CN107299103A

CN107299103A - Thick boisiana IpASR genes and its encoding proteins and application

Info

Publication number: CN107299103A
Application number: CN201710661281.9A
Authority: CN
Inventors: 张美�; 张会; 简曙光; 夏快飞
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2017-08-04
Filing date: 2017-08-04
Publication date: 2017-10-27
Anticipated expiration: 2037-08-04
Also published as: CN107299103B

Abstract

The present invention relates to engineering bacteria saccharomyces cerevisiae, Escherichia coli and field of plant molecular biology.The invention provides thick boisiana salt stress response related gene IpASR, the amino acid sequence that it is encoded is as shown in SEQ ID NO.1, and its cDNA reading frame sequence is as shown in SEQ ID NO.2.Abscisic acid/stress/Maturation induction protein I pASR of IpASR gene codes of the present invention is related to improving saccharomyces cerevisiae, Escherichia coli and the drought-and salt-tolerance of plant.By building yeast, Escherichia coli and the plant transgene overexpression vector of IpASR genes, IpASR genes are overexpressed in yeast, Escherichia coli and arabidopsis, the tolerance of yeast, Escherichia coli and arabidopsis to salt stress and drought stress can be improved.The gene can be applied to engineering bacteria and plant is directed to the genetic engineering breeding of high salt drought stress, with very big application value.

Description

Thick boisiana IpASR genes and its encoding proteins and application

Technical field

The invention belongs to biological gene engineering field, and in particular in thick boisiana (Ipomoea pes-caprae L.) IpASR(Abscisic acid-,stress-,and rIpening-induced gene) gene and its encoding proteins and adjusting Control the application of the drought-enduring aspect of organism salt tolerant.

Background technology

ASR albumen full name be abscisic acid/stress/Maturation induction albumen (Abscisic acid-,stress-,andrIpening-induced proteins, ASR albumen), it is distinctive class hydrophilic protein family in plant, generally has and turn Record the activity of the factor.Recent study finds that they rise during growth and development of plants, ripening and senscence and abiotic stress Important effect.ASR albumen be also plant LEA protein (Late-Embryogenesis ABundant Proteins, embryo Occur late period Abundant protein) a subfamily, at present in tomato, paddy rice, corn, banana, pine tree, grape, potato, lily Deng being all cloned into the gene in higher plant, and it was found that the ASR gene family numbers of different plant species are not quite similar, but are intending In southern mustard and in the absence of such gene.Existing research shows, the expression of ASR genes is cooled, osmotic pressure, the acid treatment that comes off etc. Induction, is also induced expression in ripening of fruits.Due to the research table to different floristic multiple ASR gene members Bright, ASR genes are in abiotic stress such as some plant response high salt, arid, freezings, and such coerces the mistake with plant Water is closely related, is highlighted the resistance of the related abiotic stress of ASR gene pairs dehydration.

Plant is in its growth cycle, it will usually disturbed by a variety of environment stresses, and to the production of growing of plant Raw influence.And for crops, the disturbance of environment stress generally influences whether the yield and quality of crop.At present, using many The resistance for planting breeding technique raising crops is the major issue that modern agriculture science faces.Crop transgenosis function breeding is Breeding for stress tolerance provides a kind of effective convenient means, but the technology needs to provide the candidate with degeneration-resistant biological function Gene is operated, and separates and identify such functional gene one of research focus as plant genetic engineering.Wild plant is Important natural resources and enviroment key element, and under the pressure of natural selection, the adaptive plant under extreme adverse circumstance necessarily have compared with Strong degeneration-resistant genetic resources, the excavation and identification of its degeneration-resistant functional gene are also the one of wild plant resource protection and developmental utilization Individual importance.Thick boisiana (Ipomoea pes-caprae L.) is a kind of general tropical and subtropical zone profile strand plant, with pole Strong resistance, and its salt tolerance and drought resistance are especially pronounced.At present in terms of the exploitation of thick boisiana ASR genetic resourceses also Have no report.

The content of the invention

It is an object of the invention to provide a kind of salt tolerant, drought-enduring plant functional protein --- thick boisiana abscisic acid/stress/maturation Inducible protein IpASR and its application.Present invention discover that a gram for representing abscisic acid/stress/Maturation induction GFP IpASR Grand to improve the salt tolerance of yeast strain, the present invention has cloned the abscisic acid/stress/Maturation induction GFP IpASR, leads to Cross functional study to show, the gene can improve the salt tolerant and drought-resistant ability of organism.

Realize that the technical scheme of above-mentioned purpose is as follows.

Thick boisiana abscisic acid/stress/Maturation induction protein I pASR of the amino acid sequence as shown in SEQ ID NO.1 and/or Application of the IpASR genes in tolerance of the plant to high salt/arid is improved, the cDNA of the IpASR genes is such as SEQ ID Nucleotide sequence shown in NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or be encoding amino acid sequence The nucleotide sequence of thick boisiana abscisic acid/stress/Maturation induction albumen as shown in SEQ ID NO.1.

Thick boisiana abscisic acid/stress/Maturation induction protein I pASR of the present invention, its amino acid sequence such as SEQ ID NO.1 institutes Show, its encoding gene IpASR nucleotide sequence is as shown in SEQ ID NO.2.It should be understood that, it is contemplated that the degeneracy of codon Property, on the premise of amino acid sequence is not changed, the nucleotide sequence of above-mentioned encoding gene is modified, the present invention is fallen within Protection domain in.

Thick boisiana abscisic acid/stress/Maturation induction protein I pASR of the amino acid sequence as shown in SEQ ID NO.1 and/or IpASR genes improve crops to high salt/drought tolerance, and/or regulation and control crops to oxygen in crops transgenic breeding Change the application of stress tolerance, the cDNA of the thick boisiana abscisic acid/stress/Maturation induction GFP IpASR is such as SEQ ID Nucleotide sequence shown in NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or be encoding amino acid sequence The nucleotide sequence of thick boisiana abscisic acid/stress/Maturation induction albumen as shown in SEQ ID NO.1.

It is another object of the present invention to disclose one group of thick boisiana IpASR Protein reconstitutions expression vector and its application, the restructuring is carried Body inserts IpASR genes.Recombinant vector conversion is entered in saccharomyces cerevisiae, the gene is induced in yeast by galactolipin Overexpression, yeast can be improved to the tolerance of salt stress and oxidative stress under high salt and Hydrogen Peroxide Stress processing.

