CN101775070B - Plant stress tolerance correlative protein, encoding gene and application thereof - Google Patents

Abstract

The invention discloses a plant stress tolerance correlative protein, an encoding gene and application thereof. The protein provided by the invention refers to the protein of a) or b): a) the protein formed by an amino acid sequence shown by a first sequence in a sequence table; or b) the plant stress tolerance correlative protein which is formed through the substitution and/or lack and/or addition of one or a plurality of amino acid residue radicals on the amino acid residue radical sequence of the first sequence in the sequence table and is derived from the first sequence. Experiments show that when the DNA sequence of the transcription factor 0sDRF2 of the invention for encoding and regulating the plant stress tolerance is introduced into arabidopsis for overexpression, the tolerance of the transgenic arabidopsis on the osmotic stress and the drought stress can be improved. The genome gene of the transcription factor 0sDRF2 of the invention for encoding and regulating the plant stress tolerance and the cDNA gene thereof provides the basis for culturing other tolerance improved plants with the economic value.

Description

Plant stress tolerance correlative protein and encoding sox thereof and application

Technical field

The present invention relates to a kind of plant stress tolerance correlative protein and encoding sox thereof and application.

Background technology

Along with the progress and the expanding economy of human society, shortage of water resources has become China and even the whole world says that the problem that faces, adverse circumstances such as arid are the important factors of restriction China grain-production.When plant suffers from adverse circumstance, can be caused the variation of intracellular calcium signal by the acceptor impression; Activated protein kinase causes the phosphorylation of cascade; Many transcription factors are activated and cause the expression of environment stress response gene, in plant materials, produce a large amount of differential proteins, collaborative adjusting plant physiology and biochemistry and metabolic variation; Thereby adapt to outside adverse circumstance, improve the patience of plant adverse circumstance.Transcription factor can be regulated and control a plurality of expression of gene and in the degeneration-resistant reaction of plant, bring into play important effect; Reply the expression that relevant transcription factor just possibly change a plurality of downstream anti contravariance related gene simultaneously so in plant, cross to express, thereby obtain the transfer-gen plant of resistance improvement with degeneration-resistant.

Summary of the invention

The purpose of this invention is to provide a kind of plant stress tolerance correlative protein and encoding sox thereof and application.

Plant stress tolerance correlative protein (OsDRF2 provided by the invention; Oryza sativa drought responsibleERF2); Be a kind of transcription factor; Deriving from japonica rice variety " Japan fine " (Oryza sativa subsp.japonicacv.Nipponbare), is (a) or protein (b) as follows:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by sequence 1 deutero-protein.

Sequence 1 in the sequence table is made up of 205 amino-acid residues.

In order to make the OsDRF2 in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the OsDRF2 synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.The encoding sox of OsDRF2 in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The gene (OsDRF2) of above-mentioned plant stress tolerance correlative protein of encoding also belongs to protection scope of the present invention.

Said gene can be following 1) or 2) or 3) or 4) dna molecular:

1) dna molecular shown in the sequence 2 (cDNA) in the sequence table;

2) dna molecular shown in the sequence 3 (genomic gene) in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of the stress tolerance correlative protein of encoding.

Above-mentioned stringent condition can be at 6 * SSC, and in the solution of 0.5%SDS, 2 * SSC is used in hybridization then under 65C, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The dna molecular shown in the sequence 3 is made up of 3051 Nucleotide in the sequence table, is promotor from 5 ' end 1-2000 position, and 2001-2178 is first exon, and 2179-2299 is an intron, and 2300-3051 is second exon.

The recombinant expression vector that contains above arbitrary said gene also belongs to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of said gene.

Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (like kermes synthetic enzyme Nos gene), plant gene (like soybean storage protein gene) 3 ' end to transcribe like the Agrobacterium crown-gall nodule all has similar functions.

