WO2014205596A1 - Atp hydrolase atpase-2 from cotton and coding gene and use thereof - Google Patents

Abstract

Provided are a vegetable protein, the coding gene and use thereof. Especially provided are an ATP hydrolase derived from cotton and the coding gene thereof, and the use thereof in cultivating a genetically modified plant with an improved salt tolerance.

Description

 Cotton ATP hydrolase ATPase-2 and its coding gene and application

TECHNICAL FIELD The present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived ATP hydrolase ATPase-2 and its encoding gene, and its use in the cultivation of transgenic plants with improved salt tolerance.

BACKGROUND OF THE INVENTION Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Salted soil is usually dominated by sodium salt, calcium salt or magnesium salt, and is a major factor affecting plant growth and causing food and economic crop yield reduction. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland. Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance. The selection of plant resistance to salt and alkali, drought-tolerant ability and plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land. For most crops, most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na ^{+ in the} soil will be plant Normal growth metabolism produces toxic effects. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.

The salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules. Scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the higher model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, making breakthroughs in research in this field ( Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53 : 1247- 1273 ; Zhang ZL. 201 1. Arabidopsis Floral Initiator SKB 1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing Through Altering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cell, 23 : 396-41 1 ). Higher plant cells can sense changes in physicochemical parameters in the external environment through various pathways, thereby converting extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions, activating transcription factors, and being activated. The transcription factor acts on the functional gene and initiates the expression of the stress response gene to increase the tolerance of the plant. Although researchers have conducted a large number of studies from different sides, due to the complexity of the mechanism, many important issues in plant salt resistance remain to be explored. For example, the key to plant salt resistance The factor has not been found; the molecular mechanism of plant salt tolerance is not very clear ₍

SUMMARY OF THE INVENTION The present inventors cloned a coding gene for an ATP hydrolase (designated herein as ATPase-2) of cotton using SSH (Suppression Subtractive Hybridization) in combination with RACE (rapid amplification of cDNA ends), and determined its DNA sequence. Moreover, it was found that the transgenic plants can significantly improve the salt tolerance of the transgenic plants after they are introduced into the plants by transgenic technology, and these traits can be stably inherited.

 The first aspect of the present invention provides an ATP hydrolase ATPase-2 encoding gene (designated herein as GhA TPase-2) of cotton, the sequence of which is SEQ ID NO: 2.

A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, and the nucleotide sequence of the gene is The expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300 _; preferably, the recombinant expression vector is the 35 S-GhATPase-2-2300 vector shown in Figure 2.

 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.

 A fourth aspect of the invention provides a method for improving salt tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene Preferably, the plant is Arabidopsis thaliana.

 A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Preferably, the plant is Arabidopsis thaliana.

 A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving salt tolerance of a plant and for use in plant breeding Use; Preferably, the plant is Arabidopsis thaliana.

A seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is set forth in SEQ ID NO: 1. DRAWINGS

Figure 1 shows the construction of the plant expression vector (35S-GhATPase-2-2300) of the GhATPsae-2 gene (Fig. la-lb).

 Figure 2 is a plasmid map of the plant expression vector (35S-GhATPase-2-2300) of the GhATPase-2 gene.

 Fig. 3 shows the results of salt tolerance test of T1 Arabidopsis thaliana plants transgenic with GhATPase-2 gene. Tli5 showed significant salt tolerance, and the results of Tlil3 and T1U6 were similar thereto, and are not shown here.

 Figure 4 shows the results of molecular level detection of the transcription level of GhATPase-2 gene in 1\ generation transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. M is DNA Ladder Marker ( DL2000 ), 1-8 is salt-tolerant T1 transgenic Arabidopsis plants (Tli5, Tlil3, Tlil6, respectively), 9 is a plasmid PCR positive control (35S-GhATPase-2-2300) Plasmid), 10-13 is a non-transgenic control Arabidopsis plant.

BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

 The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1. Cotton SSH library construction under salt stress:

 The specific method is:

A method according to PCR-select ^TM cDNA Clontech kit Subtraction Kit's instructions shown by suppression subtractive hybridization libraries constructed SSH (suppression subtractive library). The mRNA extracted from the cotton root tissue treated with salt was used as a sample (Tester) during the experiment, and the mRNA extracted from the untreated cotton root tissue was used as a control. Specific steps are as follows:

 (1) Test materials:

African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) Seeded on sterilized vermiculite, cultured at 25 °C, light dark cycle 16h/8h, poured 1 per week /2MS medium ( 9. 39 mM KN0 ₃ , 0. 625 mM KH2P0 ₄ , 10. 3 mM NH ₄ N0 ₃ , 0.75 mM MgS0 ₄ , 1. 5 mM CaCl ₂ , 50 μ M KI , 100 μ MH ₃ B0 ₃ , 100 μ M MnS0 ₄ , 30 μ M ZnS0 ₄ , 1 μ M Na ₂ Mo0 ₄ , 0. 1 μ M CoCl ₂ , 100 μ M Na ₂ EDTA, 100 μ M FeS0 ₄ ) once. It was used for experiments when the seedlings were as long as 25-30 cm.

 (2) Material handling:

 The test seedlings were divided into 2 groups of 4 plants each. The first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium. The second group was treated with 25 °C, light culture, placed in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treated for 6 hours. After the treatment, the roots of the two groups were cut out in time. After rapid freezing with liquid nitrogen, it was stored in a -70 °C refrigerator.

 (3) Total RNA extraction:

0.5 g of cotton roots of the control and salt treatment groups were taken, and total RNA was extracted using a plant RNA extraction kit (purchased from Invitrogen). The absorbance of total RNA at 260 nm and 280 nm, OD ₂₆ , was determined using a HITACHI UV spectrophotometer U-2001. /OD ₂₈ . The ratio of 1.8-2.0 indicates that the total RNA purity is high. The integrity of total RNA is detected by 1.0% agarose gel electrophoresis. The brightness of the 28S band is about twice that of the 18S band, indicating that the RNA integrity is good. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

The method according to Clontech's PCR-select ^TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization. Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and 2 μg of Tester cDNA and 2 μg of Driver cDNA were used as starting materials for subtractive hybridization. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. The products of the two first forward subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed genes were amplified by two inhibitory PCR amplifications (PCR). Before, the second forward subtraction hybridization product was end-filled).

 (5) Construction and preliminary screening, cloning and identification of subtractive libraries

The second inhibitory PCR amplification product of the second forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) according to the instructions of the pGEM-T Easy kit (purchased from Promega) The specific steps are linked to the pGEM-T Easy vector as follows: The following components are sequentially added to the 200 μl PCR tube: Purified combined positive subtractive hybridization cDNA fragment of the second inhibitory PCR product 3 μ 1 , 2 X T4 DNA ligase buffer 5 μl, pGEM-T Easy vector 1 μl, Τ4 DNA ligase 1 μl, ligated overnight at 4 °C. Then take 10 μ ΐ to connect the reaction product and add to the 100 μ 1 competent colon Bacillus JM109 competent cells (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, then add 250 μl of LB liquid medium (containing 1% tryptone (purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Sinopharm) were placed in a 37 ° C shaker, shaken at 225 rpm for 30 minutes, and then 200 μl of the bacterial solution was taken therefrom. Inoculated with 50 μg/ml ampicillin, 40 Qg/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), 24 g/mL IPTG (isopropyl- β-D-thiogalactopyranoside (X-gal/IPTG was purchased from TAKARA) LB (ibid.) was cultured on a solid plate for 18 hours at 37 °C. Count the clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 300 white colonies (number: Gh-S2-001 to Gh-S2-300). The white colonies picked were inoculated into LB liquid medium (same as above) containing 50 μg/ml ampicillin in 96-well cell culture plate (CORNING), and cultured overnight at 37 °C, and glycerol was added to the final concentration of glycerol. 20% (by volume), then keep at -80 °C

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use. Nested PCR primers Primer 1 and Primer 2R (PCR-select ^TM cDNA Subtraction Kit from Clontech) were used to verify the bacteriological activity of the cultured bacteria.