Meanwhile, open to build thick boisiana IpASR Protein reconstitutions prokaryotic expression carrier and its utilization, the recombinant vector is inserted IpASR genes, carrier conversion is entered in e. coli strain bl21, the gene is induced in Escherichia coli by IPTG Overexpression, it was demonstrated that IpASR genes can improve the salt tolerant and drought tolerance of engineering bacteria Escherichia coli.

Realize that the technical scheme of above-mentioned purpose is as follows：

Saccharomyces cerevisiae recombinant expression carrier inserted with IpASR genes, the cDNA of the IpASR genes is such as SEQ ID Nucleotide sequence shown in NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or it is encoded to amino acid sequence The nucleotide sequence of thick boisiana IpASR albumen as shown in SEQ ID NO.1.

Above-mentioned saccharomyces cerevisiae recombinant expression carrier is improving tolerance of the engineered strain saccharomyces cerevisiae to high salt and oxidative stress Application in property.

Coli expression carrier inserted with IpASR genes, the cDNA of the IpASR genes is such as SEQ ID NO.2 Shown nucleotide sequence, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or for encoding amino acid sequence such as The nucleotide sequence of thick boisiana IpASR albumen shown in SEQ ID NO.1.

Above-mentioned Recombinant protein expression carrier is improving engineering bacteria Escherichia coli in high salt and drought stress tolerance Application.

In addition, existing it is another object of the present invention to disclose thick boisiana abscisic acid/stress/Maturation induction GFP IpASR Overexpression vector and its application in model plant arabidopsis, the recombinant vector is by agriculture bacillus mediated inflorescence infestation method to intending Southern mustard carries out transgenosis, it is possible to increase the salt tolerant and drought resistance of arabidopsis.

Concrete technical scheme is as follows：

The present invention extracts RNA using thick boisiana seedling as material, and by RNA reverse transcriptions into cDNA, and by cDNA respectively with PDONR222 and pYES-DEST52 is destination carrier, is usedTechnology (Invitrogen) construction cDNA expression text Storehouse, and by mutant strain AXT3 of the Library plasmid transformed yeast to salt density value, picking salt tolerant yeast clone, and plasmid is extracted, confront Grain is sequenced, and passes through sequence analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi), obtain thick boisiana and take off Fall acid/stress/Maturation induction GFP IpASR cDNA total lengths, its sequence is as shown in SEQ ID NO.2.

By the Yeast expression carrier pYES-DEST52 recombinant plasmid transformed wild-type yeasts of the cDNA total lengths containing IpASR Bacterial strain W303, using the W303 of transformed yeast expression vector pYES2 empty carriers as control, respectively in salt content 5%, 7.5% He The salt tolerance of transgenic yeast is tested on 8.8% culture medium.

With the Yeast expression carrier pYES-DEST52 weights containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Group plasmid is template, designs the cDNA total length reading frame sequences that following primer expands IpASR genes respectively, is inserted into for obtaining E. coli protein expression vector pGEX 6p-1 cDNA reading frame fragments.Primer such as SEQ ID NO.3 (IpASREEF:5’- GGGGCCCCTGGGATCCATGTCTGAGATGAAACACCA-3 ') and SEQ ID NO.4 (IpASREER:5’- GGAATTCCGGGGATCCTTAAAAGAAGTGATGCTTCT-3 ') shown in.

Above PCR is expanded using the Taq enzyme of high-fidelity, and obtained target DNA fragment is reclaimed, and is connected to On e. coli protein expression vector pGEX 6p-1 (BamHI point of contacts), recombinant vector IpASR-pGEX 6p-1 are formed, and survey Sequence.

Using CaCl₂Method the expression of above-mentioned thick boisiana abscisic acid/stress/Maturation induction albumen e. coli protein is recombinated Carrier conversion enters in e. coli bl21 competence bacterial strain, and IpASR genes are induced in large intestine bar by adding 0.2mM IPTG Overexpression in bacterium.Detection expression afterwards has the salt tolerance of the Escherichia coli of thick boisiana abscisic acid/stress/Maturation induction albumen and resisted Drought.

With the Yeast expression carrier pYES-DEST52 weights containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Group plasmid is template, designs the cDNA total length reading frame sequences that following primer expands IpASR genes respectively, is inserted into for obtaining Arabidopsis transgenosis overexpression vector pMD1 cDNA reading frame fragments, for building thick boisiana abscisic acid/stress/Maturation induction egg White gene transgenic overexpression recombinant vector IpASR-pMD1.Primer such as SEQ ID NO.5 (IpASROXF:5’- GGACTCTAGAGGATCCATGTCTGAGATGAAACACCA-3 ') and SEQ ID NO.6 (IpASROXR:5’- GTCGACCCGGGGATCTTAAAAGAAGTGATGCTTC-3 ') shown in.

Using CaCl₂Method above-mentioned thick boisiana abscisic acid/stress/Maturation induction protein transgene is overexpressed into recombinant vector IpASR-pMD1 conversions enter in Agrobacterium GV3101 competence bacterial strains, are planted by arabidopsis floral infestation method arabidopsis thaliana transformation Strain, transgenic progeny is obtained by Kan resistance screenings, and thick boisiana abscisic acid/stress/Maturation induction albumen base is overexpressed to transgenosis The arabidopsis offspring of cause carries out salt tolerance and drought resistance detection.

In the present invention, the gene of a coding abscisic acid/stress/Maturation induction albumen in thick boisiana body is we disclosed IpASR, the expression of the gene can improve tolerance of the saccharomycete to salt and oxidative stress, and there is provided the gene is applied into work The application of salt tolerant and the oxidation resistance genetic improvement of journey bacterium saccharomyces cerevisiae；Expression of the IpASR in Escherichia coli can improve engineering There is provided the engineering bacteria salt tolerant that the gene is used for including Escherichia coli for tolerance of the bacterium Escherichia coli to high salt and arid With the application in terms of the genetic improvement of dehydration tolerance.The present invention also simply elaborates that the heredity that the gene can be applied into plant changes Make, cultivate the transgenic plant for improving salt tolerant/drought tolerance.

