CN101338315B - Gene for enhancing draught-resistance of plant and its uses - Google Patents

Abstract

The invention relates to a gene for improving the stress resistance of a plant the nucleotide sequence of which is expressed in the SEQ ID NO: 1. The nucleotide sequence expressed by the SEQ ID NO:1 is inserted into a vector to obtain the recombinant plasmid of the invention; the vector can select a known eukaryotic expression in the field. The recombinant plasmid is converted into tobaccos to obtain a transgenic plant. Test shows that based on an MS culture medium additionally provided with NaCl, mannitol and ABA, the transgenic plant grows better than a wild plant. After drying the transgenic plant and the wild plant for 18 days, the transgenic plant grows better than the wild plant. The detecting data for the content of malondialdehyde shows that the damage degree of the transgenic plant under stress conditions is lower than that of the wild plant. Therefore, the gene for improving the stress resistance of a plant can be applied in the fields of anti-drought of the plant and improving the salt improvement.

Description

A kind of gene and application thereof that improves stress resistance of plant

Technical field

The invention belongs to plant genetic engineering field, particularly a kind of gene that improves stress resistance of plant, recombinant plasmid and the improvement plant to the application aspect the tolerance of arid and salt

Background technology

Environment-stress (arid, high salt, low temperature and high temperature etc.) has very big negative impact for growth and development of plant.It is long that traditional breeding way is cultivated the new plant stress-resistance kind cycle, and it is slow to take effect.Cultivate plants cycle of degeneration-resistant new variety of transgenic method is shorter, does not influence existing good proterties simultaneously again, therefore becomes the important method of adversity resistant plant breed improvement gradually.

By the transgene improvement stress resistance of plant two class methods are arranged at present: (1) overexpression transcription factor (Jaglo-OttosenK.R., Gilmour S.J., Zarka D.J., Schabenberger O., Tomashow M.F. (1998) Arabidopsis CBF1verexpression induces COR genes and enhances freezing tolerance.Science.280:104-106.Kasuga M, Liu Q., Miura S., Yamaguchi-Shinozaki K., Shinozaki K. (1999) Improving plantdrought, salt and freezing by gene transfer of a single stress--inducible transcription factor.Nature Biotech.17:287-291.Hsieh T.H, Lee J.T, Charng Y.Y, Chan M.T. (2002) Tomatoplants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficitstress.Plant Physiol.130:618-626.).(2) overexpression single structure gene (Kumar S, DhingraA, Daniell H. (2004) Plastid-expressed betaine aldehyde dehydrogenase gene incarrot cultured cells, roots, and leaves confers enhanced salt tolerance.PlantPhysiology 136:2843-2854.Zhu J-K. (2001) Plant salt tolerance.Trends in PlantScience 6:66-71.).Therefore, the gene of developing the different raising stress resistance of plant in more source has great significance.

Summary of the invention

The objective of the invention is to be to provide gene, the recombinant plasmid of a new raising stress resistance of plant, the overexpression of described gene can obviously strengthen the tolerance of transgenic plant to arid and salt.

Technical scheme of the present invention is as follows:

The gene of raising stress resistance of plant of the present invention, called after ZmALDH22A1, its nucleotide sequence are shown in SEQ ID NO:1 in the sequence table, and expressed amino acid sequence of polypeptide is shown in SEQ ID NO:2 in the sequence table.The clone of this gene: the derivation aminoacid sequence with Arabidopis thaliana AtALDH22A1 carries out homologous sequence comparison search with the tBlastn program in the Maize Gene of TIGR website (www.tigr.org) Index database, obtain an EST splicing sequence (TC322649) that contains the high homology (consistence of derivation aminoacid sequence is 77%) of complete open reading frame (ORF), sequences Design primer in view of the above, utilize reverse transcription PCR (RT-PCR) technology from corn, to be separated to the cDNA of ZmALDH22A1, shown in SEQ ID NO:1 in the sequence table.

Recombinant plasmid of the present invention is the described nucleotide sequence of SEQ ID NO:1 to be inserted in the carrier for expression of eukaryon obtain, and above-mentioned carrier can be selected carrier for expression of eukaryon known in the art (as pHB or PBI121) for use.

With above-mentioned recombinant plasmid transformed tobacco, obtain transfer-gen plant.Test shows: be added with outside on the MS substratum of NaCl, N.F,USP MANNITOL, dormin (ABA), transfer-gen plant is better than wild-type plant strain growth, especially the highest transgenic lines of gene expression amount of the present invention, to compare phenotypic difference more obvious with the wild-type plant.With transfer-gen plant and wild-type plant after arid is handled 18 days, transfer-gen plant is better than wild-type plant strain growth, (transfer-gen plant impaired degree under drought condition is lower than the wild-type plant to mda for malonaldehyde, MDA) Determination on content data presentation.Therefore, the gene of described raising stress resistance of plant of the present invention can be drought-resistant plant, use in the high salt tolerance improvement.

The present invention has following beneficial effect:

1, the present invention plant drought-resistant for cultivating, high salt tolerance provides a kind of new recombinant plasmid, helps the Sustainable development and the increases in grain production of agricultural.

2, the clone of gene of the present invention and plant transgene are ordinary method, and material requested is easy to obtain.

Description of drawings

Figure 1A is the expression pattern figure of Real-time pcr analysis ZmALDH22A1 gene in milpa different tissues, organ; Figure 1B is Real-time pcr analysis (arid, high salt and dormin ABA) ZmALDH22A1 gene abduction delivering mode chart in maize seedling root under different stress conditions.

Fig. 2 is the RT-PCR analysis chart of ZmALDH22A1 transgene tobacco positive-selecting plant.

Fig. 3 is that wild-type tobacco and ZmALDH22A1 transgene tobacco seed germinate and one-tenth seedling experimental result picture at the MS flat board that contains Totomycin.

Fig. 4 is the resistance comparative result figure of ZmALDH22A1 transgenic tobacco plant and wild-type adjoining tree.

Fig. 5 is the drought resistance comparative result figure of ZmALDH22A1 transgenic tobacco plant and wild-type adjoining tree, and among the figure, A is the normal growth condition, and B is a drought condition.

Embodiment

Below in conjunction with embodiment, the invention will be further described.Among the following embodiment, all unreceipted concrete experiment conditions, be according to normal condition well known to those skilled in the art Sambrook for example, the molecular cloning of Russell; Condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1: the clone who improves the gene of stress resistance of plant

1, reagent

Restriction enzyme, Taq archaeal dna polymerase, T4 dna ligase, PrimeStar warm start high-fidelity DNA polymerase, pMD18-T cloning vector etc. are available from Dalian Bao Bio-Engineering Company; Trizol reagent is Time Technology company limited available from sky, Beijing; The reverse transcription test kit is available from Japanese ToYoBo company; Plasmid extracts and DNA reclaims test kit available from company of the excellent brilliant biotechnology in Anhui company limited; The PCR primer is synthetic by the handsome biotech firm in Shanghai; All the other reagent are import packing or homemade analytical pure product.