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Sex clones, and then all positive clones were sent to Yingjie Jieji (Shanghai) Trade r, Ltd. for sequencing.

 (6) cDNA sequencing analysis of differential clones:

 After the DNA sequencing results were removed from the vector and the ambiguous sequence and the redundant cDNA i.- , a total of 203 Expressed sequence tags ( EST ) ( Unigene ) were obtained .

 I.- Example 2 Cotton ATP hydrolase gene GhATPase-2 clone

 After the cloned from the colony Gh-S2-176 in the identified cotton SSH library was detached from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the ATP hydrolase. In this paper, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhATPase-2, and its corresponding protein was named ATPase-2 o.

 SEQ ID No: 3

 1 ACTGTTTATA AGCCAAACTC TAGATCACTT ΓΡ ΠΑΑΑΤΤΤ CTGGTAGTCC AACTGAGAAA

61 CTTGGGCTGT CTCAGATTTG GGGATGAACC TAGATGACCC AAAGCAAAAC

121 TACGAACTCA TCCAAGTAGA TCAGAGAAGA AGAGGAGTGG AGTTCTGATA

181 GTGAGAGTGG CGGTGCTACA GGA GGGTGC TGCTGAGATG

241 ATCTTGGCCA ATATTATGAT AGAAGTGGTG TAGTT ^GC CATTGACGAG

301 TAGAGATGGG GAGGATATGG CAGCCAAAAG

 361 ATAGCATTTG CACACACACA AAATCCAAAA GACAATGAAC GAGTTTTGCA AGA^ GTGGA

421 CTCATCTTGC TAGGGCTGGT AGGTTTGA^A CAGAAAAGCA

481 GTAGAAGGTT GCATAAATGC ATCAAGAT A CAATATCTTC

541 GTATAGCAAG CGAATGAGGA TTTGAGGGAA 601 GCTGTAATAG AAGGTGT

Cloning of the full-length coding gene of GhATPase-2

 Based on the sequence of SEQ ID No: 3 that has been obtained, the following two specific primers were designed as the 5'-end specific primer for 3' RACE.

 GhATPase-2 GSP1 : SEQ ID No: 4:

 GTAGAAGCTT GCATAAATGC TG GhATPase-2 GSP2: SEQ ID No: 5:

 ACAGCAAAGG CTATAGCAAC TGA

 The experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).

 The first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal primer AUAP (provided with the kit), and the cDNA obtained by reverse transcription of the mRNA extracted from the salt-treated group was used as a template. Specific steps are as follows:

 50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primers SEQ ID NO: 4 and AUAP Each 2.0 μ ΐ and 35 μ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 2 minutes), 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:

 50 yl PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 5 and P AUAP Each of 2.0 μ 1 and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

A 1300 bp fragment (Gel Extraction Kit from OMEGA) in the second round of PCR product was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method as above) The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin and 40 μgmL X-gaK 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. . Using SEQ ID NO: 5 with the universal primer AUAP The PCR amplification of the bacterial solution confirmed that 4 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained.

 Based on the obtained GhATPase-2 gene fragment, the following three specific primers were designed as the 3'-end specific primer of 5 ' RACE.

 GhATPase-2 GSP3: SEQ ID No: 6:

 ATCATAATAT TGTGAGCACA TG

GhATPase-2GSP4: SEQ ID No: 7:

 GGTTCATCCC CAAATCTGAG AC

GhATPase-2 GSP5 : SEQ ID No: 8:

 The GGACTACCAG AAATTTCAGG AAG test procedure was performed according to the kit instructions (5 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).