Beneficial effects of the present invention are as follows：

In the present invention, the thick boisiana that we are obtained by screening encodes the IpASR of abscisic acid/stress/Maturation induction albumen The progress application sex exploration of gene, finds the following application mode of the gene：(1) overexpression energy of the gene in saccharomyces cerevisiae Yeast is enough improved to high salt and the tolerance of oxidative stress；(2) by inducing thick boisiana abscisic acid/stress/Maturation induction albumen to exist After expression in escherichia coli, it is possible to increase the salt tolerance and drought tolerance of Escherichia coli；(3) excess table of the gene in arabidopsis Danone enough improves transgenic arabidopsis to high salt and the tolerance of drought stress.

The present invention also may extend to agricultural biological technical field, by changing expression of the IpASR genes in crops, adjust Tolerance of the crops to high salt and drought stress is controlled, and participates in adaptive change of the crops to a variety of environment stresses.

Brief description of the drawings

Fig. 1 shows the schematic diagrames of saccharomyces cerevisiae recombinant expression carrier IpASR-pYES-DEST 52.

Fig. 2 shows the transgenic yeast for converting IpASR-pYES-DEST 52 to the mutant strain AXT3 of salt density value to salt stress Tolerance is improved.

Fig. 3 shows that conversion IpASR-pYES-DEST 52 transgenosis wild-type yeast W303 is carried to the tolerance of salt stress It is high.

Fig. 4 shows conversion IpASR-pYES-DEST 52 transgenic yeast to H₂O₂Sensitive mutant strain yap1 Δs (A) and Skn7 Δs (B) are improved to the tolerance of oxidative stress.

Fig. 5 shows Escherichia coli expression of recombinant proteins carrier IpASR-pGEX 6p-1 schematic diagrames.

Fig. 6 shows conversion IpASR-pGEX 6p-1 transgenic escherichia coli to high-salt stress (A) and desiccation stress (B) Tolerance is improved.

Fig. 7 shows model plant arabidopsis transgenosis overexpression vector IpASR-pMD1 schematic diagrames.

Embodiment

For the ease of understanding the present invention, the present invention will be described more fully below.The present invention can be with many not With form realize, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to this The understanding of the disclosure of invention is more thorough comprehensive.

The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook People, molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) in institute The condition stated, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are commercially available in embodiment Product.

Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality The purpose of example is applied, the limitation present invention is not used in.Term "and/or" used in the present invention includes one or more related listed The arbitrary and all combination of project.

Embodiment 1：Thick boisiana abscisic acid/stress/Maturation induction albumen is obtained by thick boisiana cDNA Yeast expressions library screening Gene IpASR cDNA total lengths

The structure of 1.1 thick boisiana full-length cDNA expression libraries

The structure Primary Reference CloneMiner II cDNALibrary Construction Kit of thick boisiana cDNA library Specification, use(Invitrogen) technology is carried out.Specifically include following steps：Total RNAs extraction, mRNA The structure of separation, the structure of cDNA primary libraries and cDNA secondary libraries.Each step overview is as follows：

(1) Total RNAs extraction

Thick boisiana blade, leaf bud, rattan and the young root of 2g equivalent are taken, with liquid nitrogen grinding into powder in the mortar of precooling, by powder End is moved in multiple RNase-free 1.5mL centrifuge tubes in right amount, and each centrifuge tube adds 1mL Trizol Reagent, rapidly Concussion is mixed, and is operated according to reagent specification, obtains the total serum IgE aggregate sample of thick boisiana different tissues.Dried total serum IgE is molten In 100 μ L RNase-free water.The concentration of total serum IgE is determined with ultraviolet specrophotometer and 1% agarose gel electrophoresis And quality.

(2) mRNA separation

The thick boisiana total serum IgE more than 500 μ g is taken, is separated according to FastTrack MAG mRNA isolation Kit specifications MRNA is purified, with determined by ultraviolet spectrophotometry mRNA concentration, mRNA quality is detected with 1% agarose gel electrophoresis.

(3) structure of cDNA primary libraries

About 3 μ g thick boisiana mRNA is taken, water to the cumulative volume for adding RNase-free is 9 μ L；Then biotin- is added AttB2-Oligo (dT) Primer (30pM) 1uL and dNTPs (10mM each) 1uL is mixed, 70 DEG C of 5min, 45 DEG C of incubations 2min；Add SuperScript III First-Strand Synthesis System for RT-PCR 5 × First Strand Buffer 4 μ L and DTT (0.1M) 2 μ L, are eventually adding 3 μ L SuperScript III reverse transcriptases to middle reaction Volume is 20 μ L, and 45 DEG C of 20min of reaction in PCR instrument are placed in after mixing；50℃20min；55℃20min.Above step is synthesis The chains of cDNA first.

E.coli DNA Ligase, E.coli DNA Polymerase are sequentially added in cDNA the first chain reaction liquid I, E.coli RNaseH and T4DNA Polymerase etc. synthesize the second chain, now obtain thick boisiana double-strand cDNA.By double-strand CDNA is connected with attB1 restructuring joints after purification, and the thick boisiana double-strand cDNA for connecting joint uses CloneMiner II cDNA Library Construction Kit classification separation chromatographic column, removes the small fragment cDNA that molecular weight is less than 500bp, retains CDNA fragments more than 500bp are used for the structure of follow-up cDNA library.

BP recombining reactions are carried out with reference to CloneMiner II cDNA Library Construction Kit specification Construction cDNA primary libraries.Specific steps include：CDNA (100ng/ μ L) 13 μ L are added successively in clean PCR pipe, PDONR222 (250ng/ μ L) 2 μ L, BPThe μ L of II enzyme mix 5 after being well mixed, are placed in the μ L of cumulative volume 20 16~20h of BP recombining reactions is carried out at 25 DEG C.BP recombining reactions product after purification is imported by Escherichia coli by electroporation apparatus It is rapid after electric shock that 2ml SOC culture mediums are added into electric revolving cup in DH10B competent cells, it is primary text after preculture Storehouse bacterium solution.

(4) structure of cDNA secondary libraries

The structure of cDNA secondary libraries is carried out using LR recombining reactions, specific steps include：Primary libraries LB culture mediums After amplification cultivation, PureLink is then used^TMHQ Mini Plasmid DNA Purification Kit extract plasmid.It is primary CDNA library plasmid is dissolved using appropriate TE, and uses determined by ultraviolet spectrophotometry concentration.By primary libraries plasmid concentration Adjust to 300ng/ μ L, take 1 μ L Library plasmids to sequentially add μ L, the LR Clonase II of pYES-DEST 52 (300ng/uL) 1 Mix1 μ L, add the μ L of distilled water 12 to the μ L of cumulative volume 20.It is placed at 25 DEG C and carries out 16~20h of LR recombining reactions.Afterwards using electricity Punching instrument imports LR recombining reactions product after purification in Escherichia coli DH10B competent cells, rapid after electric shock to turn to electricity 2mL SOC culture mediums, after preculture, as secondary library bacterium solution are added in cup.

