CN103361362A - Method for culturing drought-resistant salt-tolerant transgenic cotton by using AtEDT1 gene - Google Patents

Abstract

The invention discloses a method for culturing stress tolerance transgenic cotton. According to the method, the AtEDT1 gene is led into cotton through a plant expression vector, and cotton with improved stress tolerance is obtained by screening, wherein the AtEDT1 gene is a protein (a) or protein (b), the protein (a) consists of an amino acid residue sequence of a SEQ ID NO:2 in a sequence table, and the protein (b) is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the amino acid residue sequence of the SEQ ID NO:2 in the sequence table, has the same activity with the protein (a) and is derived from the protein (a). The drought resistance and salt tolerance of an AtEDT1 transgenic cotton strain (transformant) are obviously higher than those of an acceptor comparison (non-transgenic cotton) strain.

Description

Use the AtEDT1 gene and cultivate the method for drought resistance and salt tolerance transgene cotton

Technical field

The present invention relates to a kind of method of cultivating the drought resistance and salt tolerance transgene cotton.

Background technology

Any discomfort environmental factors in the plant growth and development process all can be described as environment-stress, generally is divided into biology and coerces (such as disease and pest) and abiotic stress.The harm that environment-stress brings to agriculture production is global, and abiotic to coerce the harm that causes particularly serious, makes crop production reduction 50% to 82% (Boyer JS.Plant productivity and environment.Science 1982 (218): 443-448).Drought is the most serious in abiotic stress, also is modal environment-stress, is the main limiting factor of agriculture production in the world wide, and it is the basis of sustainability agricultural that response environment is coerced.China is large agricultural country, existing 1,500,000,000 mu of nonirrigated farmlands, and at least 5 hundred million mu of saltingss are subjected to abiotic stress harm serious, and wherein arid is the principal element of restriction China Agricul tural Sustain able Development with salting of soil, seriously hinders the development of China's sustainability agricultural.A lot of scientists all will study and by genetic engineering means enhancing plant for the resistance of various abiotic stress as research emphasis.The various abiotic stress such as arid, flood, salting of soil are day by day serious to the destruction of physical environment, and coming the expression of regulatory gene by transcription factor is key link in the plant Stress response reaction.

The anti-contrary gene of research abiotic stress biology and separating clone is extremely important, global plant biological scholar has done a large amount of work for this reason, and obtain a lot of progress (utilizing high model plant Arabidopis thaliana to study aspect the plant salt tolerance molecular mechanism especially), make the research in this field that breakthrough progress arranged.Number of patent application is that 200410000682.2 Chinese invention patent application discloses an Arabidopis thaliana transcription factor AtEDT1 (being the gene of SEQ ID NO.1 in the sequence table of the present invention), and it is relevant with drought resistance and the salt resistance of Arabidopis thaliana.

Summary of the invention

The objective of the invention is to cultivate the resisting drought saving water new cotton variety with adversity gene transcription factor AtEDT1, a kind of method of cultivating anti-Reversion gene cotton is provided.

The method of the anti-Reversion gene cotton of cultivation provided by the present invention is that Arabidopis thaliana AtEDT1 gene is imported in the cotton by plant expression vector, and screening obtains the cotton that resistance improves.

In one embodiment, described AtEDT1 is following (a) or protein (b):

(a) protein that is formed by the amino acid residue sequence of SEQ ID NO:2 in the sequence table;

(b) with the amino acid residue sequence of SEQ ID NO:2 in the sequence table through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have with (a) identical activity by (a) derivative protein.

Described AtEDT1 is preferably the protein that the amino acid residue sequence by SEQ ID NO:2 in the sequence table forms.

The amino acid residue sequence of SEQ ID NO:2:

Met Ser Phe Val Val Gly Val Gly Gly Ser Gly Ser Gly Ser Gly Gly

1 5 10 15

Asp Gly Gly Gly Ser His His His Asp Gly Ser Glu Thr Asp Arg Lys

20 25 30

Lys Lys Arg Tyr His Arg His Thr Ala Gln Gln Ile Gln Arg Leu Glu

35 40 45

Ser Ser Phe Lys Glu Cys Pro His Pro Asp Glu Lys Gln Arg Asn Gln

50 55 60

Leu Ser Arg Glu Leu Gly Leu Ala Pro Arg Gln Ile Lys Phe Trp Phe

65 70 75 80

Gln Asn Arg Arg Thr Gln Leu Lys Ala Gln His Glu Arg Ala Asp Asn

85 90 95

Ser Ala Leu Lys Ala Glu Asn Asp Lys Ile Arg Cys Glu Asn Ile Ala

100 105 110

Ile Arg Glu Ala Leu Lys His Ala Ile Cys Pro Asn Cys Gly Gly Pro

115 120 125

Pro Val Ser Glu Asp Pro Tyr Phe Asp Glu Gln Lys Leu Arg Ile Glu

130 135 140

Asn Ala His Leu Arg Glu Glu Leu Glu Arg Met Ser Thr Ile Ala Ser

145 150 155 160

Lys Tyr Met Gly Arg Pro Ile Ser Gln Leu Ser Thr Leu His Pro Met

165 170 175

His Ile Ser Pro Leu Asp Leu Ser Met Thr Ser Leu Thr Gly Cys Gly

180 185 190

Pro Phe Gly His Gly Pro Ser Leu Asp Phe Asp Leu Leu Pro Gly Ser

195 200 205

Ser Met Ala Val Gly Pro Asn Asn Asn Leu Gln Ser Gln Pro Asn Leu

210 215 220

Ala Ile Ser Asp Met Asp Lys Pro Ile Met Thr Gly Ile Ala Leu Thr

225 230 235 240

Ala Met Glu Glu Leu Leu Arg Leu Leu Gln Thr Asn Glu Pro Leu Trp

245 250 255

Thr Arg Thr Asp Gly Cys Arg Asp Ile Leu Asn Leu Gly Ser Tyr Glu

260 265 270

Asn Val Phe Pro Arg Ser Ser Asn Arg Gly Lys Asn Gln Asn Phe Arg

275 280 285

Val Glu Ala Ser Arg Ser Ser Gly Ile Val Phe Met Asn Ala Met Ala

290 295 300

Leu Val Asp Met Phe Met Asp Cys Val Lys Trp Thr Glu Leu Phe Pro

305 310 315 320

Ser Ile Ile Ala Ala Ser Lys Thr Leu Ala Val Ile Ser Ser Gly Met

325 330 335

Gly Gly Thr His Glu Gly Ala Leu His Leu Leu Tyr Glu Glu Met Glu

340 345 350

Val Leu Ser Pro Leu Val Ala Thr Arg Glu Phe Cys Glu Leu Arg Tyr

355 360 365

Cys Gln Gln Thr Glu Gln Gly Ser Trp Ile Val Val Asn Val Ser Tyr

370 375 380

Asp Leu Pro Gln Phe Val Ser His Ser Gln Ser Tyr Ar Phe Pro Ser

385 390 395 400

Gly Cys Leu Ile Gln Asp Met Pro Asn Gly Tyr Ser Lys Val Thr Trp

405 410 415

Val Glu His Ile Glu Thr Glu Glu Lys Glu Leu Val His Glu Leu Tyr

420 425 430

Arg Glu Ile Ile His Arg Gly Ile Ala Phe Gly Ala Asp Arg Trp Val

435 440 445

Thr Thr Leu Gln Arg Met Cys Glu Arg Phe Ala Ser Leu Ser Val Pro

450 455 460

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

465 470 475 480

Lys Arg Ser Met Met Arg Leu Ala Gln Arg Met Ile Ser Asn Tyr Cys

485 490 495

Leu Ser Val Ser Arg Ser Asn Asn Thr Arg Ser Thr Val Val Ser Glu

500 505 510

Leu Asn Glu Val Gly Ile Arg Val Thr Ala His Lys Ser Pro Glu Pro

515 520 525

Asn Gly Thr Val Leu Cys Ala Ala Thr Thr Phe Trp Leu Pro Asn Ser

530 535 540

Pro Gln Asn Val Phe Asn Phe Leu Lys Asp Glu Arg Thr Arg Pro Gln

545 550 555 560

Trp Asp Val Leu Ser Asn Gly Asn Ala Val Gln Glu Val Ala His Ile

565 570 575

Ser Asn Gly Ser His Pro Gly Asn Cys Ile Ser Val Leu Arg Gly Ser

580 585 590

Asn Ala Thr His Ser Asn Asn Met Leu Ile Leu Gln Glu Ser Ser Thr

595 600 605

Asp Ser Ser Gly Ala Phe Val Val Tyr Ser Pro Val Asp Leu Ala Ala

610 615 620

Leu Asn Ile Ala Met Ser Gly Glu Asp Pro Ser Tyr Ile Pro Leu Leu

625 630 635 640

Ser Ser Gly Phe Thr Ile Ser Pro Asp Gly Asn Gly Ser Asn Ser Glu

645 650 655

Gln Gly Gly Ala Ser Thr Ser Ser Gly Arg Ala Ser Ala Ser Gly Ser

660 665 670

Leu Ile Thr Val Gly Phe Gln Ile Met Val Ser Asn Leu Pro Thr Ala

675 680 685

Lys Leu Asn Met Glu Ser Val Glu Thr Val Asn Asn Leu Ile Gly Thr

690 695 700

Thr Val His Gln Ile Lys Thr Ala Leu Ser Gly Pro Thr Ala Ser Thr

705 710 715 720

Thr Ala

722

Wherein, the SEQ ID NO:2 in the sequence table is comprised of 722 amino-acid residues, is homeobox (homeo-box) structural domain from the 31st of N-terminal to the 88th amino acids residue, and major function is and the DNA combination; Be that the lipid of steroid sensitivity is regulated albumen (START from the 236th of N-terminal to the 457th amino acids residue, steroidogenic acute regulatory protein related lipid transfer) structural domain, regulating between the protein structure domain (N-terminal the 89th to the 235th amino acids residue) at the lipid of homeodomain and steroid sensitivity is leucine zipper (LZ, leucine zipper) structural domain may be relevant with dimerization.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation refer to replace outside the lipid adjusting protein structure domain of the above-mentioned homeodomain of SEQ ID NO:22 and steroid sensitivity and/or lack and/or add.

The encoding sequence of described AtEDT1 gene is preferably the SEQ ID NO:1 in the sequence table.

The sequence of SEQ ID NO:1:

atgagtttcg tcgtcggcgt cggcggaagt ggtagtggaa gcggcggaga cggtggtggt 60

agtcatcatc acgacggctc tgaaactgat aggaagaaga aacgttacca tcgtcacacc 120

gctcaacaga ttcaacgcct tgaatcgagt ttcaaggagt gtcctcatcc agatgagaaa 180

cagaggaacc agcttagcag agaattgggt ttggctccaa gacaaatcaa gttctggttt 240

cagaacagaa gaactcagct taaggctcaa catgagagag cagataatag tgcactaaag 300

gcagagaatg ataaaattcg ttgcgaaaac attgctatta gagaagctct caagcatgct 360

atatgtccta actgtggagg tcctcctgtt agtgaagatc cttactttga tgaacaaaag 420

cttcggattg aaaatgcaca ccttagagaa gagcttgaaa gaatgtctac cattgcatca 480

aagtacatgg gaagaccgat atcgcaactc tctacgctac atccaatgca catctcaccg 540

ttggatttgt caatgactag tttaactggt tgtggacctt ttggtcatgg tccttcactc 600

gattttgatc ttcttccagg aagttctatg gctgttggtc ctaataataa tctgcaatct 660

cagcctaact tggctatatc agacatggat aagcctatta tgaccggcat tgctttgact 720

gcaatggaag aattgctcag gcttcttcag acaaatgaac ctctatggac aagaacagat 780

ggctgcagag acattctcaa tcttggtagc tatgagaatg ttttcccaag atcaagtaac 840

cgagggaaga accagaactt tcgagtcgaa gcatcaaggt cttctggtat tgtcttcatg 900

aatgctatgg cacttgtcga catgttcatg gattgtgtca agtggacaga actctttccc 960

tctatcattg cagcttctaa aacacttgca gtgatttctt caggaatggg aggtacccat 1020

gagggtgcat tgcatttgtt gtatgaagaa atggaagtgc tttcgccttt agtagcaaca 1080

cgcgaattct gcgagctacg ctattgtcaa cagactgaac aaggaagctg gatagttgta 1140

aacgtctcat atgatcttcc tcagtttgtt tctcactctc agtcctatag atttccatct 1200

ggatgcttga ttcaggatat gcccaatgga tattccaagg ttacttgggt tgaacatatt 1260

gaaactgaag aaaaagaact ggttcatgag ctatacagag agattattca cagagggatt 1320

gcttttgggg ctgatcgttg ggttaccact ctccagagaa tgtgtgaaag atttgcttct 1380

ctatcggtac cagcgtcttc atctcgtgat ctcggtggag tgattctatc accggaaggg 1440

aagagaagca tgatgagact tgctcagagg atgatcagca actactgttt aagtgtcagc 1500

agatccaaca acacacgctc aaccgttgtt tcggaactga acgaagttgg aatccgtgtg 1560

actgcacata agagccctga accaaacggc acagtcctat gtgcagccac cactttctgg 1620

cttcccaatt ctcctcaaaa tgtcttcaat ttcctcaaag acgaaagaac ccgtcctcag 1680

tgggatgttc tttcaaacgg aaacgcagtg caagaagttg ctcacatctc aaacggatca 1740

catcctggaa actgcatatc ggttctacgt ggatccaatg caacacatag caacaacatg 1800

cttattctgc aagaaagctc aacagactca tcaggagcat ttgtggtcta cagtccagtg 1860

gatttagcag cattgaacat cgcaatgagc ggtgaagatc cttcttatat tcctctcttg 1920

tcctcaggtt tcacaatctc accagatgga aatggctcaa actctgaaca aggaggagcc 1980

tcgacgagct caggacgggc atcagctagc ggttcgttga taacggttgg gtttcagata 2040

atggtaagca atttaccgac ggcaaaactg aatatggagt cggtggaaac ggttaataac 2100

ctgataggaa caactgtaca tcaaattaaa accgccttga gcggtcctac agcttcaact 2160

acagcttga 2169

Described plant expression vector can be existing any plant expression vector.The promotor that starts transcription of foreign genes in the described plant expression vector is preferably cauliflower mosaic virus (CaMV) 35S promoter or figwort mosaic virus (FMV) 35S promoter or Arabidopis thaliana constitutive gene TUBULIN2 promotor.As, described AtEDT1 gene can import in the cotton by plant expression vector pCB2004, and described AtEDT1 gene can import in the cotton by plant expression vector pCB3000, and described AtEDT1 gene can import in the cotton by plant expression vector pCB3000T.In one embodiment, described cauliflower mosaic virus (CaMV) 35S promoter sequence is the sequence shown in the SEQ ID NO:3.In one embodiment, described Arabidopis thaliana constitutive gene TUBULIN2 promoter sequence is the sequence shown in the SEQ ID NO:4.Described resistance can be salt resistance and/or drought resistance and/or oxidation-resistance.