When using said gene constructed recombinant plant expression vector; Before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter; Like the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can use separately or be used in combination with other plant promoter; In addition; When using gene constructed plant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) or the anti-chemical reagent marker gene (like anti-weedkiller gene) of resistance etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Said recombinant expression vector is that said gene is inserted the recombinant plasmid that the MCS of pCAMBIA1300 ' obtains; Said pCAMBIA1300 ' is connected the dna fragmentation first and obtains with dna fragmentation second; Said dna fragmentation first is: cut pRT105 with restriction enzyme PstI enzyme, reclaim the fragment that comprises 35S promoter, MCS and poly (A), with the dna fragmentation that obtains behind the end-filling; Said dna fragmentation second is: cut pCAMBIA1300 with restriction enzyme EcoRI and HindIII enzyme, reclaim skeleton carrier, the dna fragmentation that end-filling is obtained.

The expression cassette, transgenic cell line and the reorganization bacterium that contain above arbitrary said gene (OsDRF2) all belong to protection scope of the present invention.

Said gene (OsDRF2) total length that increases or arbitrary segmental primer are to also belonging to protection scope of the present invention.

Another object of the present invention provides a kind of method of cultivating transgenic plant.

The method of the drought-enduring transgenic plant of cultivation provided by the present invention can import the gene of the said plant stress tolerance correlative protein of coding in the purpose plant (like vegetable cell or tissue), obtains the transgenic plant that resistance of reverse is higher than said purpose plant.Specifically, can said recombinant expression vector be imported in the purpose plant, obtain the transgenic plant that resistance of reverse is higher than said purpose plant.Said resistance of reverse specifically can be anti-osmotic stress and/or drought-resistant.

Utilize any carrier that can guide foreign gene in plant, to express,, can obtain drought-resistance ability enhanced transgenic cell line and transfer-gen plant the gene transfered plant cell of encoding said proteins.Carry that said expression carrier can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated through using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: tobacco, Root or stem of Littleleaf Indianmulberry, Arabidopis thaliana, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.

Experiment shows that the dna sequence dna of coding of the present invention being regulated and control the transcription factor OsDRF2 of stress resistance of plant imports Arabidopis thaliana and crosses expression, can improve the patience of transgenic arabidopsis to osmotic stress and drought stress.The genomic gene of the transcription factor OsDRF2 of coding regulation and control stress resistance of plant of the present invention and cDNA gene thereof provide the foundation for the plant that the resistance of cultivating other economically valuables improves.

Description of drawings

Fig. 1 crosses the RT-PCR detected result of expression for transfer-gen plant OsDRF2.

Fig. 2 is the phenotype photo of plant during the osmotic stress of transgenic plant is tested.

Fig. 3 is the fresh weight MV of plant during the osmotic stress of transgenic plant is tested.

Fig. 4 coerces the phenotype photo of plant in the experiment thoroughly for the arid of transgenic plant.

Fig. 5 coerces in the experiment fresh weight MV of rehydration plant after a day thoroughly for the arid of transgenic plant.

Fig. 6 coerces in the experiment for the arid of transgenic plant thoroughly, and arid is handled leaf roll rate and the rehydration surviving rate of rehydration after one day after 18 days.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Among the following embodiment, experiment all is provided with three repetitions, results averaged.

PRT105: Biological Technology institute, Chinese Academy of Agricultural Sciences; Topfer, R., Maas; C., Horicke-Grandpierre, C.; Schell, J., and Steinbiss; H.H. (1993) .Expressionvectors for high-level gene expression in dicotyledonous and monocotyledonousplants.Methods Enzymol 217,67-78.

The discovery of embodiment 1, OsDRF2 and clone

One, the clone of OsDRF2 cDNA

Get 0.2g japonica rice variety " Japan is fine " (Oryza sativa subsp.japonica cv.Nipponbare) (Institute of Crop Science, Chinese Academy of Agricultural Science, numbering WD-10576; Primary source is unclear) blade, liquid nitrogen grinding, the TRIzol method is extracted total RNA.Get the total RNA of 2 μ g and carry out reverse transcription with the M-MLV ThermoScript II, synthetic cDNA first chain as template, carries out the PCR reaction with special primer to first.The PCR product is after electrophoretic separation reclaims, and (precious biotechnology (Dalian) ltd D102A), checks order to be cloned into pMD19-T.

Special primer is following to first:

Upstream primer: 5 '-ATGGTACAGCCAAAGAAGAAG-3 ';

Downstream primer: 5 '-TCAGATGACAAAGCTACCCTC-3 '.

Sequencing result shows, this segmental nucleotide sequence shown in the sequence 2 of sequence table, the protein shown in the sequence 1 of code sequence tabulation.