2, coli strain and vegetable material

The escherichia coli cloning bacterial strain is E.coli JM109, available from Clontech company.Corn material is self-mating system 616-1, is provided by the academy of agricultural sciences, Sichuan Province.

3, substratum and solution

LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L.Transfer pH to 7.0, autoclaving with NaOH.

SOB substratum: Tryptones 20g/L, yeast powder 5g/L, NaCl 0.58g/L, KCl 0.19g/L, 100 * Mg ²⁺10mL.Transfer pH to 7.0, autoclaving with NaOH.

SOC substratum: SOB+20mM glucose.

TB buffer (face and use preceding configuration): 1M KCl 4mL, 0.45M MnCl ₂2.4mL, 0.50M CaCl ₂0.6mL, 0.50M K-MES 0.5mL, ddH ₂O 12.5mL (cumulative volume 20mL).

100 * Mg ²⁺Solution: 20.33g MgCl ₂.6H ₂O and 24.65g MgSO ₄.7H ₂The O constant volume is in 100mL H ₂O, autoclaving.

20% glucose solution: 20g glucose constant volume is in 100mL H ₂O, filtration sterilization.

1M KCl solution: 7.45g KCl constant volume is in 100mL H ₂O, autoclaving.

0.45M MnCl ₂Solution: 8.9g MnCl ₂.4H ₂The O constant volume is in 100mL H ₂O, autoclaving.

0.50M CaCl ₂Solution: 7.35g CaCl ₂.2H ₂The O constant volume is in 100mL H ₂O, autoclaving.

0.50M K-MES solution: 9.76g MES constant volume is in 100mL H ₂O transfers pH to 6.3 with KOH, and filtration sterilization is distributed into the every pipe of 0.5mL ,-20 ℃ of storages.

DMSO: the fresh DMSO of packing 200 μ l ,-20 ℃ of storages.

4, experimental technique

4.1 the plasmid trace extracts

1) the E.coli JM109 that will have a cloning vector pMD18-T plasmid is inoculated in and is equipped with in the 5ml LB nutrient solution test tube of (containing an amount of microbiotic), and 37 ℃ Shake beds are cultivated 12～16hr, with the amplification plasmid.

2) get the bacterium liquid of 1.5～5ml, under room temperature 10, the centrifugal 1min of 000xg.The evacuation substratum adds the mixed liquid of ZL-I/RNaseA of 250 μ l in the precipitation, and vortex oscillation suspends cell fully again.

3) add 250 μ l ZL-II in resuspended mixed liquid, 4～6 mixed solution of test tube of overturning gently are to obtain a clarifying lysate.

4) in above-mentioned mixed liquid, add 350ul ZL-III, and leniently turn upside down centrifuge tube for several times, until forming white flocks.Under room temperature 10, the centrifugal 10min of 000xg.

5) getting a clean Mu-Pu plasmid trace separator column is contained on the 2ml collection test tube (being equipped with).Careful sucking-off supernatant, and it is gone in pillar Inner, guaranteeing to go in the supernatant of post Inner does not have the cell contamination precipitation.Under room temperature 10, the centrifugal 1min of 000xg makes lysate flow through pillar fully.

6) discard the centrifugal liquid that throws away, the ZL damping fluid that adds 500 μ l is to pillar, and under the room temperature 10, the centrifugal 1min washing of 000xg pillar guarantees to remove remaining protein to obtain the required high quality DNA of back operation.

7) discard collection liquid, add the DNA lavation buffer solution washing pillar of 720 μ l with the dehydrated alcohol dilution, room temperature 10, the centrifugal 10min of 000xg discards washings.

8) the optional step of doing: repeating step 7, the DNA lavation buffer solution with 720 μ l washs pillar once again.

9) under the room temperature 10, the centrifugal void column 1min of 000xg is to dry pillar matrix.

10) pillar is placed on the clean little centrifuge tube of 1.5ml, directly add 50～100 μ l sterilization deionized water or TE damping fluid liquid on pillar matrix (added amount depends on expection end product concentration), 10,000xg is centrifugal, and 1min goes out DNA with wash-out.

4.2DNA recovery scheme

1) handles sepharose-EB electrophoresis miscellany with difference DNA isolation fragment.The agarose of any kind or grade can use.

2) when the spacing of band between line reached enough amount, the dna fragmentation required careful on ultraviolet lamp scaled off.So can guarantee gel that handle contains DNA taking out as much as possible

3) by the gel thin slice being contained in the method that claims its weight in the little centrifuge tube of 1.5ml, determine its volume approx.Suppose that its density is 1g/ml, so the volume of gel just can obtain by the following method: the weight of gel thin slice be 0.2g then its volume be 0.2ml.Adding volume is the NJ damping fluid of 3～4 times of gel volumes, and miscellany is placed 55～65 ℃ of water-bath temperature bath 7min, or melts fully to gel.

4) the DNA-agarose solution of 750 μ l is added to a Mu-Pu DNA and reclaims on the purification column, and pillar is contained in the 2ml collection tube of a dried Net, in 10, the centrifugal 1min of 000xg discards liquid under the room temperature.

5) step is done in choosing: wash pillar with 300 μ l NJ damping fluids, and 10, centrifugal 1min under the 000x g.

6) the DNA lavation buffer solution with the dilution of 750 μ l dehydrated alcohols washs pillar.Under the room temperature 10, the centrifugal 1min of 000xg.

7) discard effluent liquid, repeated for the 6th step once.

8) discard liquid, void column 10, the centrifugal 1min of 000xg is to dry the liquid of base for post matter remnants.

9) pillar is contained on the 1.5ml centrifuge tube of a dried Net, add 30～50 μ l 10, the sterilization deionized water of 000xg centrifugal (the end product concentration that specifically depends on expection) (the perhaps TE damping fluid of PH8.0) on base for post matter, 10, the centrifugal 1min of 000xg is with eluted dna.

4.3 the total RNA of corn extracts

1) liquid nitrogen grinds the maize leaf tissue rapidly, and press 50-100mg tissue/ml Trizol and add Trizol, concuss, room temperature is placed 5min.

2) 12, the centrifugal 5min of 000rpm.

3) get supernatant, press 200ul chloroform/ml Trizol and add chloroform, concuss 15s, room temperature is placed 3min.

4) 4 ℃ 12, the centrifugal 15min of 000g.