 Using SEQ ID NO: 7 and the universal primer AAP (provided with the kit), the cDNA obtained by reverse transcription of the mRNA extracted from the salt-treated group (reverse transcription primer SEQ ID NO: 6, dCTP plus tail) was used as a template. One round of PCR amplification, the specific steps are as follows:

 50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primers SEQ ID NO: 7 and AAP Each of 2.0 μ 1 and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50 times with double distilled water, and 2.0 μ ΐ was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 8 and the primer AUAP. The specific steps are as follows:

 50 l PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 8 and P AUAP Each of 2.0 μ 1 and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

A 2700 bp band (Gel Extraction Kit from OMEGA) was recovered from the second round of PCR product and ligated into pGEM-T Easy vector, which was then transformed into E. coli JM109. In the cells (the specific method is the same as above), the transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin and 40 μgmL X-gaK 24 g/mL IPTG for screening. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin. After incubation at 37 °C overnight, glycerol was added to a final concentration of glycerol of 20% (volume c ratio), and stored at -80 °C. spare. Using SEQ ID NO: 8 and primer AUAP for bacterial PCR amplification (reaction system and reaction conditions as above), 6 positive clones were obtained, and 4 clones were selected and sent to Yingjie Jieji (Shanghai) Trade _) r limited The company sequenced and obtained the 5' end of the cDNA of the gene. Income

 ['-- 5 'RACE product was cloned and sequenced, and D was sequenced with the above 3' RACE product and SEQ ID

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No: 3 sequence was spliced to obtain G L JPove-2 full-length cDNA sequence SEQ ID No: 9

1 CTTCTTTTAC TTCCTTTCCA TCACTCATAG TTTTAACCCT TCTTTCCTAC CGTTTTA3⁄4CA

61 TGTCGTTAAG ACACCGC^AC ACAGGTTACC TAGAACCAGC, - GGAG^AGATA

121 CTCCA TAGT GA^ATGCCAG CACCGAAGGT GGCGTTCGGT ATATATTCTG

181 CCAGGATTTT TGTTTCTTTG T oTATCAACAA GAAACAGATA TTGCGTAGTC

 ['--

241 TTTCCTACAT GTCCATGACA GTGACTCGAG TGATGATCGC

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301 _T n-nTGTTGA TCAGAA^ CA >

 361 GTAAACTTGG AGGCGTGAAA CAGATTGCTG 3⁄4AGTCTTGA AACTGATAAG AAAGATGGTA

421 TCAGTGCTA TGAAGCTGAT GGGTCAATGT A side TTCGGTGCC AACAGGTATC

481 AGAAACCACC TA^AAAAAGC TTTT TAGCT TTGTTTATGA AGCTTT 3⁄4AA GATACCACCA

541 TTATCATACT CTTGGTTTGT CTCTGGGCTT TGGCATCAAA CAGCATGGCA

601 TAACGGATGG GGTGGGAGTA GTTGTTGCTG

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 661 AAGCAAAACA GACAATTCGA AAGGAGAGTA