Two, the acquisition of OsDRF2 genomic gene

The CTAB method is extracted the genomic dna of Japanese fine blade, is template with this genomic dna, with special primer second is reacted.The PCR product is cloned into pMD19-T after electrophoretic separation reclaims, check order.

Special primer is following to second:

Upstream primer: 5 '-GACAGGGGCAACGAACTTCC-3 ';

Downstream primer: 5 '-GTAACAAAGTATGTTGATGG-3 '.

Sequencing result shows that this segmental nucleotide sequence is shown in the sequence 3 of sequence table.

With the protein called after OsDRF2 shown in the sequence 1 of sequence table, with the encoding sox called after OsDRF2 of OsDRF2.Nucleotide shown in the sequence 2 of sequence table is connected the recombinant plasmid called after pMD19-T-OsDRF2 that obtains with pMD19-T.

Acquisition of embodiment 2, transgenic plant and evaluation

One, the structure of pCAMBIA1300 '

1, cuts pRT105 with the PstI enzyme, reclaim the fragment that comprises 35S promoter, MCS and poly (A), end-filling.

2, with restriction enzyme EcoRI and HindIII enzyme cut pCAMBIA1300 (Cambia, GPO Box 3200, Canberra, ACT 2601, Australia), reclaim skeleton carrier, with end-filling.

3, the fragment that step 1 is obtained is connected with the fragment that step 2 obtains, and obtains pCAMBIA1300 '.

Two, the acquisition of transgenic plant

1,, reclaims OsDRF2 cDNA fragment with the recombinant plasmid pMD19-T-OsDRF2 of restriction enzyme BamHI and SalI double digestion embodiment 1.

2,, reclaim skeleton carrier with restriction enzyme BamHI and SalI double digestion pCAMBIA1300 '.

3, step 1 is obtained dna fragmentation and be connected, obtain recombinant expression vector with the skeleton carrier that step 2 obtains.

4, with recombinant expression vector import Agrobacterium LBA4404 ( Http:// www.cbs.knaw.nl/, NCCBbacteria/plasmids database, NCCB number 2760).

5, utilize then agriculture bacillus mediated; Through flower infusion method (Clough, S.J., and Bent; A.F. (1998) .Floral dip:a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana.Plant J 16; 735-743.) the OsDRF2 gene is imported Arabidopis thaliana Col-0, and (Arabidopsis Biological Resource Center CS70000), obtains T1 for seed.

T1 is transplanted to resistant plant in the soil for seed results back screening resistant plant in MS substratum (containing the 30mg/L Totomycin), and the T2 of results transgenic plant is for seed.

TRIzol (Invitrogen; Cat.No.15596-026) extract the total RNA of T2 for plant leaf; Get the total RNA of 2ug, with M-MLV ThermoScript II (Invitrogen, Cat.No.28025-013) synthetic cDNA first chain; Be contrast then with Actin, first carried out pcr amplification with the special primer of embodiment 1.As shown in Figure 1, the expression of OsDRF2 is arranged in the positive plant really, and do not express in the wild-type contrast, prove to have obtained the OsDRF2 transfer-gen plant.

PCAMBIA1300 ' is imported Agrobacterium LBA4404, arabidopsis thaliana transformation then, step is the same.The T2 that obtains changeing the empty carrier control plant is for seed.

Two, resistance of reverse is identified

1, to the resistance of reverse of osmotic stress

7,8 and 18), the T2 that changes the empty carrier control plant is for seed and wild-type Arabidopis thaliana (WT) seed (three strains systems:, the seedling that sprouted 4 days is handled (each strain is 100 strains) to the T2 that gets transgenic plant respectively in MS substratum (containing 200mM N.F,USP MANNITOL) for seed; 7,8 and 18) and wild-type Arabidopis thaliana seed (three strains systems:, the seedling that sprouted 4 days is handled (each strain is 100 strains) in the MS substratum, as contrast for seed with the T2 of transgenic plant.Photo when handling a week is seen Fig. 2.The fresh weight MV of plant is seen Fig. 3 when handling a week.