5) get supernatant, press 0.5ml Virahol/ml Trizol and add the Virahol mixing, room temperature is placed 10min.

6) 4 ℃ 12, the centrifugal 10min of 000g abandons supernatant, and RNA is sunken to the pipe end.

7) add 75% ethanol by 1ml 75% ethanol/ml Trizol, gentle vibration centrifuge tube, precipitation suspends.

8) 4 ℃ 8, the centrifugal 5min of 000g abandons supernatant as far as possible.

9) (the RNA sample is too not dry, otherwise is difficult to dissolving for drying at room temperature 5-10min.)。

10) use 50ul DEPC-H ₂O dissolving RNA sample, 55-60 ℃, 5-10min.

4.4RT-PCR

1)RT

In one 200 μ l EP pipe, add following component on ice:

5×RT?Buffer 4μl

DNTP Mixture (each 10mM) 2 μ l

RNase?Inhibitor(10U/μl) 1μl

Oligo(dT) ₂₀(10pmol/μl) 1μl

Total?RNA Xμl

RNase-Free?H ₂O (11-X)μl

ReverTra?Ace 1μl

Carry out reverse transcription by following program: 42 ℃ of 20min (reaction); 99 ℃ of 5min (enzyme denaturation); 4 ℃ of 5min (preservation).

2)PCR

In one 200 μ l EP pipes, add following component on ice:

5×PrimeStar?Buffer 10μl

DNTP Mixture (each 2.5mM) 4 μ l

RT product 1 μ l

TC306087-f3 1μl

TC306087-r3 1μl

ddH ₂O 32.5μl

PrimeStar 0.5μl

Increase by following program: 98 ℃ of 3min (pre-sex change); 98 ℃ of 10s (sex change), 55 ℃ of 15s (renaturation), 72 ℃ of 2.5min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

With above-mentioned PCR product is template, carries out second with nested primer TC306087-f4 and TC306087-r4 and takes turns high-fidelity PCR, extends time 2min, and other condition is the same.

Primer sequence is as follows:

TC306087-f3：5’-CGGCTGGATTCCTCACTTTAGT-3’；

TC306087-r3：5’-GGCAGCACATTGACAGTTCCTA-3’(2180bp)；

TC306087-f4：5’-AGAGCAGGTGTCGGTGTCATG-3’；

TC306087-r4：5’-TGGGGAATGGGGAGGTTTAC-3’(1878bp)。

4.5 high-fidelity PCR product tailing and be connected with cloning vector pMD18-T

Take turns second and to add 1 μ lTaq archaeal dna polymerase in the high-fidelity PCR product, 72 ℃ of reaction 15min get final product.If the PCR product is impure, then must reclaim the purification of target fragment earlier, add an amount of PCR Buffer, dNTPs and Taq archaeal dna polymerase again and carry out tailing.

PCR product behind the tailing is connected than 3/1 by the mole molecule number with cloning vector pMD18-T, and reaction system is as follows:

10×ligase?Buffer 1μl

pMD?18-T(50μg/μl) 1μl

The PCR product of tailing (～150 μ g/ μ l) 1 μ l

Ligase(350U/μl) 1μl

ddH ₂O 6μl

16 ℃ of connections are spent the night.

4.6 intestinal bacteria transform

1) preparation of competent cell

A) inoculate the single bacterium colony of intestinal bacteria in 2mL SOB nutrient solution, 37 ℃ of incubated overnight;

B) transfer the 0.5mL overnight culture to 50mL SOB nutrient solution, 18 ℃ of concuss 18～24h to A ₆₀₀≈ 0.55;

C) nutrient solution is changed in the 50mL centrifuge tube over to ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm;

D) remove supernatant, add the TB damping fluid suspension cell (noting: rotate gently) of 16mL precooling, ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm not with vibrator or pressure-vaccum mixing;

E) remove supernatant, add the TB damping fluid suspension cell of 4mL precooling, add the DMSO of 280 μ l, soft mixing, ice bath 10min;

F) be sub-packed in precooling and get in the 1.5mL EP pipe liquid nitrogen cryopreservation.

2) transform

A) taking out a pipe competent cell ice bath from liquid nitrogen thaws;

B) 10 μ l are connected product and competent cell mixing gently, ice bath 30min;

C) 42 ℃ of thermal shocking 90s, ice bath 1-2min immediately;

D) add the SOC of 0.8mL, mixing, 37 ℃ of gentle shaking table 1h.

E) room temperature 13, and the centrifugal 1min of 000rpm outwells a part of supernatant liquor, stays the supernatant liquor of about 200 μ l, with supernatant liquor and cell mixing, are coated with LB+amp (50 μ g/ml) flat board, 37 ℃ of overnight incubation with the rifle head.

4.7 the quick cracking process of cell is identified recombinant vectors

1) the single transformant of picking is inoculated in 500 μ l and contains in the corresponding antibiotic LB nutrient solution, and 37 ℃ of shaking culture are to A ₆₀₀Be 0.6～0.8.

2) get 200 μ l bacterium liquid to 0.5ml EP pipe, 13, the centrifugal 1min of 000rpm removes supernatant, stays about 20 μ l supernatants.

3) add 20 μ l 2 * quick lysate [0.2mol/L NaOH 50mL+SDS 0.5g, sucrose 27.2g adds distilled water to 200ml], thermal agitation.

4) the centrifugal 15min of 13000rpm.

5) get the direct electrophoresis of 5 μ l supernatants.With contrast ratio, what electrophoresis band lagged behind promptly may be recombinant vectors.

4.8 bacterium colony PCR identifies recombinant vectors

Be bacterium colony PCR again to determine that inserting fragment is target fragment through the recombinant vectors that quick cracking process is identified, reaction system is as follows:

10×PCR?Buffer 2μl

Mg ²⁺(1.5mM) 1.2μl

DNTP Mixture (each 2.5mM) 1.6 μ l

Bacterium liquid 1 μ l

TC306087-f4 0.4μl

TC306087-r4 0.4μl

ddH ₂O 12.4μl

Taq archaeal dna polymerase 1 μ l

Reaction conditions: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 55 ℃ of 30s (renaturation), 72 ℃ of 2min (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

The recombinant vectors that bacterium colony PCR is determined checks order, and sequencing result is the nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.

Embodiment 2:Real-time PCR (real-time quantitative PCR) analyzes the expression of gene pattern that improves stress resistance of plant

1, experiment material, reagent and instrument

Conventional reagent is identical with embodiment 1 with corn material.The Real-timePCR test kit is the SYBR Premix Ex Taq of Dalian Bao Bio-Engineering Company ^TM(Perfect RealTime), quantitative real time PCR Instrument are hundred happy companies

Quantitative real time PCR Instrument (Bio-Rad Laboratories, USA).