721 GTGATATAAA AGTGGAAGTA GTGAGAGATG A TTA ATCA GTCTTTGAAG

781 TGCTTGAAAA TTGGTGATGA AATTCGAGCG

 841 TCT GGA GG AAGGTGGATG AATGTAGCAT GACCGGTGAA ZiGCGACCATG

901 TTGAGATGAA TGGCAGCAAC TTCTTTCTGG TACAAAGGTC ACAAATGGGT

961 TCGGTTCAAT TGAATACGGC A GGGGGGAG ZiTGATGAGTT

1021 CCA A^\CCG GAAGAAACTC TGGCCTCAAG AAGCTGACTT

1081 CTGCAAT GG GAAGA TGGA TCTTGGAGTG CTGCTTATTC

AGAATATAT CGAGGCAAGA 1141 'TGGAAACACA AAAGATGACC

AGAATATAT CGAGGCAAGA

 CAAAGTT GA CAGCA GATG AACTCAGTCG TGGAGATAAT TTCAGCAGCA AT ACCATCG

 1261 AATTCCAGAA GGTCTTCCTT TGGCAGTGAC CCTAACTTTG GCCTATTCTA

1321 TGAAGCAAAT GATGGCTGAT TCAGA kCT TTCAGCTTGC GAGACGATGG

1381 GTTCAGCCAC AACAATCTGC ACAGATAAGA CAGGTACCCT TACTTTA ^A GAAATGAAGG

"ϊ 4 1 TTATGGAGTT TTGGC AGGG AAAGAATTAA TTCCTCAGAA ATAGCACCTA

1501 ATG CCATAA GTTACTGCAA CAAGCGGTTG CTC CAACAC AACCGGTAC GTTTATAAGC

1561 CAAACTC AG ATCAC TCCT GAAATTTCTG GTAGTCCAAC TGAGAAAGCA AT CTCTCTT

1621 GGGCTGTCTC AGATTTGGGG ATGAACCTAG ATGACCCAAA GCAi^-ACTAC GAACTCA CC

1681 AAGTAGAGGC CTTCAATTCA GAGAAGAAGA GGAGTGGAGT TCTGA AAGG AGGAAGAGTG

1741 AGAGTGGCGG TGCTACACAG AGGGTGCTGC TGAGATGATC TTGGCCATGT

GCATTTGCAC1801 GCTCACAATA TTATGATAGA AGTGGTGTAG TTA AGCCAT TGACGAGGA , GAAAGAGTAG 1861 AGATGGGGAA AGTTATTGAG GATATGGCAG

GCATTTGCAC

1921 ACACAC A ^ATGAACGAG TTTTG3⁄4AGA AAGTGGACTC ATCTTGCTAG

1981 TTTGAAAGAC CCATGTCGTC CTGGTGTCAG A AAGCAGTA GAAGCTTGCA

2041 TAAATGCTGG AGT^A3⁄4CATC ^AGATTAT^A CTGGAGACAA TATCTTCACA GC AAGGCTA

21 01 TAGCAACTGA ATGTGGCATT TTGC^ACCCA A T GAGGAT T T GAGGGAAGCT GTAATAGAAG

2161 GTGTACAGTT CCG^A3⁄4TTAC TCACCAGAAG A^AGAATGGC TAAGATCAAC AA ATTTGTG

2221 TAATGGCCAG ATCCTCACCT TTTGACA^ C TTCTGATGGT ACAGTGTCTA AAGCAGAATG

2281 GTCATGTTGT AGCAGTCACT GGGGACGGCA CCAATGATGC ACCAGCTTTA AAGGAAGCAG

23 1 ATATTGGACT TTCGATGGGA ATCCAAGGAA CAGAAGTTGC CAAGGAAAGC TCAGACATA

2401 TCATTTTGGA TGATAACTTT ACTTCTGTGG TGACTGTTTT AAGGTGGGGT AGGTGTGTCT

2461 TCAATAACAT TCAGAAATTC ATTCAGTTTC AGCTTACAGT GAATATAGCA GCACTAGTCA

2521 TCAACTTCAT TGCTGCAGTA TCTTCTGGTG A^ATCCCATT GACAGCAGTC CAACTATTGT

2581 GGGTGAACTT AATAATGGAT ACGT TGGGG CTTTAGCTTT GGCCACTGAG CGACCTACCA

2641 ATGATCTCAT GAC3⁄4AAGCCA CCTGTGGGTC GATCCAAGCC CCTCATATCC AACATCATGT

2701 GGAGGAACCT CATTGCTCAA GCCTTGTATC AGGTTGCAGT TCTGCTAACC CTTCAGTTCA

2761 GGGGAAAATT TATCT TGAT GTGGATAAAA AGGTCAATAA CACAC TATT TTGAACACTT