Sequence shown in the SEQ ID NO:3:

gattagcctt ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc 60

agcaggtctc atcaagacga tctacccgag caataatctc caggaaatca aataccttcc 120

caagaaggtt aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa 180

agatatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 240

caaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa 300

ggccatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 360

acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 420

ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 480

ggcaattgag acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc 540

agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 600

tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 660

tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 720

gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 780

ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 840

tatttttaca acaattacca acaacaacaa acaacaacaa cattacaatt actatttaca 900

attacaat

Sequence shown in the SEQ ID NO:4

tgcagagcaa atgagatcaa actgagagaa aatatcagag gaatataaaa aggaatcaca 60

aggagaaaga taagagaagg attacaagac atttgtgttt gggtggatga ggttttaaga 120

agagagtatc tcgttcatca aagttgtggt tatcgttatt tgacttgagg aagctatata 180

tctcttttgt tttttttcct ctaaatatct tgtgtgtata aaatacgaaa atcaactcaa 240

aacataagtt ttaacacatg ttttttttat attaaaaccc cataattata ttgttttttc 300

attgctttgc acttttttat tttccagaac tattttaccc tcaattaagt ttcaaatctc 360

attagacaca atccaattgt atttaaaatt ttaattataa agcacaccaa aaatatgatg 420

tggtcaagct ttatacaagg gccctttcct ttaaatatac tttacaatgt tttatttttg 480

gacaagatat tatcacccat aactttccta cctgatcaaa attatatcat attttaccat 540

ttcacttttc aatcaatgcc tatatatcca catttgcaaa atgatgaatg tatatacaaa 600

attgaagttg tggattgttt tgttttaggg aatctagttt acatatggag atgatgtcac 660

ttccaagaga gttggtacta tccaacgctc tctcttgttt ccaagatgtt tcgtctatcg 720

gttgcatcac ctgaactata cgcgagacgg tcccttttag actgtcccga atgctatatc 780

tatgttttca tgttgaacct cccagctaaa ctcagctggt atgttctcga ccggaaaaca 840

tcaggtccta gtacagctaa gagctaacaa tctcttggtg gctgttccct tgctccactt 900

acatagttat atgtctctac gttcaagctt agccgtcgtt ggttcaacaa tctttgtcat 960

gagtgcttgt ctgtattgct catggactgc ccatctaaca tgtgtgccac gcagatgact 1020

gtgtctagta gcatagaaaa aacttgtgtg taccccttga ttgtgtcgaa taaaacagaa 1080

agctcaaagt ttacatgaat tactaaattg tttctcttta aaatccttaa actatgtacc 1140

atatatgcat atgggattaa gataattact acgcaataat cgaagtctaa ttcagggaga 1200

ctaatccaag taattcacaa tcttgattaa gagatcattg gtagttagaa gtttcagtgc 1260

ataacagttt ctcaattggt caatgtattt actttattgt ttcggtcatc tccacaaggt 1320

cgtcaaacca tatacagata tactataaag tagtataaca caaccaattc cataaatctc 1380

ttttctcaat atgaaaaacg aactagatta actacaaatc aacaagagtt tggattaaaa 1440

caaaaacaaa ggagatttgg gaaagaagaa agtttatcat cagagaggag tgatcggtac 1500

atcaatcggc cacaaaaggc ctcagacgac cgtcgtgaga attttagatc aaatctcatc 1560

acttgaattt aacttcttca catcaaaaaa tcgattactt tagcaaagta aaacaatatc 1620

aaagatgaaa gctacaaaaa cacgaagaaa aaagagacga atatagccgg cgattatggg 1680

tactgaagac tagtttgcct ttttatatag gatgatggag caaatctagg gtttagtttg 1740

acaattaaag catacgacac cgtttatttt taaatgggct cgtctattta tctacatgtc 1800

tggcccatgt atgactttac tagatctagt tctgaaattg aaagttataa atttttgtat 1860

agattacttt cttcttcctt atttttaaca cacaatttat tatatgaaaa caatttaact 1920

acattaatga aaaaaataca attttttcaa taatacaatt ttgcataacg tgctcccata 1980

tctctactct acaactggga 2000

Described AtEDT1 gene can be building up in the existing plant expression vector with existing method, can add any promotor that comprises constitutive promoter, strengthens promotor, inducible promoter, tissue-specific promoter, etap specificity promoter before its transcription initiation Nucleotide.For the ease of identifying and screen turning described AtEDT1 gene plant cell or plant, can process employed carrier, as the antibiotic marker (gentamicin, kantlex etc.) that adds plant screening property mark (Bar gene, gus gene, luciferase genes etc.) or have resistance.Carry the expression vector of described AtEDT1 gene can be by using the conventional biological method converting cotton cell or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, agriculture bacillus mediated, pollen tube channel, and the cotton that transforms become plant through tissue cultivating, obtain the cotton plants that resistance improves.

Experiment showed, that cotton turns the drought tolerance of AtEDT1 gene strain (transformant) apparently higher than contrast (not transgene cotton) strain.Pore is an important determinative of arid tolerance because transpiration dries out, cotton turns the stomatal frequency of AtEDT1 gene strain blade lower epidermis and compares with contrast, reduced by 32%, the decrease of stomatal frequency, so that the Efficiency Decreasing that plant dries out from blade by transpiration can improve plant greatly to the hold facility of moisture.

Description of drawings

Fig. 1 is plasmid pCB2004 carrier collection of illustrative plates.

Fig. 2 is SacI single endonuclease digestion and the HindIII of pCB2004/AtEDT1, and the EcoRI double digestion is identified collection of illustrative plates.

Fig. 3 is plasmid pCAMBIA2300 carrier collection of illustrative plates.

Fig. 4 swimming lane 1-5 is that the EcoRI enzyme is cut pCB3000/AtEDT1, and swimming lane 6-10 is that the enzyme that the DraIII enzyme is cut pCB3000T/AtEDT1 is cut the evaluation collection of illustrative plates.

Fig. 5 is agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain.

The bacterium colony PCR of C58C1/pCB3000/AtEDT1 and agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain C58C1/pCB3000T/AtEDT1 identifies electrophorogram.

Fig. 6 is transgene cotton genetic transformation schema.

Fig. 7 is that transformant and acceptor contrast PCR detect electrophorogram.

Fig. 8 is that to make up that different transgenosiss isozygoty be the result that arid was processed 40 days to two kinds of pCB3000/AtEDT1 and pCB3000T/AtEDT1.

Fig. 9 is that to make up that different transgenosiss isozygoty be the result that salt stress was processed 60 days to two kinds of pCB3000/AtEDT1 and pCB3000T/AtEDT1.

Figure 10 is pCB3000/AtEDT1 transgenosis T2 for homozygous lines-122 result after the arid in various degree.