2 methods

2.1 the processing of corn material

Corn seed 37 ℃ of soaked overnight of tap water.Seed was placed in the culture dish that is covered with moistening filter paper in second day, issues seedling, add water every day and keep filter paper moistening in 25 ℃, 16h illumination/8h dark condition.After 10 days, be that maize seedling is when just having grown the 3rd leaf, maize seedling is handled with different stress conditions: maize seedling is soaked 4h respectively in 250mM NaCl and 100uM ABA solution, processed then places maize seedling 4h on the dried filter paper, and control group is about to maize seedling and soaks 4h in water.Collect root, stem or the leaf of at least 5 strain maize seedlings under every kind of treatment condition after the processing respectively, quick-frozen and put into-80 ℃ of cryogenic refrigerators and preserve in liquid nitrogen immediately is up to use.

2.2RNA extraction, electrophoresis and reverse transcription

Root, stem, spire, climax leaves, pollen, filigree and the rataria of getting the maize seedling of various conditions processing respectively extract total RNA, and total RNA of extraction handles with DNaseI.According to electrophoresis result, get about 1 μ g RNA and be used for reverse transcription, obtain cDNA (concrete grammar is seen embodiment 1).

2.3 Real-time PCR primer design

The primer of ZmALDH22A1 gene is as follows, expection amplified fragments 145bp.

087RT-f：5’-CAGAAGCGTGCCATCAGAATA-3’；

087RT-r：5’-AGCCTTCTACGCCAGCAAAT-3’；

Internal control gene is corn actin 1 gene (GenBank accession no.J01238), and its primer is as follows, expection amplified fragments 143bp.

Mac1RT-f：5’-TTCCTAGCAGCATGAAGGTTAAA-3’；

Mac1RT-r：5’-CGGACCAGTTTCGTCATACTCT-3’.

2.4Real-time?PCR

Following all operations must be worn the disposable plastic gloves, the detection that influences fluorescence to avoid polluting EP pipe outer wall.CDNA and primer add after preferably all diluting again, to reduce sampling error as far as possible.Each sample and gene redundancy 3 times.

In the EP of one 200 μ l pipe, add following component (cumulative volume 25 μ l) on ice:

2×SYBR?Premix?Ex?Taq ^TM 12.5μl

The cDNA 6.5 μ L of dilution in 1: 15

087RT-f/r (10 μ M) the 6 μ L of dilution in 1: 12

By following program (the Real-timePCR test kit SYBRPremix Ex Taq that adopts Dalian Bao Bio-Engineering Company to provide that increases ^TMReaction conditions): 95 ℃ of 40s; 95 ℃ of 5s, 60 ℃ of 20s (40 circulations); 95 ℃ of 60s; 55 ℃ of 60s; 55 ℃ of 10s (each circulation increases by 0.5 ℃, 80 circulations).

2.5Real-time PCR data analysis

Real-time PCR data analyze (2 with relative expression software tool (REST) software ^{-Δ Δ Ct}Method).Concrete operations with REST software analysis Real-time PCR data:

1) opens REST software;

2) import target gene (ZmALDH22A1) and internal control gene (actin 1) name in the pink zone of the introduction of handbook page or leaf;

3) be used to do the Ct Value Data of typical curve (PCR efficient) to calculate their PCR efficient separately at PCR efficiency page or leaf input internal control gene and target gene.As adopt 2 ^{-Δ Δ Ct}Method, the PCR efficient of gene is approximately 1, and this step can omit;

4) at the Ct Value Data of CP input+randomization test page or leaf by prompting input internal control gene and target gene, click " run Pair Wise Fixed reallocation Randomisation Test " key (redness), the expression ratio in computing sample and the control sample behind the target gene normalizing, P value and standard error;

5) access expression rate and P Value Data at CP input+randomization test page or leaf, access the standard error data at CP variation page or leaf;

6) in excel software, make histogram (seeing the excel software manual for details) and promptly finish data analysis.

3, result

Real-time PCR data analysis result shows that ZmALDH22A1 all has expression in root, stem, spire, climax leaves, pollen, filigree and rataria, see Figure 1A.In root, the expression amount of coercing ZmALDH22A1 in the treatment samples is untreated relatively, and the expression amount of ZmALDH22A1 all has significance to raise in the sample.Wherein, the ZmALDH22A1 expression amount is be untreated 1.59 times (p＜0.01) of ZmALDH22A1 expression amount in the sample in the 250mM NaCl treatment samples; The ZmALDH22A1 expression amount is be untreated 1.32 times (p＜0.05) of ZmALDH22A1 expression amount in the sample in the processed sample; The ZmALDH22A1 expression amount is to be untreated 2.86 times (p＜0.01) of ZmALDH22A1 expression amount in the sample in the 100 μ M ABA treatment samples, sees Figure 1B.

Embodiment 3: expression and the resistance test of gene in tobacco that improves stress resistance of plant

1, material

1.1 reagent and material

Conventional reagent is identical with embodiment 1.

Being used for genetically modified agrobacterium strains is EHA105, available from Clontech company; Transforming with tobacco bred is ' the big gold dollar of safflower ', is provided by the biological study of Chengdu Branch of the Chinese Academy of Sciences; Plant expression vector is pHB, and institute provides by the Shanghai plant physiology.

1.2 substratum and solution

The YEB substratum: yeast extract 1g/L, extractum carnis 5g/L, peptone 5g/L, sucrose 5g/L,

MgSO ₄.7H ₂O?0.5g/L。Transfer pH to 7.0, autoclaving with NaOH.YEP substratum: yeast extract 10g/L, peptone 10g/L, NaCl 5g/L.Transfer pH to 7.0, autoclaving with NaOH.

The conventional tissue culture medium (TCM) of using:

2, method

2.1 the structure of ZmALDH22A1 gene plant overexpression recombinant vectors

The ZmALDH22A1 gene primer that is used for plant overexpression vector construction is as follows:

087-f5：CGG AAGCTTAGAGCAGGTGTCGGTGTCATG(HindIII)

087-r5：TTT GGATCCTGGGGAATGGGGAGGTTTAC(BamHI)

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 58 ℃ of 30s (renaturation), 72 ℃ of 40s (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

High-fidelity PCR product is used HindIII and BamHI double digestion after electrophoresis reclaims, be connected (connection site: HindIII and BamHI) with the pHB carrier of cutting with same restrictions enzyme enzyme then, obtain the overexpression recombinant vectors that contains the ZmALDH22A1 gene, called after pHB-087.

