 ~ 0.5.

4 conversion of plants

By agriculture bacillus mediated flower-dipping method transformation mode plant Arabidopsis thaliana, operate as follows:

1. the OD that 200mL step 3 obtains is got ₆₀₀be the Agrobacterium bacterium liquid of 0.2 ~ 0.5, the centrifugal 3min of 10000g, gets precipitation 200mL permeabilization buffer resuspended; Transform permeabilization buffer used to contain: 0.5 × MS macroelement, 0.5 × MS trace element, 0.5mg/L VB5,5% sucrose, 44nM6-BA(Sigma company, the U.S.) and 0.03%Silwet L-77(LEHLE SEEDS company, the U.S.);

2. permeabilization buffer 200mL being contained object Agrobacterium is placed in a container, upset kind has the flowerpot of Arabidopis thaliana, in the permeabilization buffer that plant is immersed containing Agrobacterium to be transformed, soak 5 minutes, slow taking-up flowerpot, is sidelong in pallet, covers black plastic cloth lucifuge 24 hours, within second day, take off plastic cloth, upright placing flowerpot;

3. MS screening dull and stereotyped (the additional 80 μ g/mL Totomycin of MS substratum and 50 μ g/mL penbritins) is prepared, the T1 transforming results is seeded in MS screening flat board for seed after sterilization, the flat board of each diameter 15cm can be sowed the Arabidopis thaliana seed of 100 μ about g;

4. MS is screened flat board and be placed in 4 DEG C of vernalization after 3 days, lie in growth case and cultivate (22 DEG C of constant temperature, 24h illumination), select root system and overground part on MS screening flat board after 7-10 days and grow normal positive plant, move into after normal MS substratum delays seedling 3-5 days and be implanted into soil, T2 is for seed for individual plant results; Breeding is also identified to T3 generation, obtains 48 transgenic lines isozygotied.

5 Function Identification

(1) salt resistant function qualification

Get T3 and use 1% hypochlorite disinfectant respectively for transgenic line and wild-type (ecotype Columbia) Arabidopis thaliana seed, vernalization 3 days in 4 DEG C of refrigerators, be then placed in temperature 22 DEG C, the incubator of humidity 50%, continuously 24h illumination cultivates; After 7d, observe growth of seedling situation;

After seedling cotyledon launches completely, move into transgenic line and wild type seedlings containing on 200mMNaCl and normal MS solid medium (containing 1 × macroelement, 1 × trace element, 1 × molysite, 3% sucrose and 0.8% agar), the incubator being then placed in identical light and temperature condition (22 DEG C, humidity 50%, continuous 24h illumination) is cultivated; After cultivating 12-15d, observe transgenic line and the phenotype of wild type seedlings under high-salt stress.

Found that under 200mM NaCl, the whole albefaction of wild type seedlings is dead, and transgenic line seedling leaves still keeps green, illustrates that SsMBTF1 gene overexpression can alleviate Arabidopis thaliana salt damage, significantly improves the resistance to high salt of Arabidopis thaliana.

(2) drought-enduring Function Identification

The T3 that picking broadcasts rear 8-10 days from culture dish for the normal seedling plantation of transgenic line and wild-type in the little basin that the 80-100g flower cultivating soil ratio 1:1 of vermiculite (soil with) is housed in advance, every little basin kind 4 seedlings;

Then little basin is placed in rectangular tray, each tray puts 12 little basins, adds a certain amount of clear water in tray, then covers transparent plastic film on 12 little basins, is placed in temperature 20 DEG C, the greenhouse of the dark 8h of illumination 16h/ cultivates;

Film is taken off, according to the suitable moisturizing of soil dry-wet situation after 5-6 days; A situation arises to observe growth of seedling and disease and pest later every day, and carry out suitable moisturizing and laxative and prevent the popular of soil overdrying and disease and pest;

After growth 3-4 week, now Arabidopsis plant not yet bolting bloom, stop watering, allow its soil natural dehydration, one week afterwards Arabidopsis plant blade start to occur wilting dehydration that then rehydration after 3 days allows its restoration ecosystem.

Found that WT lines blade wilting dehydration is serious, can not recover until dead after rehydration, and turn SsMBTF1 gene strain blade table and reveal temporary transient dehydration, can restore normal growth after rehydration, illustrate that SsMBTF1 gene overexpression can improve Arabidopis thaliana drought-resistance ability.

Claims (9)

1. a Suaeda salsa albumen, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.

2. the encoding gene of Suaeda salsa albumen as claimed in claim 1, it is characterized in that, base sequence is as shown in SEQ ID No.2.

3. contain expression unit or the recombinant plasmid of encoding gene as claimed in claim 2.

4. Suaeda salsa gene, improving the application in plant stress tolerance, is characterized in that as claimed in claim 2, and described resistance of reverse is at least one in salt tolerance and drought tolerance.

5. apply as claimed in claim 4, it is characterized in that, described plant is dicotyledons.

6. apply as claimed in claim 4, it is characterized in that, comprising:

(1) extract Suaeda salsa total serum IgE reverse transcription is cDNA, be template with cDNA, utilize Suaeda salsa gene described in primer amplification;

(2) utilize the gene constructed recombinant vectors of this Suaeda salsa, and this recombinant vectors is imported in target plant by agrobacterium-mediated transformation, obtain conversion of plant;

(3) from conversion of plant, screening obtains resistance of reverse plant;

The base sequence of described primer is:

Upstream primer: 5 '-TCTAGAATGGTGAGAGAAAAGATTAA-3 ';

Downstream primer: 5 '-GGTACCTTAATATATCCCCCTGTTTC-3 '.

7. apply as claimed in claim 6, it is characterized in that, in step (2), the initial carrier of described recombinant vectors is Super1300 or pCAMBIA1300.

8. apply as claimed in claim 7, it is characterized in that, in step (3), by the T1 of conversion of plant for planting seed in containing in the screening flat board of hygromycin B, get positive plant many generations breeding, obtain the resistance of reverse plant of genetic stability.

9. apply as claimed in claim 8, it is characterized in that, in described screening flat board, the concentration of hygromycin B is 80 ~ 100 μ g/mL.