Figure 11 be pCB3000/AtEDT1 transgenosis T2 for the arid different time of homozygous lines-122 after the result of rehydration.

Figure 12 be pCB3000/AtEDT1 transgenosis T2 for the arid different time of homozygous lines-122 after the survival rate statistics of rehydration different time.

Figure 13 is that transformant pCB3000/AtEDT1T2 is for 200 times of light microscopic photos of homozygous lines-122 and acceptor contrast blade lower epidermis pore.

Figure 14 a is that transformant pCB3000/AtEDT1T2 is for the statistics of homozygous lines-122 and acceptor contrast stomatal frequency and epidermal cell density.

Figure 14 b is that transformant pCB3000/AtEDT1T2 is for homozygous lines-122 and acceptor contrast photosynthetic rate statistics relatively.

Figure 14 c is that transformant pCB3000/AtEDT1T2 is for homozygous lines-122 and acceptor contrast stomatal conductance statistics relatively.

Figure 14 d is that transformant pCB3000/AtEDT1T2 is for homozygous lines-122 and acceptor contrast efficiency of water application statistics relatively.

Figure 15 is that transformant pCB3000/AtEDT1T2 is for the light saturation curve measurement result figure of homozygous lines-122 and acceptor contrast R15.

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1, turn the cultivation of AtEDT1 gene cotton

One, the structure of transgene expression vector

1, the cDNA gene of AtEDT1 obtains

Utilize SuperScript ^TMIII Reverse Transcriptase test kit (Invitrogen company, Cat.No.18080-044) (can be from Arabidopis thaliana Biological resources center (ABRC from the environmental Arabidopis thaliana of Columbia, Arabidopsis Biological Resource Center) buy) in RT-PCR amplification obtain the cDNA gene that nucleotide sequence is the AtEDT1 of SEQ ID NO:1 in the sequence table, detailed process is as follows:

Trizol reagent is adopted in the extracting of total amount RNA, and (Invitrogen company Cat.No.15596-018), uses the total RNA of 1 microgram as the template of reverse transcription reaction, oligo d (T) ₂₅As the primer of reverse transcription, each reverse transcription reaction uses the SuperScript of 200 units ^TM(Invitrogen company Cat.No.18080-044) and according to ThermoScript II product operation instruction carries out the III ThermoScript II.RT-PCR the primer sequence is according to the sequences Design of AtEDT1 gene, 5 ' end primer is 5 '-atgagtttcgtcgtcggcgt-3 ', 3 ' end primer is 5 '-tcaagctgtagttgaagctgt-3 ', the PCR reaction uses 1 microlitre inverse transcription reaction liquid to be template, other carries out according to the operation instruction of the ExTaq enzyme of Takara company fully, annealing temperature is 56 ℃, and the extension time is 100 seconds, 30 circulations.Pcr amplification product separates by 1% agarose gel electrophoresis, reclaim and be cloned on the pMD18-T Vector carrier, sample presentation Shanghai is given birth to worker biotech firm and is carried out sequencing analysis, the result shows, this pcr amplification product has the nucleotide sequence of SEQ ID NO:1 in the sequence table, is the cDNA gene of AtEDT1.

2, the structure of transgene expression vector pCB2004/AtEDT 1, pCB3000/AtEDT 1, pCB3000T/AtEDT1

(1) structure of pCB2004

PCB2004 makes up as follows---

PCAMBIA3301 (CAMBIA) is digested with BstXI and SmaI, between the BstXI of pCAMBIA3301 and SmaI recognition site, insert by the BstXI of series connection successively, SstI, DraIII (a), AscI, Avr II, SwaI, DraIII (b), the multiple clone site fragment that the SmaI recognition sequence forms obtains recombinant vectors pCB2002.Wherein the SmaI recognition site is that following two synthetic polynucleotide: 5 '-AGCTCACGGGGTGGCGCGCCTAGGATTTAAATCACAAAGTGCCC-3 ' and 5 '-GGGCACTTTGTGATTTAAATCCTAGGCGCGCCACCCCGTGAGCTCATG-3 ' obtained 66 ℃ of annealing in 60 seconds.

With Gateway Conversion A test kit (Invitrogen, Gateway vector Conversion system, Cat.No.11828-019) in Conversion A obtain carrier is carrier pCB2003 by flat terminal the connection between the SmaI that is inserted into pCB2002 and the PmlI recognition site.Take pCAMBIA3301 (CAMBIA) as template, use primer: 5 ' end is 5 '-GGGCACGGGGTGGATTAGCCTTTTCAATTTCAGAAA-3 ', 3 ' end is 5 '-GGGCACTTTGTGATTGTAAATAGTAATTGT-3 ', obtain cauliflower mosaic virus (CaMV) 35S promoter sequence by pcr amplification, after cauliflower mosaic virus (CaMV) the 35S promoter PCR product usefulness DraIII restriction endonuclease digestion that obtains, directly be connected the DraIII enzyme to cut the linearized pCB2003 large fragment of digestion to carry out the T4 connection, obtain plasmid pCB2004 (carrier figure sees Fig. 1).

(2) structure of pCB2004/AtEDT1

The cDNA gene of the AtEDT1 that obtains in the step 1 is utilized GatewayR BP ClonaseTMEnzyme MixII test kit (Invitrogen company, Cat.No.11789-020) by GatewayTM recombinant clone technology, shuttle back and forth between the attR1 and attR2 of pCB2004, obtain containing the recombinant expression vector pCB2004/AtEDT1 of the cDNA sequence of AtEDT1 gene.

Utilize the SacI single endonuclease digestion to carry out the evaluation of positive colony, the result as shown in Figure 2, the SacI enzyme is cut carrier framework part and the size that pCB2004/AtEDT1 obtains 8427bp and is the fragment of 2944bp (the AtEDT1cDNA major part fragment that comprises 35S promoter part and 1990bp); Utilize HindIII and EcoRI double digestion to carry out the evaluation of positive colony, the result as shown in Figure 2, HindIII and EcoRI double digestion pCB2004/AtEDT1 obtain 10705 carrier framework part and size is the AtEDT1cDNA middle portion fragment of 666bp, and the cDNA gene of the presentation of results AtEDT1 that two kinds of enzymes are cut has been inserted between the attR1 and attR2 of pCB2004.Among Fig. 2, swimming lane 1 is the result of SacI single endonuclease digestion pCB2004/AtEDT1, and M is Fermentas DNA ladder (250bp-10Kb); Swimming lane 2 is the result of HindIII and EcoRI double digestion pCB2004/AtEDT1.