2.2 the preparation of Agrobacterium competent cell

1) the single bacterium colony of picking Agrobacterium is in the YEB of 2ml liquid nutrient medium (containing Rif 50 μ g/ml), and 28 ℃ of shaking culture are spent the night;

2) get incubated overnight liquid 500 μ l and transfer in 50ml YEB (containing Rif50 μ g/ml) liquid nutrient medium, 28 ℃ of shaking culture are to OD ₆₀₀=0.5;

3) 4 ℃ of centrifugal 5min of 5000rpm collect thalline, add the NaCl solution suspension thalline of 10ml 0.15M, ice bath 10min;

4) 4 ℃ of centrifugal 5min of 5000rpm collect thalline, with the 20mM CaCl of 1ml precooling ₂Solution suspension thalline, ice bath 10min;

5) cell for preparing uses immediately, or is distributed into 200 μ l/ pipe, quick-frozen 1min in the liquid nitrogen, and it is standby to put-70 ℃ of preservations.

2.3 the conversion of Agrobacterium

1) gets 200 μ l competent cells, thaw on ice;

2) add the pHB-087 recombinant vectors that 1 μ g builds, flick mixing, ice bath 30min;

3) quick-frozen 1min in the liquid nitrogen, 37 ℃ of water-bath 5min add 1ml YEB substratum then, 28 ℃ of shaking culture 4h at a slow speed;

4) culture is coated on the YEB flat board that contains 50 μ g/ml Kan and 50 μ g/ml Rif, cultivated about 48h for 28 ℃.

2.4 the evaluation of Agrobacterium positive colony

The single bacterium colony that grows on the picking flat board is inoculated in the YEB liquid nutrient medium that contains 50 μ g/ml Kan and 50 μ g/ml Rif, and 28 ℃ of shaking culture are spent the night, and is that template is carried out the pcr amplification evaluation with bacterium liquid.

2.5 agrobacterium-mediated transformation transformation of tobacco

2.5.1 the preparation of tobacco aseptic seedling

With the tobacco mature seed with 70% alcohol rinsing 1min; Be soaked among the chlorine bleach liquor of active chlorine content 2～4% vibration 15～20min then; With sterilized water washing 4～5 times, blot redundant moisture with sterilization filter paper, be inoculated on the MS minimum medium.25 ℃ of dark 5d that cultivate, illumination then (16h illumination/8h is dark) is cultured to and grows aseptic seedling.Aseptic seedling can and be preserved by tissue-culturing quick-propagation.

2.5.2 transform preparation with Agrobacterium bacterium liquid

Picking Agrobacterium positive colony is in YEB (or YEP) liquid nutrient medium (containing 50mg/L Rif and 50mg/L Kan), and 28 ℃, 180rpm are cultured to OD ₆₀₀=0.6～0.8.Get bacterium liquid, the ratio in 1～2% changes in the liquid nutrient medium of fresh antibiotic-free, adds the AS (can add and can not add) of 100～500 μ M, and 28 ℃, 180rpm are cultivated about 6h to OD ₆₀₀=0.2～0.5 promptly can be used for transforming.

2.5.3 transform and cultivate

Get the tobacco aseptic seedling, blade is cut into the fritter of about 0.5cm * 0.5cm size, in above-mentioned Agrobacterium bacterium liquid, soak 5min.

Take out tobacco leaf, on aseptic filter paper, blot; Change MS substratum (containing 2.0mg/L 6-BA and 0.5mg/LIAA) over to, cultivate 2～3d altogether.

Take out tobacco leaf from being total to substratum, on aseptic filter paper, inhale and remove unnecessary thalline, change MS substratum (containing 2.0mg/L 6-BA, 0.5mg/L IAA, 50mg/L hyg and 200mg/L Tim) then over to, 25 ℃ of illumination (16h illumination/8h is dark) screening differentiation culture.Screened for 2 generations, per 2 weeks of generation.

Change the tobacco young shoot of screening and culturing after 4 weeks over to 1/2MS substratum (containing 50mg/L hyg and 100mg/L Tim) and carry out root culture.Tobacco plant after taking root moves into the flowerpot cultivation.

2.6 the evaluation of transfer-gen plant

2.6.1 the extraction of tobacco gene group DNA

1) tobacco leaf is used liquid nitrogen grinding in mortar, every 0.1g vegetable material adds 2 * CTAB damping fluid that 600 μ L are preheated to 90 ℃.Mixing is placed 60min for 65 ℃ gently.

2) after thing to be mixed is chilled to room temperature, add equal-volume chloroform/primary isoamyl alcohol (24: 1), put upside down centrifuge tube mixing several times gently, the centrifugal 5min of room temperature 10000rpm.

3) get supernatant, add the Virahol of 0.7 times of volume, mixing, room temperature is placed 15min deposit D NA.

4) the centrifugal 10min of room temperature 13000rpm.70% washing with alcohol precipitation once.

5) dry air 5～10min, precipitation is dissolved in 50 μ l aqua sterilisas.

6) get 5 μ l genome dna electrophoresis and detect quality.

2.6.2 genomic dna PCR detects

In order to determine that the ZmALDH22A1 gene has been incorporated into the genome of positive-selecting tobacco plant, we are template with tobacco gene group DNA, and ZmALDH22A1 full length gene cDNA and hygromycin gene fragment have been carried out pcr amplification.The ZmALDH22A1 gene specific primer is 087-f5 and 087-r5, and the hygromycin gene special primer is:

Hyg-f：5′-TCGTTATGTTTATCGGCACTTTG-3′；

Hyg-r：5′-GCGTCTGCTGCTCCATACAAG-3′

Increase by following program: 94 ℃ of 5min (pre-sex change); 94 ℃ of 40s (sex change), 56 ℃ of 30s (renaturation), 72 ℃ of 40s (extension), described denaturation renaturation-30 circulations of extension; 72 ℃ of 5min (extending eventually).

2.6.3RT-PCR detect

For determining that further the ZmALDH22A1 gene has been incorporated into the genome and the expression of positive-selecting tobacco plant, we are template with tobacco leaf cDNA, and the ZmALDH22A1 gene has been carried out pcr amplification, and primer is 087RNAi-f1 and 087RNAi-r1.

2.6.4 hygromycin resistance experiment

Picking three strains detect tobacco transfer-gen plant (the called after ZmALDH22A1-Overexpression1,2,3 that determines through above-mentioned RT-PCR at random, abbreviate transfer- gen plant 1,2,3 as), its first-generation (T1) seed and wild-type tobacco seed (each about 200) surface sterilization are placed on the MS flat board that contains the 50mg/L Totomycin, and 25 ℃ of 16h illumination/8h secretly cultivate.Write down its percentage of germination and seedling rate.