The screening of 1.2 thick boisiana cDNA libraries

The method that the screening of thick boisiana cDNA library uses the mutant strain AXT3 of plasmid library transformed yeast salt density value.AXT3 exists It can not be grown on the culture medium of NaCl containing 75mM, and wild-type yeast then can be with normal growth.The inverted zymogens of AXT3 Behind grain library, on the culture medium for being coated on the NaCl containing 75mM, cultivated 3 to 7 days through 30 DEG C, can obtain several can recover To the yeast clone of salt density value.To these clonal expansion cultures and extract plasmid, you can obtain multiple thick boisiana resistant gene of salt, wherein One cDNA full length sequence for coding thick boisiana abscisic acid/stress/Maturation induction protein I pASR.Detailed step is as follows：

(1) acquisition of thick boisiana cDNA library plasmid

The acquisition of Library plasmid uses solid LB media flat board TRAP, i.e., according to total clone's number in library, by thick boisiana Secondary library bacterium solution is coated on the culture of 37 DEG C of 100 LB plate overnights containing Amp antibiotic 14cm, it is ensured that library it is each Clone is obtained for the amplification of equivalent.After bacterium colony is grown, plus LB liquid medium elution, alkaline lysis method of extracting plasmid.(2) it is thick The mutant strain AXT3 of rattan cDNA library plasmid transformed yeast salt density value

The secondary library plasmid concentration of adjustment after purification is dashed forward to 1 μ g/ μ L using lithium acetate method transformed yeast to salt density value Mutant AXT3.Picking yeast monoclonal is inoculated in liquid YPD medium, and 30 DEG C of concussion and cultivates are stayed overnight.Afterwards according to 1:100 Ratio transfers yeast liquid in fresh liquid YPD medium, 30 DEG C of concussion and cultivates to OD₆₀₀For 0.5 or so, lithium salts is molten Liquid is resuspended, plus the smart carrier DNA of appropriate salmon, 50%PEG.Thalline is diluted according to precipitation capacity, is coated on the NaCl's containing 75mM Cultivated on yeast screening assay culture medium (yeast minimal medium adds adenine, lacks uracil), until there is transformant.

The acquisition and sequence analysis of 1.3 thick boisiana abscisic acids/stress/Maturation induction GFP IpASR cDNA total lengths

The picking yeast monoclonal on the yeast screening assay culture medium of the NaCl containing 75mM, connects bacterium in the liquid for being not added with NaCl Amplification cultivation is carried out in body screening and culturing medium.It is collected by centrifugation after yeast thalline, according to HiPure Yeast Plasmid Mini Kit specification extracts yeast plasmid.Because yeast plasmid content is relatively low, it is impossible to directly carry out sequencing analysis, therefore by zymogen In grain conversion bacillus coli DH 5 alpha competent cell, after picking monoclonal amplification cultivation, using alkaline lysis method of extracting plasmid, carry out Sequencing analysis.Sequencing is completed by Guangzhou Ai Ji biotech firms.

CDNA nucleotide sequence comparisons, translation after sequencing etc. are carried out on DNAStar7.0 biosoftwares, sequence homology Property analysis use blast program, in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) website is enterprising OK.

Sequencing result shows that cDNA of one of clone containing coding thick boisiana abscisic acid/stress/Maturation induction albumen is complete Long, its structural representation is as shown in Figure 1.Remove after the carrier sequences of pYES-DEST 52, thick boisiana abscisic acid/stress/Maturation induction The cDNA full length sequences of albumen are as follows, and reading frame is the nucleotide sequence as shown in SEQ ID NO.2, and its most long reading frame is volume Thick boisiana IpASR albumen of the code amino acid sequence as shown in SEQ ID NO.1.

SEQ ID NO.1

Amino acid sequence

>IpASR protein

MSEMKHHHHFGHHKDNEEERQSSCGENTYGTDEKPYGQTGYGEESYERKNTYGDDSYERKNTYGDDSSC ERKNTYGDDSSYERKNTYGDDSYYERKNTYGDDSYGQIDKYGSEGVTGGIEPEGKTHEDYEKEKKHHKHLEQLGGLG TVAAGAYALYEKHEAKKDPENAHKHKIAEEVAAVAAVGSGAFAFHEHHEKKEAKEEEEEAEGKKKHHFF

(painting dash area is cDNA reading frame SEQ ID NO.2 to cDNA sequence, and upstream is 5 ' non-translational regions, and downstream is 3 ' Non-translational region)

>IpASR

AACATATTTTAGCTTAGCATTCATATTTCGTCCACTACCAGAAACCATCCCAAAAAACTAAAATAATCATGTCTGAG ATGAAACACCACCACCACTTTGGTCACCACAAAGACAATGAGGAAGAGAGGCAGTCTTCCTGTGGTGAAAACACTTA TGGCACTGATGAGAAACCTTATGGCCAAACAGGCTATGGGGAAGAGTCTTATGAGAGGAAAAACACCTATGGTGATG ACTCTTATGAGAGGAAAAACACCTATGGAGATGACTCTTCTTGTGAGAGGAAAAACACCTATGGAGATGACTCTTCT TATGAGAGGAAAAACACTTATGGAGATGACTCTTATTATGAGAGGAAAAACACTTATGGAGATGACTCTTACGGCCA AATCGACAAGTATGGGAGTGAAGGCGTGACCGGCGGCATAGAACCGGAAGGGAAAACTCATGAGGATTACGAAAAGG AGAAAAAACACCACAAGCATCTCGAGCAGCTCGGCGGGCTTGGCACCGTCGCCGCCGGTGCCTATGCCTTGTACGAG AAGCACGAGGCAAAGAAAGACCCAGAGAATGCGCACAAGCATAAGATAGCAGAAGAGGTGGCGGCAGTGGCTGCCGT TGGATCAGGTGCATTTGCATTCCATGAGCATCATGAGAAGAAGGAAGCTAAGGAGGAAGAAGAAGAGGCTGAGGGAA AGAAGAAGCATCACTTCTTTTAATTACATACTATATATGTGTTTTTATTATTAAGATCAAACTAAATAATCCGATCC TTAGCTTGTACGACGTGTGTGAATAATGGGTTTGGGCTTATGCGTGTTGAGGTACGAGGAGGGTTTCTTTGTACCTA GCTAAGCTACCTATCTACTCTATTTGTGTCGACTGAGATGTTTTATTATTCTCTAGTGTATGTGGATTGTAATAATA CTAATACTGATATAAAGTTTATTAAAAAAAAAAAAAAA。