Visible by Fig. 2 and Fig. 3: in the treatment with mannitol, WT compares with wild-type, and the suffered inhibition of OsDRF2 transgenic line 7,8,18 leaf growths is less, and fresh weight is 1.5-2 a times of wild-type; In control treatment, the growth of transgenic line 7,8,18 and wild-type WT does not have significant difference; Phenotype of commentaries on classics empty carrier plant and fresh weight and wild-type do not have significant difference.This showed that expressing the OsDRF2 gene can improve the patience of Arabidopis thaliana to osmotic stress.

2, to the resistance of reverse of drought stress

7,8 and 18), the T2 that changes the empty carrier control plant is for seed and wild-type Arabidopis thaliana (WT) seed the T2 that gets transgenic plant respectively is for seed (three strains systems:; The seedling (each strain is 100 strains) that sprouted for 2 weeks is transplanted in the soil; Arid is handled 18 days (not watering), rehydrations then.(three strains systems: 7,8 and 18) with wild-type Arabidopis thaliana seed, sprout 2 all seedling replantings in soil, water every day in right amount, as contrast for seed with the T2 of transgenic plant.

Respectively after arid was handled 18 days and rehydration take pictures after one day, see Fig. 4.After the rehydration one day, the fresh weight MV of plant is seen Fig. 5, and leaf roll rate and the rehydration surviving rate of rehydration after one day that arid was handled after 18 days are seen Fig. 6.

Visible by Fig. 4, Fig. 5 and Fig. 6: in the control treatment, transgenic line 7,8,18 and wild-type WT Arabidopis thaliana growth of seedling indifference; After arid was handled 18 days, wild-type WT blade 85% was crispaturaed, leaf look browning, and cessation of growth cessation, and transgenic line 7,8,18 blades only have 10-20% to crispatura, and about 80% leaf color deepens, and 20% leaf color is normal; Rehydration one day after, 95% above transgenic line 7,8,18 leaf looks recover light green, and 75% wild-type WT leaf look does not recover; The phenotype of changeing the empty carrier plant does not have significant difference with each detection index and wild-type.This showed that expressing the OsDRF2 gene can improve the patience of Arabidopis thaliana to drought stress.

Sequence table

< 110>Biological Technology institute, Chinese Academy of Agricultural Sciences

< 120>plant stress tolerance correlative protein and encoding sox thereof and application

<130>CGGNARY102028

<160>3

<210>1

<211>205

<212>PRT

< 213>Japanese fine paddy rice (Oryza sativa subsp.japonica cv.Nipponbare)

<400>1

Met Val Gln Pro Lys Lys Lys Phe Arg Gly Val Arg Gln Arg His Trp

1 5 10 15

Gly Ser Trp Val Ser Glu Ile Arg His Pro Leu Leu Lys Arg Arg Val

20 25 30

Trp Leu Gly Thr Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp

35 40 45

Glu Ala Ala Val Leu Met Ser Gly Arg Asn Ala Lys Thr Asn Phe Pro

50 55 60

Val Gln Arg Asn Ser Thr Gly Asp Leu Ala Thr Ala Ala Asp Gln Asp

65 70 75 80

Ala Arg Ser Asn Gly Gly Ser Arg Asn Ser Ser Ala Gly Asn Leu Ser

85 90 95

Gln Ile Leu Ser Ala Lys Leu Arg Lys Cys Cys Lys Ala Pro Ser Pro

100 105 110

Ser Leu Thr Cys Leu Arg Leu Asp Pro Glu Lys Ser His Ile Gly Val

115 120 125

Trp Gln Lys Arg Ala Gly Ala Arg Ala Asp Ser Asn Trp Val Met Thr

130 135 140

Val Glu Leu Asn Lys Glu Val Glu Pro Thr Glu Pro Ala Ala Gln Pro

145 150 155 160

Thr Ser Thr Ala Thr Ala Ser Gln Val Thr Met Asp Asp Glu Glu Lys

165 170 175

Ile Ala Leu Gln Met Ile Glu Glu Leu Leu Ser Arg Ser Ser Pro Ala

180 185 190

Ser Pro Ser His Gly Glu Gly Glu Gly Ser Phe Val Ile

195 200 205

<210>2

<211>618

<212>DNA

< 213>Japanese fine paddy rice (Oryza sativa subsp.japonica cv.Nipponbare)