2.7 transfer-gen plant ZmALDH22A1 genetic expression component analysis experiment

Extract the RNA of tobacco transfer-gen plant transfer-gen plant 1, transfer-gen plant 2 and transfer-gen plant 3 and wild-type tobacco adjoining tree blade, the synthetic cDNA of counter-rotating.With cDNA is template, (GenBankaccession no.AJ133422) is internal control gene with tobacco GAPDH gene, and with real-time RT-PCR method ZmALDH22A1 mRNA being carried out deciding ZmALDH22A1 gene real time RT-PCR primer is 087RT-f and 087RT-r (seeing embodiment 2);

Tobacco GAPDH gene real time RT-PCR primer is: (expection clip size 133bp)

NgapRT-f：5’-CTCAACATTGTTTCAAATGCTAGCT-3’

NgapRT-r：5’-CAGTTTTCTGTGTGGCTGTGATG-3’

2.8 transgenosis and the wild-type seedling resistance experiment on the MS substratum

The sterilization of the first-generation (T1) seed-coat of wild-type tobacco seed and transgene tobacco is placed on the MS flat board of the NaCl (0mM, 50mM, 100mM and 250mM) that contains different concns, N.F,USP MANNITOL (0mM, 150mM, 300mM and 500mM), dormin (ABA) (0 μ M, 0.5 μ M, 1 μ M and 2 μ M) (every ware is put 50 seeds), secretly cultivates in 25 ℃ of chambers of group training, 16h illumination/8h.Add up germination rate every day.Treat that dull and stereotyped going up when transgene tobacco and wild-type tobacco seedling have enough difference take a picture, measure the long and fresh weight (surveying the mean value of 10 strain seedling, the triplicate measurement) of root.

2.9 mda (malonaldehyde, MDA) Determination on content

1) get plant leaf 0.2g, liquid nitrogen grinds; Add 2ml 0.1%TCA (trichoroacetic acid(TCA)) solution, the concussion mixing places on ice.

2) homogenate is in 4 ℃ 13, and the centrifugal 10min of 000rpm, supernatant liquor are sample extracting solution.

3) get supernatant liquor 0.5ml (contrast adds 0.5ml 0.1%TCA solution), add the 20%TCA solution that 1ml contains 0.5% (w/v) TBA (thiobarbituricacid), behind the mixing in boiling water bath 30min.

4) ice bath cooling rapidly, 4 ℃ 13, the centrifugal 10min of 000rpm.Get supernatant liquor and survey A ₅₃₂And A ₆₀₀

5) calculate MDA content as follows:

MDA content (nmol/g)=155 ^-1* (A ₅₃₂-A ₆₀₀) * extension rate ÷ plant tissue fresh weight (g)

2.10 transgenosis and the experiment of wild-type plant soil drought resistance

The first-generation (T1) the seed-coat sterilization of transgene tobacco is placed on the MS flat board that contains the 50mg/L Totomycin, and the sterilization of wild-type tobacco seed-coat is placed on the MS flat board.25 ℃ of 16h illumination/8h secretly cultivate.After 4 weeks, get the close seedling replanting (vermiculite: nutrition soil: common soil=1: 1: 1), watered water one time in per two days, water the 1/8MS nutrient solution weekly a time to soil of size.After 4 weeks, a part of seedling stops to water, and another part normally waters to compare, and up to obvious difference, takes pictures, and surveys MDA content.

3, result

3.1 the evaluation of transfer-gen plant

3.1.1 transgene tobacco positive-selecting plant RT-PCR qualification result

The RT-PCR analysis is carried out in picking 9 strains at random in transfer-gen plant, and the result expands the product that the expection size in 6 strains, as shown in Figure 2.

3.1.2 transgenosis and wild type seeds be the hygromycin resistance experimental result on the MS flat board

Containing on the MS flat board of Totomycin, wild-type tobacco and transgene tobacco seed can both rudiments.But, but can not Cheng Miao after the wild-type tobacco seed germination; And the not only rudiment of transgene tobacco seed, and most Cheng Miao, the seedling rate of transfer-gen plant 1, transfer-gen plant 2 and transfer-gen plant 3 is respectively 77%, 80% and 78%, as shown in Figure 3.This has proved that again transfer-gen plant 1, transfer-gen plant 2 and transfer-gen plant 3 are the ZmALDH22A1 transgene product really.

3.2 the resistance of transgenosis and wild-type tobacco seedling

As can be seen from Figure 4, on the MS flat board that contains NaCl (100mM), N.F,USP MANNITOL (300mM), ABA (0.5 μ M), the seedling of three strain tobacco transgenic lines looks better than wild-type seedling, shows that the resistance of transgenic lines is better than wild-type.

On the MS of different concns NaCl flat board, the fresh weight of transgenosis and wild-type seedling, root length, MDA value see the following form:

As can be seen from the table:

1) fresh weight: after 50mM NaCl handled, wild-type was reduced to 68.8% of control group; Transfer-gen plant 1 is reduced to 68.9% of control group; Transfer-gen plant 2 is reduced to 77.4% of control group; Transfer-gen plant 3 is reduced to 74.8% of control group.After 100mM NaCl handled, wild-type was reduced to 36.8% of control group; Transfer-gen plant 1 is reduced to 38.4% of control group; Transfer-gen plant 2 is reduced to 40% of control group; Transfer-gen plant 3 is reduced to 39.9% of control group.After 250mM NaCl handled, wild-type was reduced to 3.3% of control group; Transfer-gen plant 1 is reduced to 14.3% of control group; Transfer-gen plant 2 is reduced to 31.4% of control group; Transfer-gen plant 3 is reduced to 31.5% of control group.This shows that under 50mM and 100mMNaCl treatment condition, the difference between the fresh weight of transgenosis and wild-type seedling is very little.But under 250mM NaCl treatment condition, the fresh weight of transgenosis seedling is apparently higher than the fresh weight of wild-type seedling, especially higher transgenic lines transfer-gen plant 2 and the transfer-gen plant 3 of ZmALDH22A1 expression amount.

2) root is long: after 50mMNaCl handled, wild-type was reduced to 92.1% of control group; Transfer-gen plant 1 is reduced to 91.3% of control group; Transfer-gen plant 2 is 96.0% of a control group; Transfer-gen plant 3 is reduced to 96.0% of control group.After 100mM NaCl handled, wild-type was reduced to 86.1% of control group; Transfer-gen plant 1 is reduced to 91.0% of control group; Transfer-gen plant 2 is reduced to 95.4% of control group; Transfer-gen plant 3 is reduced to 90.4% of control group.After 250mM NaCl handled, wild-type was reduced to 2.5% of control group; Transfer-gen plant 1 is reduced to 4.3% of control group; Transfer-gen plant 2 is reduced to 7.8% of control group; Transfer-gen plant 3 is reduced to 8.5% of control group.Can see that under lower concentration (50mM) NaCl treatment condition, the difference between the root of transgenosis and wild-type seedling is long is very little.But under 250mM NaCl treatment condition, the root of transgenosis seedling is kept burning day and night to show long greater than the root of wild-type seedling, especially higher transgenic lines transfer-gen plant 2 and the transfer-gen plant 3 of ZmALDH22A1 expression amount.Result and fresh weight measuring result are similar.