(3) pCB3000/AtEDT1 Vector construction

PCB3000/AtEDT1 makes up as follows---

On the pCB2004/AtEDT 1 plasmid basis that has obtained, upstream and downstream is introduced respectively SmaI and XbaI enzyme cutting site, the design primer, 5 ' end is 5 '-TCC CCCGGG GCGGACGTTTTTAATGTACTGA-3 ', 3 ' end is 5 '-TGC TCTAGA CCTGTCAAACACTGATAGTTTA-3 ', and pcr amplification obtains 35S promoter and adds AtEDT1cDNA part (purpose clip size 5043bp); After the PCR product that obtains digests with SmaI and XbaI enzyme cutting, with stand-by after the recovery of 30%PEG precipitation;

PCAMBIA2300 (CAMBIA) plasmid (carrier figure sees Fig. 3) comprises multiple clone site (EcoRI-SacI-KpnI-SmaI-BamHI-XbaI-SalI-PstI-SphI-HindIII), same with SmaI and XbaI restriction endonuclease two cut digest pCAMBIA2300 (CAMBIA) plasmid after, reclaim large fragment with the 30%PEG precipitation, cutting back to close rear stand-by PCR fragment with above-mentioned enzyme is connected by T4, between the SmaI of pCAMBIA2300 and XbaI site, insert and contain the fragment that cauliflower mosaic virus 35 S promoter sequence (SEQ ID NO:3) adds AtEDT1cDNA gene (SEQ ID NO:1), obtain recombinant expression vector pCB3000/AtEDT1.Utilize the EcoRI single endonuclease digestion to carry out the evaluation of positive colony, the result is shown in Fig. 4 swimming lane 1-5, and the EcoRI enzyme is cut carrier framework part and the size that pCB3000/AtEDT1 obtains 10132bp and is the fragment of 2061bp (the AtEDT1cDNA major part fragment that comprises part 35S promoter and 1074bp); Worker biotech firm is given birth in pCB3000/AtEDT1 sample presentation Shanghai check order, sudden change and frameshit phenomenon do not illustrate that the pCB3000/AtEDT1 construction of recombinant vector is correct.

(4) pCB3000T/AtEDT1 Vector construction

PCB3000T/AtEDT1 makes up as follows---

On the pCB3000/AtEDT1 plasmid basis that has obtained, 35S promoter is replaced as the promotor of Arabidopis thaliana constitutive expression gene TUBULIN2.The promoter sequence of selected TUBULIN2 gene is seen SEQ ID NO:4).The design primer adds respectively DraI II restriction enzyme site at two ends, 5 ' end primer is 5 '-TCCG cacggggtg tgcagagcaaatgagatcaa-3 '; 3 ' end primer is 5 '-TCCG cactttgtgtcccagttgtagagtagagat-3 '; Pcr amplification obtains the promoter sequence part (purpose fragment 2000bp) of Arabidopis thaliana TUBULIN2 gene; After the PCR product that obtains was cut digestion with the DraIII enzyme, the 30%PEG precipitation reclaimed stand-by;

Same with behind the DraIII endonuclease digestion digestion pCB3000/AtEDT1 plasmid, reclaim large fragment with the 30%PEG precipitation, cutting back to close rear stand-by PCR fragment with above-mentioned enzyme is connected by T4, between two DraIII sites of pCB3000/AtEDT1, excise 35S promoter (SEQ ID NO:3), add the fragment (SEQ ID NO:4) of the promoter sequence of TUBULIN2 gene, obtain recombinant expression vector pCB3000T/AtEDT1.The pCB3000T/AtEDT1 that builds is carried out the DraIII enzyme cut evaluation, obtain the carrier framework part of 11276bp and size for the fragment of 2009bp (SEQ ID NO:4), illustrate that the pCB3000/AtEDT1 construction of recombinant vector is correct.Among Fig. 4, swimming lane 6-10 is that the DraIII enzyme is cut the result who identifies pCB3000T/AtEDT1, and M is Fermentas DNA ladder (250bp-10Kb).

Two, agriculture bacillus mediated genetic transformation

1, contains the preparation of agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain of pCB3000/AtEDT1 and pCB3000T/AtEDT1

The preparation of C58C1 competent cell: agrobacterium tumefaciens (Agrobacterium tumefaciens) the bacterial strain C58C1 (D.J professor Oliver of Iowa State University is so kind as to give) of-70 ℃ of preservations is coated on LB (containing gentamicin 50mg/L) the solid ware, cultivates activation for 28 ℃.Get 4-5 single colony inoculation after 2 days in 3ml LB liquid nutrient medium (containing gentamicin 50mg/L) incubated overnight.The bacterium liquid of incubated overnight is inoculated in the LB liquid nutrient medium (containing gentamicin 50mg/L) of 500ml by 1: 100 volume ratio, 250rpm, is cultured to OD by 28 ℃ ₆₀₀Then the centrifugal collection thalline of=0.6,5000rpm uses isopyknic aseptic deionized water of precooling resuspended, repeats seven times, and thalline is resuspended in 10% glycerine of 0.5ml the most at last.

The pCB3000/AtEDT1 of above-mentioned preparation and pCB3000T/AtEDT1 plasmid electricity are transformed Agrobacterium C58C1: (agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1 competent cell is according to English process plate " Arabidopis thaliana laboratory manual " the .Detlef Weigel and Jane Glazebrook. .2004 of Chemical Industry Press March the 1st edition with 50ul agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1 competent cell respectively to get the pCB3000/AtEDT1 of 10ng and pCB3000T/AtEDT1 plasmid, the method preparation of the 1st printing) mix after, join (0.2cm in the electric shock cup of precooling, Invitrogen company, Cat no:P450-50).(specification sheets of instrument is: MicroPulser to use Agrobaterium (Agrobacterium) the electric shock program that carries on the Biorad company electroporation apparatus ^TMElectroporation Apparatus Operation Instruction and Applications Guide, Catalog NO:165-2100).Add the recovery of SOC liquid nutrient medium after the electric shock after 4 hours, coating contains the LB solid culture plate of gentamicin 50mg/L and kantlex 50mg/L, is inverted for 28 ℃ and cultivates two days.The fragment (primer sequence 5 ' end is 5 '-GCA ACA TGA GAG AGC AGA TAA TA-3 ', and 3 ' end is 5 '-TCA AGA AGC AAA TCT TTC ACA CAT T-3 ') of getting in single bacterium colony amplification AtEDT1 gene is carried out bacterium colony PCR checking.It is the bacterial strain that contains pCB3000/AtEDT1 that PCR obtains agrobacterium tumefaciens (Agrobacterium tumefaciens) the bacterial strain C58C 1 that the pCB3000/AtEDT1 of the Gene Partial fragment band of 1091bp transforms, called after agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000/AtEDT1.It is the bacterial strain that contains pCB3000T/AtEDT1 that PCR obtains agrobacterium tumefaciens (Agrobacterium tumefaciens) the bacterial strain C58C1 that the pCB3000T/AtEDT1 of the AtEDT1 Gene Partial fragment band of 1091bp transforms, called after agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000T/AtEDT1.The bacterium colony PCR qualification result of agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000/AtEDT1 and agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000T/AtEDT1 as shown in Figure 5, swimming lane 1-8 is the PCR result of agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000/AtEDT1, swimming lane 9-14 is the bacterium colony PCR result of agrobacterium tumefaciens (Agrobacterium tumefaciens) C58C1/pCB3000T/AtEDT1, all obtain the band of 1091bp AtEDT1 Gene Partial fragment, M is Fermentas DNA ladder (250bp-10Kb).

2, the genetic transformation of cotton

The concrete grammar of genetic transformation is with reference to the method for (2007) such as (1994) such as Chen Zhixian and Wu Shenjie, and the implementation step is as follows:

2.1 aseptic seedling preparation

Cotton seeds (come from regeneration that academy of agricultural sciences, Shanxi Province Cotton Research Institute oneself cultivates isozygoty strain R15 and WC) is with drying after vitriol oil stoste (general 98%) lint, then use 75% ethanol surface disinfection 10-30 after second, outwell ethanol, disinfectant with hydrogen peroxide 1-2 with 15% hour, then outwell hydrogen peroxide, behind aseptic water washing 3 times, be immersed in the sterilized water 18-24 hour, by the time seed shows money or valuables one carries unintentionally; Peel off the seed skin under aseptic condition, be inoculated on the seedling substratum, (Fig. 6 a) in 5 days in 25 ℃～28 ℃ cultivations.