Embodiment 2：IpASR genes overexpress the salt tolerance and the tolerance to oxidative stress for improving yeast in yeast

The transformed yeast bacterial strains of 2.1IpASR-pYES-DEST 52

The recombinant plasmids of IpASR-pYES-DEST 52 that sequencing result is verified adjust concentration to 0.1 μ g/ μ L, use The method of lithium acetate method distinguishes mutant strain AXT3 and corresponding wild-type yeast strain W303 of the transformed yeast to salt density value, and Transformed yeast is to H₂O₂Sensitive mutant strain yap1 Δs and skn7 Δs and corresponding wild-type yeast strain WT.Use yeast simultaneously Expression vector empty carrier pYES2 is compareed, and above-mentioned yeast strainss are converted respectively.

2.2IpASR genes express improve in yeast Salt tolerant mutant AXT3 and wild-type strain W303 to be turned The salt tolerance of gene yeast

Picking yeast strain AXT3 conversion empty carrier pYES2 and IpASR gene overexpression vectors IpASR-pYES-DEST 52 monoclonal, is inoculated in the yeast minimal fluid nutrient medium that 2ml with the addition of galactolipin, 30 DEG C of constant-temperature table (200rpm) trainings Support to bacterium solution OD₆₀₀It is worth up to 2.According to 1:1、1:10、1:100、1:1000 are diluted bacterium solution step by step, and 2 μ l are drawn respectively step by step The bacterium solution of dilution is dropped to added with 50mM, on 75mM and 200mM NaCl yeast minimal solid medium flat board.30 DEG C of cultures 7 My god, observe yeast growth situation.

As shown in Fig. 2 the transgenic yeast of overexpression IpASR genes is compared to conversion empty carrier pYES2 AXT3 ferment For female mutant strain, it can be grown on addition 50mM and 75mM NaCl culture medium flat plate, and addition 200mM NaCl's Can also slightly it be grown on culture medium flat plate；And the yeast for not expressing thick boisiana abscisic acid/stress/Maturation induction protein I pASR (turns Change empty carrier pYES2) it can not then grow, show that expression of the IpASR genes in yeast can improve yeast Salt tolerant mutant Tolerances of the AXT3 to salt stress.

Meanwhile, picking wild-type yeast strain W303 conversion empty carrier pYES2 and IpASR gene overexpression vectors IpASR- PYES-DEST 52 monoclonal, carries out fluid nutrient medium culture according to the method described above, and drops to and be added with after diluting step by step On 5%, 7.5% and 8.8% (being mass volume ratio) NaCl yeast minimal solid medium flat board.30 DEG C are cultivated 7 days, are seen Examine yeast growth situation.

As shown in figure 3, the transgenic yeast of overexpression IpASR genes is compared to conversion empty carrier pYES2 W303 ferment For female mutant strain, it can be grown on the culture medium flat plate for adding 5% and 7.5%NaCl, and upgrowth situation is good, and adding Plus can also slightly be grown on 8.8%NaCl culture medium flat plate；And thick boisiana abscisic acid/stress/Maturation induction albumen is not expressed IpASR yeast (conversion empty carrier pYES2) can not then grow, and show that expression of the IpASR genes in yeast can improve open country Tolerances of the raw type yeast strainss W303 to salt stress.

2.3IpASR genes are in yeast to H₂O₂Transgenosis can be improved by being expressed in sensitizing mutation strain yap1 Δs and skn7 Δs Tolerance of the yeast to oxidative stress

Picking yeast is to H₂O₂Sensitizing mutation strain yap1 Δs and skn7 Δs conversion empty carrier pYES2 and IpASR gene overexpression Carrier IpASR-pYES-DEST 52 monoclonal, while also picking wild-type yeast WT converts empty carrier pYES2 monoclonal As control, above-mentioned clone is inoculated in the yeast minimal fluid nutrient medium that 2ml with the addition of galactolipin respectively, 30 DEG C of constant temperature shake Bed (200rpm) is cultivated to bacterium solution OD₆₀₀It is worth up to 2.According to 1:1、1:10、1:100、1:1000 are diluted bacterium solution step by step, point The bacterium solution that 2 μ l dilute step by step is not drawn drops to the H added with 0.75mM₂O₂Yeast minimal solid medium flat board on.30 DEG C of trainings Support 7 days, observe yeast growth situation.

As shown in figure 4, the transgenic yeast of overexpression IpASR genes is compared to conversion empty carrier pYES2 yap1 Δs , can be in addition 0.75mM H for yeast mutant₂O₂Culture medium flat plate on grow；And thick boisiana abscisic acid/side of body is not expressed Urgent/Maturation induction protein I pASR yeast yap1 Δs (conversion empty carrier pYES2) can hardly then grow (Fig. 4 A).Equally, surpass The transgenic yeast skn7 Δs for expressing IpASR genes also can be in addition 0.75mM H₂O₂Culture medium flat plate on grow；And not Expression thick boisiana abscisic acid/stress/Maturation induction protein I pASR yeast skn7 Δs (conversion empty carrier pYES2) then can hardly Grow (Fig. 4 B).These results show IpASR genes in H₂O₂Expression in sensitive yeast mutant yap1 Δs and skn7 Δs can Improve the tolerance that yeast is coerced oxidative stress.