3) MDA content: under the various concentration NaCl treatment condition, the relative comparison group, the MDA content of transgenosis and wild-type has all raise, but genetically modified MDA content is all obviously little than the value of wild-type rising.After 50mMNaCl handled, wild-type was upgraded to 169.7% of control group; Transfer-gen plant 1 is upgraded to 147.8% of control group; Transfer-gen plant 2 is upgraded to 115.0% of control group; Transfer-gen plant 3 is upgraded to 131.9% of control group.After 100mM NaCl handled, wild-type was upgraded to 204.9% of control group; Transfer-gen plant 1 is upgraded to 182.8% of control group; Transfer-gen plant 2 is upgraded to 122.0% of control group; Transfer-gen plant 3 is upgraded to 147.6% of control group.After 250mM NaCl handled, wild-type was upgraded to 249.1% of control group; Transfer-gen plant 1 is upgraded to 225.3% of control group; Transfer-gen plant 2 is upgraded to 135.6% of control group; Transfer-gen plant 3 is upgraded to 166.5% of control group.Transgenic lines transfer-gen plant 2 and transfer-gen plant 3 are under any concentration NaCl condition, and the lift-off value of MDA amount all is starkly lower than wild-type.Difference between transfer-gen plant 1 and the wild-type is also not obvious.

Experimental result shows, under high density NaCl stress conditions, the transgenosis seedling is obviously better than wild-type growth of seedling ground, and the expression amount of ZmALDH22A1 has good dependency in the tolerance of NaCl and transgenic line.

3.3 the drought resistance of transgenosis and wild-type tobacco

Experimental result is seen Fig. 5, as can be seen from the figure, is stopping to water back the 18th day, and here the wild-type plant obviously withers, and transfer-gen plant 1 plant phenotype is similar to wild-type, but transfer-gen plant 2 and transfer-gen plant 3 plant are without any the sign here that withers.Experimental result shows that transgenic tobacco plant is more drought-resistant than wild-type tobacco plant.

Sequence table

＜110〉Sichuan University

＜120〉a kind of gene and application thereof that improves stress resistance of plant

<160>2

<170>PatentIn?Version?3.2

<210>1

<211>1896

<212>DNA

＜213〉corn (Zea mays)

<220>

<221>mRNA

<222>(1).....(1896)

＜223〉gene of raising stress resistance of plant

<400>1

cggaagctta?gagcaggtgt?cggtgtcatg?gccttctggt?ggccgctgct?ggtgctcgcc?60

gccgcatacg?cgctctgtcg?cctgctcctc?ttcctcatcc?cgcccaccgt?cccatccatc?120

gacgtcgacg?cctccgacgt?gttggtcaag?gaggacagct?tcatctacat?accgagaagg?180

ggtaaatcaa?ctcagactga?caaggtccaa?tgctatgaac?cagcgaccat?gaaatacttg?240

gggtacttcc?cagtggtgac?tcctgatgag?gtcaaggaac?atgttgcaca?atccaggaaa?300

gctcaaagga?tatgggcaaa?aagcaacttc?aagcaaaggc?gccagtttct?tcgaattctt?360

ctcaagtata?tacttgaaca?tcaggatctc?atatgcgagg?tatcatctcg?ggacactgga?421

aagactatgg?ttgatgcttc?attaggagag?ataatgacca?cctgtgagaa?gattacctgg?480

cttttggatg?agggcgagaa?gtggctgaaa?cctgaataca?gatctactgg?gagatcaatg?540

ctgcataaga?gagcaaaagt?tgagttttac?cctcttggag?tgatcggtgc?gattgtatct?600

tggaattatc?ccttccataa?tgtttttaat?ccagtgctag?cagcagtatt?ctccggtaat?660

gcagcagtta?taaaggtctc?agaacatgcg?acttggtcgg?gctgctttta?ttttcgtatt?720

atacaagcag?ctctttcagc?tgttggtgca?cctgagaatc?tggtgcacat?aataactggt?780

tttgcggaaa?caggccaagc?tcttgtatca?tcagtagaca?aaataatatt?tgtgggatca?840

ccaggtgttg?gaaaaatgat?catgaaaagg?gcgtcggaga?ctctaatacc?tgtaactctg?900

gagcttggtg?gcaaagattc?atttattgtg?tgtgaagatg?tagatttacc?cagtgttgtt?960

caagttgcta?ctagagctgc?tctgcaatct?agtgggcaga?attgcgctgg?tgctgaaaga?1020

ttttatgttc?acgatgacat?ctattctgcc?tttgtttcac?agatagtgaa?gacagtcaaa?1080

tctataagtg?ttgggccccc?attatcaggc?agatatgaca?tgggtgcaat?ctgtatgatt?1140

gagcactctg?agaaacttca?gaatcttgtt?aatgatgctc?tagacaaagg?tgccgaaatt?1200

gctgtcagag?ggagctttgg?caatcttggt?gaagatgcag?ttgatcaatt?tttcccacca?1260

actgtcctgg?tgaatgttga?tcacacaatg?aaaattatgc?aagaagagac?atttggacct?1320

attataccaa?tcatgaaatt?cagttctgat?gaagaggcta?tcaaacttgc?aaacgactca?1380

aaatatggtc?ttggctgtgc?tgttttttct?ggcaaccaga?agcgtgccat?cagaatagcg?1440

tcccagttac?actgtggggt?agcagcaatc?aatgattttg?cttcaagtta?catgtgccag?1500

tctttgccat?tcggtggtgt?taaagacagc?ggattcggga?gatttgctgg?cgtagaaggc?1560

ttgcgagctt?gctgccttgt?gaagtctgtt?gtagaagata?gattgtggcc?atatattaga?1620

acagtgatcc?cgaagcctat?ccagtatccc?gtctcagagc?atggatttga?gttccagcag?1680

ttgcttgtgg?agactctgta?tggctatagc?gtgtgggaca?ggttgcggtc?ccttgtgaat?1740

cttataaaga?tggttaccga?gcagaattct?gccccggctt?caaatgcgac?cacgaagaaa?1800

aggcgatgag?acgaatgccc?ggggggttca?ttgaatctgt?ttatggtaag?tctgatctct?1860

ggacgctgta?aacctcccca?ttccccagga?tccaaa 1896

<210>2

<211>593

<212>PRT

＜213〉corn (Zea mays)