<400>2

atggtacagc caaagaagaa gtttcgtgga gtcaggcagc ggcactgggg ctcctgggtc 60

tctgagatca gacaccccct ccttaaaagg agggtgtggc tgggcacctt tgagacggcc 120

gaggaggctg cgcgagccta cgatgaggct gctgtgctga tgagtggccg caacgccaag 180

accaacttcc ccgtgcagag gaactccacc ggtgatctcg ccacggccgc agaccaggac 240

gcccgtagca atggcggtag caggaactcc tccgcgggca acctgtcaca gattctcagt 300

gctaagctcc gcaagtgctg caaggcgcca tctccgtcct taacctgcct ccgcctcgac 360

cccgagaagt cccacattgg cgtgtggcaa aagcgcgcag gggcccgtgc tgactccaac 420

tgggtgatga cggtggagct caacaaagag gtagaaccaa ctgaacctgc agctcagccc 480

acatcaacag caacagcttc gcaagtgaca atggatgatg aggaaaagat tgcgctgcaa 540

atgatcgagg agttgctgag caggagcagt ccagcttcac cctcacatgg agagggagag 600

ggtagctttg tcatctga 618

<210>3

<211>3051

<212>DNA

< 213>Japanese fine paddy rice (Oryza sativa subsp.japonica cv.Nipponbare)