Embodiment 3：IpASR genes overexpress the salt tolerance and dehydration tolerance for improving Escherichia coli in Escherichia coli

3.1 Escherichia coli expression of recombinant proteins carrier IpASR-pGEX 6p-1 structure

With the Yeast expression carrier pYES-DEST 52 containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Recombinant plasmid is template, using SEQ ID NO.3 and SEQ ID NO.4 as primer, is expanded using the Taq enzyme of high-fidelity by PCR The cDNA reading frame total lengths of IpASR genes.The PCR system of use refers to TaKaRa companies PrimeSTAR HS DNAPolymerase with GC Buffer specifications.The DNA fragmentation of amplification is according to Magen companies HiPure Gel Pure DNAKits specifications.Reclaiming obtained fragment is used to be inserted into Escherichia coli expression of recombinant proteins vector pGEX 6p-1. PGEX6p-1 plasmids are handled through BamHI single endonuclease digestions, reclaim linearization plasmid.The cDNA reading frame PCR fragments of IpASR after recovery Concentration is determined through Nanodrop companies ultraviolet specrophotometer with linearisation pGEX 6p-1 plasmids, using TaKaRa (Clontech) companyHD Cloning Kit carry out the homologous recombination connection of DNA fragmentation and carrier.According to saying Reaction product is converted e. coli jm109 competence bacterial strain by the method for bright book.Picking monoclonal, extracts plasmid, is reflected through sequencing It is set to after correct positive colony, preserves plasmid standby.After sequencing analysis, IpASR cDNA nucleotide sequences such as SEQ ID Shown in NO.2, coded protein amino acid sequence is as shown in SEQ ID NO.1.The Escherichia coli recombinant protein table built It is as shown in Figure 5 up to carrier IpASR-pGEX 6p-1.

3.2 thick boisiana abscisic acids/stress/induced expression of the Maturation induction protein I pASR albumen in Escherichia coli

Using alkaline lysis method of extracting IpASR-pGEX 6p-1 recombinant plasmids, and convert into e. coli bl21 competence In bacterial strain, picking monoclonal induces IpASR genes in Escherichia coli into LB liquid medium by adding 0.2mM IPTG In overexpression.

3.3 expression have the salt tolerance detection of thick boisiana abscisic acid/stress/Maturation induction protein I pASR Escherichia coli

Picking conversion thick boisiana abscisic acid/stress/Maturation induction protein expression vector IpASR-pGEX 6p-1 Escherichia coli Monoclonal, the Escherichia coli clones to convert pGEX 6p-1 empty carriers are inoculated in LB liquid medium respectively as control (the final concentration of 100 μ g/ml of addition ampicillin), culture 12 hours in constant-temperature table (37 DEG C, 200rpm).Press afterwards According to 1:100 ratio is transferred in fresh LB liquid medium (the final concentration of 100 μ g/ml of addition ampicillin), training 2 to 4 hours are supported to OD₆₀₀Value adds final concentration of 0.2mM IPTG Fiber differentiations 2 to 4 hours to OD up to 0.5 afterwards₆₀₀Value reaches 1.According to 1:1、1:10、1:100、1:1000 are diluted bacterium solution step by step, the bacterium solution that 2 μ l dilute step by step is drawn respectively and is dropped to add Added with 1%NaCl (control, 145mM NaCl) and 4%NaCl (high-salt stress, 680mM NaCl) LB culture mediums (in culture medium Add final concentration of 0.2mM IPTG and 100 μ g/ml ampicillin) on, 37 DEG C are cultivated 12 to 24 hours, observe large intestine Bacillus growing state.

As shown in Figure 6A, under the normal growing conditions of addition Low Concentration NaCl (LB culture mediums, 1%NaCl), conversion is empty Vector pGEX 6p-1 and expression IpASR albumen (conversion IpASR protein expression vector IpASR-pGEX 6p-1) Escherichia coli Upgrowth situation it is consistent, but under the normal growing conditions of high salt concentration (LB culture mediums, 4%NaCl), translation table reaches IpASR eggs The upgrowth situation of Bai Zaiti (IpASR protein expression vector IpASR-pGEX 6p-1) Escherichia coli far superior to will be converted only Empty carrier pGEX 6p-1 Escherichia coli, show thick boisiana abscisic acid/stress/Maturation induction protein I pASR energy in Escherichia coli Enough improve the salt tolerance of Escherichia coli.

3.4 expression have the dehydration tolerance detection of thick boisiana abscisic acid/stress/Maturation induction protein I pASR Escherichia coli

Picking conversion thick boisiana abscisic acid/stress/Maturation induction protein expression vector IpASR-pGEX 6p-1 Escherichia coli Monoclonal, the Escherichia coli clones to convert pGEX 6p-1 empty carriers are inoculated in LB liquid medium respectively as control (the final concentration of 100 μ g/ml of addition ampicillin), culture 12 hours in constant-temperature table (37 DEG C, 200rpm).Press afterwards According to 1:100 ratio is transferred in fresh LB liquid medium (the final concentration of 100 μ g/ml of addition ampicillin), training 2 to 4 hours are supported to OD₆₀₀Value adds final concentration of 0.2mM IPTG Fiber differentiations 2 to 4 hours to OD up to 0.5 afterwards₆₀₀Value reaches 1.1 μ L each six parts of bacterium solution is taken respectively, three parts are control, three parts are placed in 37 DEG C of desiccation culture casees dry 1 hour to bacterium in addition Body is completely dried, and 1mL fresh liquid culture medium renewal cultivation is added afterwards 0.5 hour, is applied on LB culture medium flat plates, Control is then directly coated on LB culture medium flat plates, and 37 DEG C are cultivated 12 to 24 hours, observe clone's number of Escherichia coli.

As shown in the B in Fig. 6, the bacterium solution without dehydrating processing compares (conversion pGEX 6p-1 empty carriers) and expression The monoclonal bacterium colony (CFU) of the original bacterium solution formation of Escherichia coli (conversion IpASR-pGEX 6p-1) 1 μ L of IpASR albumen between 6x10⁶And 7x10⁶Between, both do not have significant difference.The 1 original bacterium solutions of μ L express IpASR albumen after dehydrating The monoclonal clump count that is formed of Escherichia coli (conversion IpASR-pGEX 6p-1) it is unloaded far more than only conversion pGEX6p-1 The Escherichia coli of body, both have extremely significant difference, show thick boisiana abscisic acid/stress/Maturation induction protein I pASR in large intestine bar The dehydration tolerance (drought tolerance) of Escherichia coli can be improved in bacterium.