＜223〉polypeptide of raising stress resistance of plant

<400>2

MET?Ala?Phe?Trp?Trp?Pro?Leu?Leu?Val?Leu?Ala?Ala?Ala?Tyr?Ala

1 5 10 15

Leu?Cys?Arg?Leu?Leu?Leu?Phe?Leu?Ile?Pro?Pro?Thr?Val?Pro?Ser

20 25 30

Ile?Asp?Val?Asp?Ala?Ser?Asp?Val?Leu?Val?Lys?Glu?Asp?Ser?Phe

35 40 45

Ile?Tyr?Ile?Pro?Arg?Arg?Gly?Lys?Ser?Thr?Gln?Thr?Asp?Lys?Val

50 55 60

Gln?Cys?Tyr?Glu?Pro?Ala?Thr?MET?Lys?Tyr?Leu?Gly?Tyr?Phe?Pro

65 70 75

Val?Val?Thr?Pro?Asp?Glu?Val?Lys?Glu?His?Val?Ala?Gln?Ser?Arg

80 85 90

Lys?Ala?Gln?Arg?Ile?Trp?Ala?Lys?Ser?Asn?Phe?Lys?Gln?Arg?Arg

95 100 105

Gln?Phe?Leu?Arg?Ile?Leu?Leu?Lys?Tyr?Ile?Leu?Glu?His?Gln?Asp

110 115 120

Leu?Ile?Cys?Glu?Val?Ser?Ser?Arg?Asp?Thr?Gly?Lys?Thr?MET?Val

125 130 135

Asp?Ala?Ser?Leu?Gly?Glu?Ile?MET?Thr?Thr?Cys?Glu?Lys?Ile?Thr

140 145 150

Trp?Leu?Leu?Asp?Glu?Gly?Glu?Lys?Trp?Leu?Lys?Pro?Glu?Tyr?Arg

155 160 165

Ser?Thr?Gly?Arg?Ser?MET?Leu?His?Lys?Arg?Ala?Lys?Val?Glu?Phe

170 175 180

Tyr?Pro?Leu?Gly?Val?Ile?Gly?Ala?Ile?Val?Ser?Trp?Asn?Tyr?Pro

185 190 195

Phe?His?Asn?Val?Phe?Asn?Pro?Val?Leu?Ala?Ala?Val?Phe?Ser?Gly

200 205 210

Asn?Ala?Ala?Val?Ile?Lys?Val?Ser?Glu?His?Ala?Thr?Trp?Ser?Gly

215 220 225

Cys?Phe?Tyr?Phe?Arg?Ile?Ile?Gln?Ala?Ala?Leu?Ser?Ala?Val?Gly

230 235 240

Ala?Pro?Glu?Asn?Leu?Val?His?Ile?Ile?Thr?Gly?Phe?Ala?Glu?Thr

245 250 255

Gly?Gln?Ala?Leu?Val?Ser?Ser?Val?Asp?Lys?Ile?Ile?Phe?Val?Gly

260 265 270

Ser?Pro?Gly?Val?Gly?Lys?MET?Ile?MET?Lys?Arg?Ala?Ser?Glu?Thr

275 280 285

Leu?Ile?Pro?Val?Thr?Leu?Glu?Leu?Gly?Gly?Lys?Asp?Ser?Phe?Ile

290 295 300

Val?Cys?Glu?Asp?Val?Asp?Leu?Pro?Ser?Val?Val?Gln?Val?Ala?Thr

305 310 315

Arg?Ala?Ala?Leu?Gln?Ser?Ser?Gly?Gln?Asn?Cys?Ala?Gly?Ala?Glu

320 325 330

Arg?Phe?Tyr?Val?His?Asp?Asp?Ile?Tyr?Ser?Ala?Phe?Val?Ser?Gln

335 340 345

Ile?Val?Lys?Thr?Val?Lys?Ser?Ile?Ser?Val?Gly?Pro?Pro?Leu?Ser

350 355 360

Gly?Arg?Tyr?Asp?MET?Gly?Ala?Ile?Cys?MET?Ile?Glu?His?Ser?Glu

365 370 375

Lys?Leu?Gln?Asn?Leu?Val?Asn?Asp?Ala?Leu?Asp?Lys?Gly?Ala?Glu

380 385 390

Ile?Ala?Val?Arg?Gly?Ser?Phe?Gly?Asn?Leu?Gly?Glu?Asp?Ala?Val

395 400 405

Asp?Gln?Phe?Phe?Pro?Pro?Thr?Val?Leu?Val?Asn?Val?Asp?His?Thr

410 415 420

MET?Lys?Ile?MET?Gln?Glu?Glu?Thr?Phe?Gly?Pro?Ile?Ile?Pro?Ile

425 430 435

MET?Lys?Phe?Ser?Ser?Asp?Glu?Glu?Ala?Ile?Lys?Leu?Ala?Asn?Asp

440 445 450

Ser?Lys?Tyr?Gly?Leu?Gly?Cys?Ala?Val?Phe?Ser?Gly?Asn?Gln?Lys

455 460 465

Arg?Ala?Ile?Arg?Ile?Ala?Ser?Gln?Leu?His?Cys?Gly?Val?Ala?Ala

470 475 480

Ile?Asn?Asp?Phe?Ala?Ser?Ser?Tyr?MET?Cys?Gln?Ser?Leu?Pro?Phe

485 490 495

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

500 505 510

Gly?Leu?Arg?Ala?Cys?Cys?Leu?Val?Lys?Ser?Val?Val?Glu?Asp?Arg

515 520 525

Leu?Trp?Pro?Tyr?Ile?Arg?Thr?Val?Ile?Pro?Lys?Pro?Ile?Gln?Tyr

530 535 540

Pro?Val?Ser?Glu?His?Gly?Phe?Glu?Phe?Gln?Gln?Leu?Leu?Val?Glu

545 550 555

Thr?Leu?Tyr?Gly?Tyr?Ser?Val?Trp?Asp?Arg?Leu?Arg?Ser?Leu?Val

560 565 570

Asn?Leu?Ile?Lys?MET?Val?Thr?Glu?Gln?Asn?Ser?Ala?Pro?Ala?Ser

575 580 585

Asn?Ala?Thr?Thr?Lys?Lys?Arg?Arg

590

Claims (4)

1. gene that improves stress resistance of plant, the nucleotides sequence that it is characterized in that it are classified as in the sequence table shown in the SEQ IDNO:1.

2. recombinant plasmid that improves stress resistance of plant is characterized in that it contains the described gene of claim 1.

3. the application of the described gene of claim 1 in the drought-resistant improvement of plant.

4. the application of the described gene of claim 1 in the plant anti-salt improvement.

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