<400>3

gacaggggca acgaacttcc atcaatctga atcactttta cagaacacat ctgtagcagc 60

aaaggacagt tgattagtga aatatgacga gccgccaatt attgcaggca gactgttcag 120

atacttcaat gttgatacgg tgtacgaagt actcttgcca tgaaatggga ctgcaagcct 180

gtaaatagat caaaggtcat agtcaggaac acctcatatc cgtagaaata cagaatccag 240

gctggatcat gtccattaca aattacatac tagaattttc ggacttacat aaaggagtga 300

catgcataca gagagcataa ttcctcaaat gtgcgaagag agcaaaaaaa gaaacagacc 360

acacctcaaa cttattacag tttatacgtt acacaaatac ttcaatggca aacaagatgc 420

gtgaaacaac atgtagctgc caaacagaaa aggtaacaaa tatgttggga tagccacaaa 480

aaggtgaact tgcagactat acgaagcaca agaattcagt tatttgatca atgaaagatt 540

ataggcacaa agagtagagt gttactcttt gctcctactt catcttcctg atgtctgcta 600

cttctgaata aaatatatta tcatgttctg cacccagtaa ggagttactt ctaactgtat 660

catgaagaaa tataaacgtg tcagaaaata ttaaaaaata taaatgattc accagctaag 720

agcgtcgagc aacactcagc agtgagcaca caccatcatc ataaccaggc tgtgtacatg 780

gagctacctt agacaagctt aaatgccaac caactccaag cagtcccaga tcaaaaagct 840

catgacaacg gaaaacttga aaaagaaaaa aaaaacttta caagtcttaa ccaaaaaaac 900

tctaaattta cacacacaca cacacacaca ctaaaggcaa cacattctta cacatttcaa 960

actccagttc tatagcgcaa aacaagactt gagtgttgaa atatgaagaa agtttcccaa 1020

taagacgaga gaaaactgaa cacgtcatca caaataaagc acgagatatt cccaaacgca 1080

gtttgcagca taagtcttgc catatgagac gcctagttaa gaacaacgtc catgcatcca 1140

ctggcactca aagctcatga ctggaccata tccacacccc accatagctg tggaagaatt 1200

atggccatgg agttgatctg acacaaatgc acaattgctt tgcagaacaa ctagcctagc 1260

ttatactaat gctctaggag ctaatgtgct aatgtgaatc aacccggggt tgccccacaa 1320

ttaatgtggc taccatgggc gtaggtgaga gtgttaatta ccaatgtcct agcagagaag 1380

gtggttatgc taatacccgc acagttcacc gacagccccc actgcgggcc ttgtggactg 1440

gaccaccttc accttcagtt gcccctcccc tgaattctct cttcacctct actacctctg 1500

tcccgaaata actttatttt tcacctatcc cgtacatacc aatacaaaga caaaaatacc 1560

ataatgtctt ctattttaac aaatcacaat gcaattttac cccactttaa caaactccaa 1620

tgcatttgtc tcccacttcc atagattcaa tataatgatt tacttaaaag ataaaagtta 1680

ttttgagaca aataagatgg taaaaaatga agttattttg ggatagaggg aagtatctcc 1740

agtcctgcct tacttttatc ccttggcaca cacctgctag ttgctactgc ttgtgaaccc 1800

agcccttggt gatgttcagt gaaaactagg ccaaacacaa tctctttgat tctctctttc 1860

tatctctgta tctctgatac gtactatttg accacctata cgtctcacca catttaacgc 1920

ggcactgtag acgcaagtac aggccgcagc agtttatatt cactcaaaca agtgctttcc 1980

tcctccccca cacctcctcc gttcagttca gaggcgccta gcaatagcag ctcattgcct 2040

catctctgcc tcccctgtcc ttctgggggc agagaatctc tccactgctg gaaaaatggt 2100

acagccaaag aagaagtttc gtggagtcag gcagcggcac tggggctcct gggtctctga 2160

gatcagacac cccctcctgt aagctcttct atcaacatcc ctctaatttt ctgctaataa 2220

tgttatgttt tgatgtgtct aatactctaa tctaataagt aataagtctc aatgcatatg 2280

gtgtttgcaa ctaatgcagt aaaaggaggg tgtggctggg cacctttgag acggccgagg 2340

aggctgcgcg agcctacgat gaggctgctg tgctgatgag tggccgcaac gccaagacca 2400

acttccccgt gcagaggaac tccaccggtg atctcgccac ggccgcagac caggacgccc 2460

gtagcaatgg cggtagcagg aactcctccg cgggcaacct gtcacagatt ctcagtgcta 2520

agctccgcaa gtgctgcaag gcgccatctc cgtccttaac ctgcctccgc ctcgaccccg 2580

agaagtccca cattggcgtg tggcaaaagc gcgcaggggc ccgtgctgac tccaactggg 2640

tgatgacggt ggagctcaac aaagaggtag aaccaactga acctgcagct cagcccacat 2700

caacagcaac agcttcgcaa gtgacaatgg atgatgagga aaagattgcg ctgcaaatga 2760

tcgaggagtt gctgagcagg agcagtccag cttcaccctc acatggagag ggagagggta 2820

gctttgtcat ctgaaaggct tggatgaaac gacggcataa cgaagtcacc actctagacc 2880

atggttccag aatttcctgc tggtacacag gcagttcaga ttacttcagt acaatagaaa 2940

atatctatta cttcagacaa gtaacaagac atataagttt gcaactatgt gtagctatat 3000

gtgtttatga aaagctatat atacgcagtc tccatcaaca tactttgtta c 3051

Claims (4)

1. method of cultivating anti-osmotic stress and/or drought-resistant transgenic plant; Be to import in the purpose plant by the proteinic encoding sox that the aminoacid sequence shown in the sequence in the sequence table 1 is formed, obtain the transgenic plant that resistance of reverse is higher than said purpose plant; Said purpose plant is an Arabidopis thaliana; Said resistance of reverse is an anti-osmotic stress and/or drought-resistant.

2. the method for claim 1, it is characterized in that: the said proteinic encoding sox of being made up of the aminoacid sequence shown in the sequence in the sequence table 1 is following 1) or 2) dna molecular:

1) dna molecular shown in the sequence 2 in the sequence table;

2) dna molecular shown in the sequence 3 in the sequence table.

3. according to claim 1 or claim 2 method, it is characterized in that: said encoding sox imports in the said purpose plant through the recombinant expression vector that contains said encoding sox.

4. method as claimed in claim 3 is characterized in that: said recombinant expression vector is that said encoding sox is inserted the recombinant plasmid that the MCS of pCAMBIA1300 ' obtains;

Said pCAMBIA1300 ' is connected the dna fragmentation first and obtains with dna fragmentation second; Said dna fragmentation first is: cut pRT105 with restriction enzyme PstI enzyme, reclaim the fragment that comprises 35S promoter, MCS and poly (A), with the dna fragmentation that obtains behind the end-filling; Said dna fragmentation second is: cut pCAMBIA1300 with restriction enzyme EcoRI and HindIII enzyme, reclaim skeleton carrier, the dna fragmentation that end-filling is obtained.

Images