2821 TTG CCTCTG GCAAGTGTT AATGAG'TTCA ATGCCAGAAA A C T T Gi-iAAAG AAGAATA'TAT

2881 TCCAGGGACT ACACAAGAAC AAACTCTTTC T/iGGGATCAT TGCCA AACC ATAATTCTTC

2941 AAG GGTAAT GGTGGAATTC TTGAAGAGGT TTGGTAACAC TGAGAGGCTG AACTGGGGGC

3001 AATGGGG AC TTGTA TGGA AT GCAGCTT TGTCCTGGGC ACTTGGCTGG CTAGTTAAGT

3061 GGA TCCAGC ATGAAAAAAA ATCCTC TCT CGG CACAAG GGTTTTGCA CCAAAAAAAA

3121 T/iCCAGT AG CTCCAATGAG ATGCTCCTTT TCA TTATAT AGAACGTTAG GAATATCAGT

3181 ATA AACCTT GGTACAATT'T CTTTTA TAT TCTAATGCAG TATTTTAAT TCCTTCTGTT

3241 CACTATT AT CTCGG TCTA GT CATTTTA AGAGTTGAAT TTGTAAGCTT CTATGTGAAC

3301 AATGAAGATA GTATGCAT A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:

 SEQ ID No: 10:

 ATGTCGTTAA GACACCGCAA CA SEQ ID No: 11:

TC ATGCTGGA ATCC ACTTAA CTA The G/L4 Pa-2 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11. The PCR reaction was carried out using the cDNA of the above cotton as a template using PrimeSTAR HS DNA polymerase of TaKaRa. 50 yl PCR reaction system: 10 yl 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 10 and SEQ ID NO: 11 Each of 2.0 μ 1 and 30 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 3 minutes), extension at 72 ° C 10 minute.

 PCR amplification product plus A tail: 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and the resulting precipitate was dissolved in 21 μl of double distilled water. Then, 2.5 μl of OXEx Buffer 0.5 μ 15 mM dATP, 1.0 lExTaq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 3000 bp was recovered (Omega recovery kit), and ligated into the pGEM T-easy vector to obtain a GhATPase-2-pGEM plasmid, and then the ligated product was transformed into E. coli JM109 competent cells (method) Same as above), and the transformed bacterial liquid was applied to LB solid medium containing 50 g/mL ampicillin and 40 g/mL X-gaK 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin. After incubation at 37 ° C overnight, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. . Using SEQ ID NO: 10 and SEQ ID NO: 11 to verify the bacterial liquid PCR amplification (reaction system and reaction conditions as above), 7 positive clones were obtained, and 4 positive clones were selected and sent to Yingjiejie (Shanghai) Trade. Sequencing, the resulting sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.