2.2 Agrobacterium is cultivated

(1) Agrobacterium activation: the Agrobacterium of carrying goal gene (C58C1/pCB3000/AtEDT1 and C58C1/pCB3000T/AtEDT1) of above-mentioned conversion is inoculated in the LB liquid nutrient medium, and 28 ℃ of shaking culture are spent the night OD ₆₀₀For subsequent use during for 0.3-0.7.

(2) rule at LB solid medium (containing 50mg/L kantlex and 50mg/L gentamicin) with the agrobacterium liquid of above-mentioned overnight incubation, cultivated 48 hours for 28 ℃.

(3) picking 3-5 single bacterium colony, overnight incubation in the LB liquid nutrient medium that contains 50mg/L kantlex, 25mg/L Rifampin, (OD when treating that bacterial strain enters logarithmic phase ₆₀₀Be about 2.0), with the common culture medium of liquid bacterium liquid is diluted to OD ₆₀₀For subsequent use during for 0.3-0.5.

2.3 infect and be total to cultivation

The hypocotyl of cotton aseptic seedlings is cut into segment about 0.5-0.7cm with knife blade in the sterilization culture dish of super clean bench, dilution bacterium liquid soaks and infects the hypocotyl section that cuts after 7-8 minute, blot bacterium liquid unnecessary on the plumular axis section with sterilization filter paper, be placed on the common culture medium that is covered with one deck sterilization filter paper, with the sealed membrane sealing, cultivate altogether 2 days (Fig. 6 b) for 22 ℃～25 ℃.

2.4 resistant calli is induced, propagation and succeeding transfer culture

Hypocotyl section after cultivating altogether is inoculated in the resistant calli inducing culture, and 30 days subcultures once.Inoculate for the first time and can observe hypocotyl about 10 days and cut off both sides and expand to some extent, have a small amount of callus to grow (Fig. 6 c) on the 20-30 days tangent planes.Treat that callus lines comparatively soft about diameter 0.5cm (Fig. 6 d) appears in the hypocotyl two ends, with tweezers the two ends callus is peeled to change over to identical substratum with scalpel and separately cultivate, can effectively remove the Agrobacterium pollution and prevent the callus browning phenomenon.

2.5 inducing of embryo callus

The callus diameter reaches 1-3cm on the resistant calli inducing culture, with its subculture in the new calli induction media of withdrawing from hormone and additional saltpetre, between cultivating, cultivate under the normal condition, left and right sides subculture identical substratum once every other month, the embryo callus subculture of the thin grain of rice shape of yellow-green colour or greyish-green begins in the bottom of callus piece and begins appearance (Fig. 6 e) on every side after 2-4 time.

2.6 regeneration

Choosing embryo callus changes in the embryo's maturation and germination medium that spreads filter paper, 0.1-0.2g/ bottle (Fig. 6 f), further form embryoid (Fig. 6 g), the embryoid of sprouting grows to (Fig. 6 h) about hypocotyl 1cm, choose and be inserted into continued growth on the Regenerated plantlet growth medium, part can grow up to very soon and is the normal plantlet of root, stem and leaf (Fig. 6 i), grows to 3-5 sheet true leaf or long to 3-5cm high (Fig. 6 j) is arranged more than the epicotyl.Get plantlet 2-3 sheet leaf under the aseptic condition, method is extracted DNA in a small amount, can be used for the follow-up pcr amplification that carries out marker gene and goal gene and detects, and positive plantlet top 3-5cm cuts, and does scion for grafting.

Three, the evaluation of transformant

1, the PCR of transformant identifies

Get Regenerated plantlet 2-3 sheet tender leaf on the super clean bench, CTAB method (Bushra et al.1999) is extracted transformant (turning the transgenic cotton plant of pCB3000/AtEDT1 or pCB3000T/AtEDT1) and contrast (not genetically modified cotton acceptor material R15 or WC) genomic dna, is used for PCR and identifies.Concrete operations are as follows:

(1) get Regenerated plantlet 2-3 sheet tender leaf on the super clean bench, add freshly prepared Extraction buffer 600 microlitres (prescription sees the following form 1) of precooling, electricity turns grinding.4 ℃, centrifugal 20 minutes of 5000rpm abandons supernatant;

Table 1 cotton DNA Extraction buffer prescription

(2) add lysis buffer (prescription sees the following form 2) 600 microlitres of 65 ℃ of preheatings in the precipitation, 65 ℃ of water-baths 30 minutes, during upset mixing 2-3 time;

The preparation of table 2 lysis buffer

Annotate: above solution is limpid, and extracting solution is in 4 ℃ of preservations, lysis buffer room temperature preservation.Press before using and add β-ME (beta-mercaptoethanol) at 1: 100.

(3) add 800 microlitre chloroforms: primary isoamyl alcohol (volume ratio 24: 1) mixed solution, overturn more than 50 times 15 ℃, centrifugal 20 minutes of 9000rpm, supernatant is changed in the centrifuge tube of 1.5ml, add the Virahol of 0.6 times of volume precooling, slowly overturn 30 times, mixing, left standstill 10 minutes, centrifugal 10 minutes of room temperature 9000rpm abandons supernatant, the washing with alcohol that in precipitation, adds 1ml 70%, centrifugal 2 minutes of 1000rpm.Outwell supernatant liquor, air seasoning 20 minutes;

(4) add 30 microlitre TE damping fluids (PH 8.0 for 10mM Tris-HCl, 1mM EDTA), dissolving genomic dna (4 ℃, more than 30 minutes); Preserve.

2, the PCR of regeneration plant detects

Do template with the 100ng genomic dna, the Partial Fragment of amplifying target genes AtEDT1, primer sequence are that 5 ' end is 5 '-atgagtttcgtcgtcggcgt-3 '; 3 ' end is 5 '-gtctgaagaagcctgagcaat-3 ', and PCR obtains the positive plant of the AtEDT1 gene fragment band of 752bp.

PCR reaction system (25 μ l system): archaeal dna polymerase Ex Taq (5U/ μ l) 0.25 μ l, 10 * ExTaq buffer, 2.5 μ l, dNTPs (four kinds of dNTP, every kind of each 10mM) 2 μ l, P ₁(10 μ M) 1 μ l, P ₂(10 μ M) 1 μ l, genomic dna template 1 μ l, ddH ₂O 17.25 μ l.

PCR response procedures: 95 ℃ of denaturation 5min of elder generation; Then 94 ℃ of sex change are 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ 30 seconds, 35 circulations; 72 ℃ are incubated 10min again; Last 4 ℃ of preservations.Pcr amplification product detects by 1% agarose gel electrophoresis, the result as shown in Figure 7, the transformant strain can amplify the AtEDT1 gene cDNA Partial Fragment of 752bp, and contrast is failed to expand and respective strap.Among Fig. 7, M is DL 2000 molecular weight Marker; 1-11 is regeneration plant; 12 negative plant; 13,14 are acceptor contrast R15; 16 is the plasmid positive control.Wherein, that change among the 1-5 is pCB3000/AtEDT1, and that change among the 6-11 is pCB3000T/AtEDT1.