Embodiment 4：IpASR genes overexpress the salt tolerance and drought tolerance for improving arabidopsis in arabidopsis

4.1 arabidopsis transgenosis overexpression vector IpASR-pMD1 structure

With the Yeast expression carrier pYES-DEST 52 containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Recombinant plasmid is template, using SEQ ID NO.5 and SEQ ID NO.6 as primer, is expanded using the Taq enzyme of high-fidelity by PCR The cDNA reading frame total lengths of IpASR genes.The PCR system of use refers to TaKaRa companies PrimeSTAR HS DNA Polymerase with GC Buffer specifications.The DNA fragmentation of amplification is according to Magen companies HiPure Gel Pure DNA Kits specifications.Reclaiming obtained fragment is used to be inserted into arabidopsis transgenosis overexpression vector pMD1.PMD1 plasmids are passed through The processing of BamHI single endonuclease digestions, reclaims linearization plasmid.The cDNA reading frames PCR fragment and linearisation pMD1 matter of IpASR after recovery Grain determines concentration through Nanodrop companies ultraviolet specrophotometer, using TaKaRa (Clontech) companyHD Cloning Kit carry out the homologous recombination connection of DNA fragmentation and carrier.Reaction product is converted large intestine by method to specifications Bacillus JM109 competence bacterial strains.Picking monoclonal, extracts plasmid, after being correct positive colony through sequencing identification, preserves plasmid IpASR-pMD1 is standby.

4.2 arabidopsis transgenosis overexpress the acquisition of plant

Plant transgene overexpression vector IpASR-pMD1 conversions are entered in Agrobacterium GV3101 using freeze-thaw method, obtained Recombinational agrobacterium.

Then recombinational agrobacterium is utilized, passes through flower infusion method (Clough SJ, Bent AF.1998.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J.16:735-743) by IpASR channel genes Columbia ecotype (Col) arabidopsis, T1 generations are obtained Seed.

T1 screens resistant plant after being harvested for seed in MS culture mediums (containing 50mg/L kanamycins), by resistant plant It is transplanted in soil, T2 is for seed for harvest.By T2 for cultivating seeds be plant (T2 is for plant), extract blade genomic DNA, use Primer pair SEQ ID NO.5 and SEQ ID NO.6 enter performing PCR identification, and the plant that PCR is accredited as the positive is that T2 plants for transgenosis Strain.T2 produces T3 for seed for transfer-gen plant selfing, and kalamycin resistance screening is carried out for seed to T3, all with card that Chloramphenicol resistance is homozygote.Randomly select the T3 of three transgenic homozygous body strains and carry out salt tolerant and drought tolerance for seed Identification.

The salt tolerance of 4.3 arabidopsis transgenosis overexpression progeny of plants and drought tolerance detection

The T3 of three transfer-gen plant homozygous lines (OX1, OX2, OX3) is randomly selected for seed (each strain 50), It is control with wildtype Arabidopsis thaliana (WT) (50), seed, following Salt-Tolerance Identification is carried out respectively：By each strain plant Seed is sowed on the MS culture medium flat plates of the NaCl containing 300mM simultaneously, and being placed in 22 illumination boxs, (illumination/8 are small within 16 hours When it is dark) in, after sprouting 6 days, count germination rate.Compared with wild type, transgenosis overexpresses the transgenic line of IpASR genes The germination rate of seed is above the seed of wildtype Arabidopsis thaliana control, shows that transgenosis overexpression IpASR arabidopsis seed is sprouted Hair process can be improved to Salt Stress Tolerance.

Equally, the T3 of three transfer-gen plant homozygous lines (OX1, OX2, OX3) is randomly selected for seedling (each strain 20 Strain), with wildtype Arabidopsis thaliana seedling, (20 plants) are control, plant (22 DEG C, 16 hours illumination/8 in Nutrition Soil and in greenhouse Hour is dark) normally cultivate 20 days, Osmotic treatment (not watering) is carried out afterwards 20 days, 7 days statistics are survived after subsequent rehydration is handled Rate.Compared with wild type, the survival rate after the transgenic line rehydration of transgenosis overexpression IpASR genes is significantly higher than wild type Control, showing transgenosis overexpression IpASR Arabidopsis plant has stronger drought tolerance.

Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

SEQUENCE LISTING

<110>South China Botanical Garden Chinese Academy of Sciences

<120>Thick boisiana IpASR genes and its encoding proteins and application

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 215

<212> PRT

<213>Thick boisiana IpASR protein

<400> 1

Met Ser Glu Met Lys His His His His Phe Gly His His Lys Asp Asn

1 5 10 15

Glu Glu Glu Arg Gln Ser Ser Cys Gly Glu Asn Thr Tyr Gly Thr Asp

20 25 30

Glu Lys Pro Tyr Gly Gln Thr Gly Tyr Gly Glu Glu Ser Tyr Glu Arg

35 40 45

Lys Asn Thr Tyr Gly Asp Asp Ser Tyr Glu Arg Lys Asn Thr Tyr Gly

50 55 60

Asp Asp Ser Ser Cys Glu Arg Lys Asn Thr Tyr Gly Asp Asp Ser Ser

65 70 75 80

Tyr Glu Arg Lys Asn Thr Tyr Gly Asp Asp Ser Tyr Tyr Glu Arg Lys

85 90 95

Asn Thr Tyr Gly Asp Asp Ser Tyr Gly Gln Ile Asp Lys Tyr Gly Ser

100 105 110

Glu Gly Val Thr Gly Gly Ile Glu Pro Glu Gly Lys Thr His Glu Asp

115 120 125

Tyr Glu Lys Glu Lys Lys His His Lys His Leu Glu Gln Leu Gly Gly

130 135 140

Leu Gly Thr Val Ala Ala Gly Ala Tyr Ala Leu Tyr Glu Lys His Glu

145 150 155 160

Ala Lys Lys Asp Pro Glu Asn Ala His Lys His Lys Ile Ala Glu Glu

165 170 175

Val Ala Ala Val Ala Ala Val Gly Ser Gly Ala Phe Ala Phe His Glu

180 185 190

His His Glu Lys Lys Glu Ala Lys Glu Glu Glu Glu Glu Ala Glu Gly

195 200 205

Lys Lys Lys His His Phe Phe

210 215

<210> 2

<211> 648

<212> DNA

<213>Thick boisiana IpASRcDNA reading frames

<400> 2

atgtctgaga tgaaacacca ccaccacttt ggtcaccaca aagacaatga ggaagagagg 60

cagtcttcct gtggtgaaaa cacttatggc actgatgaga aaccttatgg ccaaacaggc 120

tatggggaag agtcttatga gaggaaaaac acctatggtg atgactctta tgagaggaaa 180

aacacctatg gagatgactc ttcttgtgag aggaaaaaca cctatggaga tgactcttct 240

tatgagagga aaaacactta tggagatgac tcttattatg agaggaaaaa cacttatgga 300

gatgactctt acggccaaat cgacaagtat gggagtgaag gcgtgaccgg cggcatagaa 360

ccggaaggga aaactcatga ggattacgaa aaggagaaaa aacaccacaa gcatctcgag 420

cagctcggcg ggcttggcac cgtcgccgcc ggtgcctatg ccttgtacga gaagcacgag 480

gcaaagaaag acccagagaa tgcgcacaag cataagatag cagaagaggt ggcggcagtg 540

gctgccgttg gatcaggtgc atttgcattc catgagcatc atgagaagaa ggaagctaag 600

gaggaagaag aagaggctga gggaaagaag aagcatcact tcttttaa 648

<210> 3

<211> 36

<212> DNA

<213>Artificial sequence

<400> 3

ggggcccctg ggatccatgt ctgagatgaa acacca 36

<210> 4

<211> 36

<212> DNA

<213>Artificial sequence

<400> 4

ggaattccgg ggatccttaa aagaagtgat gcttct 36

<210> 5

<211> 36

<212> DNA

<213>Artificial sequence

<400> 5

ggactctaga ggatccatgt ctgagatgaa acacca 36

<210> 6

<211> 34

<212> DNA

<213>Artificial sequence

<400> 6

gtcgacccgg ggatcttaaa agaagtgatg cttc 34

Claims

1. thick boisiana abscisic acid/stress/Maturation induction protein I pASR of the amino acid sequence as shown in SEQ ID NO.1 and/or Application of the IpASR genes in tolerance of the plant to high salt/arid is improved, the cDNA reading frames of the IpASR genes are such as SEQ Nucleotide sequence shown in ID NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or be coded amino acid The nucleotide sequence of thick boisiana abscisic acid/stress/Maturation induction albumen of the sequence as shown in SEQ ID NO.1.

2. thick boisiana abscisic acid/stress/Maturation induction protein I pASR of the amino acid sequence as shown in SEQ ID NO.1 and/or IpASR genes improve crops to high salt/drought tolerance, and/or regulation and control crops to oxygen in crops transgenic breeding Change the application of stress tolerance, the cDNA reading frames of the thick boisiana abscisic acid/stress/Maturation induction GFP IpASR is such as Nucleotide sequence shown in SEQ ID NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or be coding ammonia The nucleotide sequence of thick boisiana abscisic acid/stress/Maturation induction albumen of the base acid sequence as shown in SEQ ID NO.1.

3. application according to claim 1 or 2, it is characterized in that, the crops are arabidopsis.

4. the saccharomyces cerevisiae recombinant expression carrier inserted with IpASR genes, it is characterized in that, the cDNA of the IpASR genes is such as Nucleotide sequence shown in SEQ ID NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or it is encoded to ammonia The nucleotide sequence of thick boisiana IpASR albumen of the base acid sequence as shown in SEQ ID NO.1.

5. the saccharomyces cerevisiae recombinant expression carrier described in claim 4 is improving engineered strain saccharomyces cerevisiae to high salt and the oxidation side of body Application in urgent tolerance.

6. the coli expression carrier inserted with IpASR genes, it is characterized in that, the cDNA reading frames of the IpASR genes are Nucleotide sequence as shown in SEQ ID NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or be coding The nucleotide sequence of thick boisiana IpASR albumen of the amino acid sequence as shown in SEQ ID NO.1.

7. claim 6 Recombinant protein expression carrier is improving engineering bacteria Escherichia coli to high salt and drought stress tolerance In application.

8. a kind of improve arabidopsis thaliana salt-tolerance and/or the biological agent of drought resistance, it is characterized in that, its active ingredient will from right Coli expression carrier described in saccharomyces cerevisiae recombinant expression carrier described in 4 or claim 6, or its active ingredient is asked to contain tune The biological products of thick boisiana IpASR gene expressions are controlled, the cDNA reading frames of the thick boisiana IpASR genes are such as SEQ ID NO.2 institutes The nucleotide sequence shown, or be the nucleotide sequence with SEQ ID NO.2 complementary pairings, or the IpMT genes are coded sequence Amino acid sequence such as SEQ ID NO.1 nucleotide sequence.

9. a kind of method for the salt tolerant and/or drought resistance for improving arabidopsis, it is characterized in that, comprise the following steps：

(1) thick boisiana abscisic acid/stress/Maturation induction GFP IpASR cDNA total lengths are obtained, its cDNA reading frame sequence is such as Shown in SEQ ID NO.2；

(2) by the Yeast expression carrier pYES-DEST52 recombinant plasmid transformed wild-type yeast bacterium of the cDNA total lengths containing IpASR Strain W303, tests the salt tolerance of transgenic yeast on salt content 5%-8.8% culture medium respectively；

(3) with the Yeast expression carrier pYES-DEST52 weights containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Group plasmid is template, expands the cDNA total length reading frame sequences of IpASR genes respectively with primer, and large intestine bar is inserted into for obtaining Mycoprotein expression vector pGEX 6p-1 cDNA reading frame fragments；

And reclaimed obtained target DNA fragment, it is connected on e. coli protein expression vector pGEX 6p-1, is formed Recombinant vector IpASR-pGEX 6p-1；

(4) recombinant vector IpASR-pGEX 6p-1 conversions are entered in e. coli bl21 competence bacterial strain, induction Overexpression of the IpASR genes in Escherichia coli；

(5) with the Yeast expression carrier pYES-DEST52 weights containing thick boisiana abscisic acid/stress/Maturation induction GFP cDNA Group plasmid is template, and the cDNA total length reading frame sequences of IpASR genes are expanded with primer, and being inserted into arabidopsis for acquisition turns base Because of overexpression vector pMD1 cDNA reading frame fragments, base is turned for building thick boisiana abscisic acid/stress/Maturation induction GFP Because of overexpression recombinant vector IpASR-pMD1；

Above-mentioned thick boisiana abscisic acid/stress/Maturation induction protein transgene overexpression recombinant vector IpASR-pMD1 conversions are entered In Agrobacterium GV3101 competence bacterial strains, by arabidopsis floral infestation method arabidopsis thaliana transformation plant, obtained by Kan resistance screenings Obtain arabidopsis progeny transgenic offspring.

10. method according to claim 9, it is characterized in that, the primer in step (3) is SEQ ID NO.3 and SEQ ID Primer in NO.4, and/or step (5) is SEQ ID NO.5 and SEQ ID NO.6.