After most of transfer-gen plant extracts DNA, all done the PCR positive detection, the statistics of the strain of grafting survival and PCR positive detection sees the following form 3.

Table 3 transgenic positive detection statistics table

2, the grafting of regeneration plant and T0 gather in the crops for seed

In being housed, vermiculite (1: the 30) basin of having mixed composite fertilizer (N, P, K each 15%) plants the sea island cotton kind (disease-resistant, plant is vigorous) of upper well developed root system, strong stress resistance, when treating cotton seedling 3-5 sheet true leaf stand-by (as rootstock seedling).With cleft graft and cortex grafting method transgenosis is regenerated the cotton plant grafting to rootstock seedling.Can receive 50-70%T in 6-10 month after the grafting ₀For the seed of plant, do not receive that the plant of seed is eliminated.

Embodiment 2, the drought resistance that turns AtEDT1 gene cotton and salt resistance detect

The selection of 1, isozygotying and being

The individual plant of results seed is proceeded offspring's the seed selection of isozygotying, and T1 is for plant in plantation, carries out after field resistance detects with the kantlex spraying of 4000mg/L, and the positive goal gene PCR that proceeds detects, and keeps pair positive plants; Carry out equally the plant plantation in T2 generation and detect, the kalamycin resistance of transfer-gen plant detect and the PCR detection all unseparated plant isozygoty as the candidate who has obtained this transformation event and be, can obtain isozygotying of Partial Conversion event in this generation and be.There is not the work of the transformation event continuation repetition previous generation that obtaining isozygotys is.With the plantation of continuous two generations, plant is unseparated to be regarded as to isozygoty and is.In April, 2011 is at the Yang Bao farm plantation T of Shanxi cotton institute ₁With T2 for plant, T2 identifies and the PCR detection through kalamycin resistance for plant, is not separated into standard with plant, having obtained tentatively that 4 pCB3000/AtEDT1 isozygoty is to isozygoty with 3 pCB3000T/AtEDT1 to be.

2, transformant drought resistance and salt resistance are identified

2.1pCB3000/AtEDT1 the drought resisting salt resistance detected result that transgenosiss different from pCB3000T/AtEDT1 are isozygotied and are

In the greenhouse potted plant growth each 3 homozygous lines of pCB3000/AtEDT1 and pCB3000T/AtEDT1 and 2 acceptor R15 and WC high frequency regeneration to isozygoty be that every is 3 basins, 10 seeds of every basin.Block that Resistance Identification during 3 true leaves, pull out negative plant, every basin stays 1 strain after the seedling thinning, is used for carrying out transfer-gen plant drought resistance and salt resistance detection during 7 true leaves.

Drought resisting begins to cut off the water supply when processing cotton plants from stem 7-8 leaf, continued growth was taken pictures after 40 days, the results are shown in Figure 8, acceptor contrast R15 blade major part is curling, withered, it is-122 that 3 kinds of transformant pCB3000/AtEDT1 transgenosis T2 generation isozygoty, and-119 ,-74 performances are consistent, the attitude continued growth (Fig. 8 a-c) that still keeps standing upright of most of blade is that-122 drought-enduring phenotype is the strongest especially to isozygoty.It is-39 greener than acceptor contrast R15 blade that transformant pCB3000T/AtEDT1 transgenosis T2 generation isozygotys, more smooth (Fig. 8 d), transformant pCB3000T/AtEDT1 transgenosis T2 is-14 for isozygotying, the attitude continued growth (Fig. 8 e-f) that still keeps standing upright of-44 most of blades, acceptor contrast WC blade is substantially withered.In sum, 6 of pCB3000/AtEDT1 and two kinds of structures of pCB3000T/AtEDT1 to isozygoty be all to show the drought-resistant character stronger than acceptor contrast.

Salt tolerant is identified and is begun to water 2%NaCl (mass percent concentration 2g/100ml from cotton plants stem 7-8 leaf equally, be equivalent to 0.342M) solution, 2～3 days once, each 500 milliliters, continued growth was taken pictures after 60 days, the results are shown in Figure 9, acceptor contrast R15 blade has been wilted withered substantially, it is-122 that 3 kinds of transformant pCB3000/AtEDT1 transgenosis T2 generation isozygoty, the attitude continued growth (Fig. 9 a-c) that still keeps standing upright of-119 ,-74 most of blades; Acceptor contrast R15 blade is withered, transformant pCB3000T/AtEDT1 transgenosis T2 is that-39 blades also keep deployed condition (Fig. 9 d) for isozygotying, transformant pCB3000T/AtEDT1 transgenosis T2 is-14 for isozygotying, the attitude continued growth (Fig. 9 e-f) that still keeps standing upright of-44 most of blades, the most of blade of acceptor contrast WC blade is curling, withered.

In sum, 6 of pCB3000/AtEDT1 and two kinds of structures of pCB3000T/AtEDT1 to isozygoty be all to show the salt resistance shape stronger than acceptor contrast.

2.2pCB3000/AtEDT1 transgenosis T2 is for homozygous lines-122 arids-rehydration experiment

PCB3000/AtEDT1 transgenosis T2 is soaked seed an evening (approximately 16 hours) with hot water (about 40 degree) for the cotton seeds of homozygous lines-122 and the seed of acceptor contrast R15 thereof, from water, pull out, drain, be seeded into that (the flowerpot size is identical in the flowerpot that soaks saturated moisture content soil, the soil that every basin fills is weighed, soil weight is identical, guarantee the growing environment term harmonization) as far as possible, approximately 2 centimetres of the planting seed degree of depth, cover with plastic film after sowing is good, can emerge the rear film of in time raising of emerging in 2 days.Transformant and contrast be every potted plant strain all, does not after planting rewater, and carries out drought stress and processes.Figure 10 shows that pCB3000/AtEDT1 transgenosis T2 is for homozygous lines-122 and the acceptor contrast R15 situation after the arid in various degree, top 3 basins of parallel placement are transformant pCB3000/AtEDT1 transgenosis T2 for homozygous lines-122 among the figure, below 3 basins be acceptor contrast R15.Drought stress is in the time of 18 days, and the difference of contrast and transformant plant is little, all goes back normal growth, and not wilting, (Figure 10 a); Drought stress 22 days, contrast most of blade curling, it is withered and yellow, dead that lower blade begins, and only has a small amount of blade to keep deployed condition, and transformant is the outermost layer blade begin curling, the attitude continued growth (Figure 10 b) that still keeps standing upright of most of blade; After drought stress 24-27 days, the contrast blade is almost all curling withered and yellow, the redgreen blade, although the transformant blade has also occured curlingly because of dehydration, plant leaf still be green (Figure 10 c-f).

Behind the drought stress different time, continue transformant pCB3000/AtEDT1 transgenosis T2 is carried out rehydration for homozygous lines-122 and acceptor contrast R15, observe form, the statistics survival rate.After Figure 11 a-f is arid 28 days, the situation of rehydration different time; After Figure 11 g-l is arid 29 days, the situation of rehydration different time.Top 2 basins of parallel placement are transformant pCB3000/AtEDT1 transgenosis T2 for homozygous lines-122 among Figure 11, below 2 basins be acceptor contrast R15.

After the rehydration 2 days, the acceptor contrast does not all have the plant survival, but transformant blade major part all begins (Figure 11 b-d that again stands upright; H-j); Rehydration 4 days and 6 days has more transformant blades to stand upright, and whole strain plant recovers the growth conditions before the drought stress substantially, also has indivedual individual plants dead, and contrast does not still have survival (Figure 11 e; K).After 28 days, the plant of rehydration different time carries out the survival rate statistics with drought stress, and statistics shows, rehydration is in the time of 9 days, and contrast is survival not, and transformant has 87.1% survival, rehydration 21 days and 38 days, contrast be not survival still, and (Figure 12 a) for transformant survival rate 87.1%; After 29 days, the plant of rehydration different time carries out the survival rate statistics with drought stress, and statistics shows, rehydration is in the time of 8 days, and contrast is survival not, and transformant has 62.5% survival, rehydration 20 days and 37 days, contrast be not survival still, and the transformant survival rate keeps 62.5% (Figure 12 b).Comprehensive above-mentioned arid-rehydration experimental result shows, pCB3000/AtEDT1 transgenosis T2 obviously shows than the stronger drought-resistant character of acceptor contrast for homozygous lines-122.

Embodiment 3, the associated biomolecule that turns AtEDT1 gene cotton are learned character observation

To soak seed an evening with hot water (about 40 degree) for the seed of seed and acceptor contrast R15 thereof through the T2 of the positive pCB3000/AtEDT1 transformant pure lines-122 of the PCR of embodiment 1 qualification result, from water, pull out, drain, be seeded into that (the flowerpot size is identical in the flowerpot that soaks saturated moisture content soil, the soil that every basin fills is weighed, soil weight is identical, guarantee the growing environment term harmonization) as far as possible, approximately 2 centimetres of the planting seed degree of depth, cover with plastic film after sowing is good, can emerge the rear film of in time raising of emerging in 2 days.Whenever potted plant 1 strain after seedling survives also vigorous growth fully, is chosen the good and consistent transformant of growth conditions and is contrasted respectively 40 strains and carry out the observation that associated biomolecule is learned index.

1. Stoma of Leaves density and epidermic cell number are observed

1) chose normal growth 40 days, the same position (cotyledon is not counted, from the bottom up several the 5th leaves) of the transformant that growth conditions is consistent and the same leaf of contrast position;

2) on slide glass, evenly smear permanent solid 502 super glue, selected cotton leaf zone lower epidermis is close on the slide glass that scribbles glue, firmly press;

3) after the glue solidifies, blade is peeled off slide glass, cotton leaf lower epidermis pore is namely opened up in slide glass;

4) microscopically is observed pore, averages in each 20-22 visual field of rubbing counting; Calculate each visual field area according to micrometer, calculate Stoma of Leaves density (individual/mm ²);

5) when the measurement and record of stomatal number, recorder chrotoplast number simultaneously.According to pore mean value, every Zhang Tuo's sheet is got 10 visual field countings, averages.

A and b are respectively 200 times of light microscopic photos of transformant and contrast blade lower epidermis pore among Figure 13, and wherein a is transformant pCB3000T/AtEDT1T2-122 pure lines lower epidermis pores (200 times), and b is acceptor contrast R15 lower epidermis pore (200 times).Be easy to find out that from 200 times of pore light microscopic photos contrast and transformant there are differences in stomatal frequency, after at least 50 visual field pore quantity of statistics, calculate stomatal frequency and do statistical study, find all have significant difference (p＜0.01) (Figure 14 a on the quantity of pore He on the epidermal cell density between transformant and the contrast, table 4), because the cotton leaf pore is difficult to observe, therefore can only pore be opened up to get off to observe with the method for rubbing, record, so just can't observe and cartogram chrotoplast changing conditions, also be difficult to calculate stomatal index.

The different strain stomatal frequencies of table 4 and epidermal cell density

2, the mensuration of transformant Photosynthetic Index

Choose the consistent transformant pCB3000/AtEDT1T2 of growth conditions for pure lines-122 and acceptor contrast R15 blade, measure photosynthetic rate (the μ mol CO2/m of identical leaf position ²S), efficiency of water application (mg/g), stomatal conductance (mmol/m ²S) and the light saturation curve, instrument is the photosynthetic instrument of LICOR 6400XT of U.S. LiCOR company.Seedling during mensuration in every basin all must be measured, the measurement result of statistics transformant and each 40 strain of acceptor contrast.

Statistical result showed, the T2 of transformant pCB3000/AtEDT1 is 7.98 μ mol CO2/m for the photosynthetic rate of pure lines-122 ²S is a little less than the photosynthetic rate 8.58 μ mol CO2/m of acceptor contrast R15 ²S (Figure 14 b); The stomatal conductance of acceptor contrast R15 is 0.1mmol/m ²S is than the stomatal conductance 0.06mmol/m of transformant ²S wants high (Figure 14 c), and difference has reached conspicuous level (p＜0.01).The T2 of transformant pCB3000/AtEDT1 is 7.68mg/g for the moisture content utilization ratio of pure lines-122, and than the efficiency of water application 6.21mg/g high (Figure 14 d) of acceptor contrast R15, difference has also reached conspicuous level (p＜0.01).

In addition, light saturation curve (Figure 15) result shows that the T2 of transformant pCB3000/AtEDT1 is for being sheerly-122 at 1000-1500 μ mol photons m ^-2s ^-1High illumination condition under, still can keep being significantly higher than the photosynthetic rate of contrast, the photosynthetic rate of acceptor contrast R15 is subject to obvious inhibition, illustrate that cotton turns AtEDT1 gene transformation body plant and resists high light and suppress to have certain resistance, and this is that farm crop are improved desired proterties.

Claims (10)

1. a method of cultivating the resistant transgenic cotton is that Arabidopis thaliana AtEDT1 gene is imported in the cotton by plant expression vector, and screening obtains the cotton that resistance improves;

Described AtEDT1 is following (a) or protein (b):

(a) protein that is formed by the amino acid residue sequence of sequence in the sequence table 2;

(b) with the amino acid residue sequence of sequence in the sequence table 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have with (a) identical activity by (a) derivative protein.

2. method according to claim 1, it is characterized in that: the encoding sequence of described AtEDT1 gene is the SEQ ID NO:1 in the sequence table.

3. method according to claim 2 is characterized in that: the promotor that starts transcription of foreign genes in the described plant expression vector is cauliflower mosaic virus 35 S promoter or radix scrophulariae mosaic virus 35 S promoter.

4. method according to claim 3 is characterized in that: described AtEDT1 gene imports in the cotton by plant expression vector pCB2004 and pCB3000.

5. method according to claim 4 is characterized in that: between the attR1 and attR2 of described AtEDT1 gene insertion plant expression vector pCB2004 and pCB3000.

6. method according to claim 2 is characterized in that: the promotor that starts transcription of foreign genes in the described plant expression vector is the promotor of Arabidopis thaliana constitutive expression gene TUBULIN2.

7. method according to claim 6 is characterized in that: described AtEDT1 gene imports in the cotton by plant expression vector pCB3000T.

8. method according to claim 7 is characterized in that: between the attR1 and attR2 of described AtEDT1 gene insertion plant expression vector pCB3000T.

9. arbitrary described method in 8 according to claim 1, it is characterized in that: described resistance is salt resistance and/or drought resistance and/or oxidation-resistance.

10. arbitrary described method in 8 according to claim 1, it is characterized in that: described cotton is cotton acceptor material R15 or WC.

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