 Amino acid sequence of ATPase-2 protein: SEQ ID NO: 1

 1 SLRHRNTGY LEPA SDGED

 21 TPIVKCQHRR WRSVFAAIYS

 41 ARIFVSLYKK IINKKQILRS

 61 LSYIALDVHD SDSSDDRLPS

 81 LGVDQKTLTE VVREKSLETL

 101 SKLGGVKQIA ASLETDKKDG

 121 ISANEADLAH RVNVFGANRY

 141 QKPPKKSFFS FVYEAFKDTT

 161 IIILLVCAVL SLGFGIKQHG

 181 ITDGGYDGGS IVIAVFLVVA

 201 VSAVSNFKQN RQFEKLSKES

 221 SDIKVEVVRD GRRQFISVFE

 241 VVVGDVVCLK IGDQIPADGL

 261 FLDGHSLKVD ESS TGESDH

 281 VEINGSNNPF VLSGTKVTNG

 301 FGS LVTSVG NTAWGE S

 321 SINRELDEET PLQARLNKLT

 341 SAIGKIGLAV AVLVLAVLLI

 361 RYFTGNTKDD QGNKEYIRGK

 381 TKFDS NSV VEIISAAITI

401 VVVAIPEGLP LAVTLTLAYS 421 MKQMMADHAM VRKLSACETM

 441 GSATTICTDK TGTLTLNEMK

 461 V EFWLGKEL GSISSEIAP

 481 NVHKLLQQAV ALNTTGTVYK

 501 PNSRSLPEIS GSPTEKAILS

 521 WAVSDLG NL DDPKQNYELI

 541 QVEAFNSEKK RSGVLIRRKS

 561 ESGGATQVHW KGAAE ILA

 581 CSQYYDRSGV VKAIDEEERV

 601 E GKVIED A AKSLRCIAFA

 621 HTQNPKDNER VLQESGLILL

 641 GLVGLKDPCR PGVRKAVEAC

 661 INAGVNIKII TGDNIFTAKA

 681 IATECGILQP NEDLREAVIE

 701 GVQFRNYSPE ER AKINKIC

 721 VMARSSPFDK LLMVQCLKQN

 741 GHVVAVTGDG TNDAPALKEA

 761 DIGLSMGIQG TEVAKESSDI

 781 IILDDNFTSV VTVLRWGRCV

 Q

 801 FNNIQKFIQF QLTVNIAALV

 821 INFIAAVSSG EIPLTAVQLL

 841 WVNLIMDTFG ALALATERPT

 861 NDLMTKPPVG RSKPLISNI

 881 WRNLIAQALY QVAVLLTLQF

 901 RGKFIFDVDK KVNNTLIFNT >

921 FVLCQVFNEF NARKLEKKNI

 941 FQGLHKNKLF LGIIAITIIL

 961 QVVMVEFLKR FANTQRLNWG

 981 QWGTCIGIAA LSWPLGWLVK

 1001 WIPA*

Nucleotide sequence of GhATPase-2 gene SEQ ID NO: 2

 1 ATGTCGTTAA GACACCGCA , CACAGGTTAC CTAGAACCAG CCATGTCTGA TGGAGAAGAT

61 ACTCCAATAG TGAAATGCCA GCACCGAAGG TGGCGTTCGG TTTTTGCAGC CATATATTCT

121 GCCAGGATTT GTAC^AGA^A ATTATCAACA AGA^ACAGAT '. j H '

181 CTTTCCTACA TTGCCCTTGA TGTCCATGAC AGTGACTCGA GTGATGATCG CTTACCTTCT

241 CTCGGTGTTG ATCAGAA^AC ACTCACTGAG GTGGTAAGGG AGAAGAGCCT TGAAACCCTA

301 AGTAAACTTG GAGGCGTGAA GCAAGTCTTG AAACTGATAA GA AGATGGT

361 ATCAGTGCTA ATGAAGCTGA TCTTGCACAT CGGGTCAATG TATTCGGTGC

421 CAGAAACCAC TTTGTTTATG AGATACCACC 481 ATTATCATAC TCTTGGTTTG TGCTGTTCTC TCTCTGGGCT TTGGCATCAA ACAGCATGGC

541 ATA3⁄4CGGATG GAGGGTACGA TGGTGGGAGT ATTGTCATTG CTGTCTTTCT AGTTGTTGCT

601 GTATCTGCTG TCAGTAACTT TA3⁄4GCAAAAC AGACAATTCG AGA^ACTTTC ^ ^GGAGAGT

661 AGTGATATAA AAGTGGAAGT AGTGAGAGAT , ^ A3⁄4TTTATATC AGTCTTTGAA

721 GTTGTCGTGG GTGATGTCGT GTGCTTGAi A ATTGGTGATC AAATTCCAGC GGATGGATTG

781 TTCTTGG EATG GACACTCTTT GAAGGTGGAT G^ATCTAGCA TGACCGGTGA AAGCGACCAT

84 1 GTTGAGATCA ATGGCAGCAA CAATCCTTTT GTTCTTTCTG G ACAAAGGT CACA^ATGGG

 9

 901 TTCGGTTCAA T EHGCTTGTTAC ATGAATACGG CATGGGGGGA GATGATGAGT

961 TCCATAAACC GTGAGTTAGA TGAAGAAACT CCATTACAGG CTCGCCTCAA CAAGCTGACT

 TCTG 3⁄4ATTG GGAAGATTGG ATTGGCAGTG GCTGTACTAG

 1 081 CGTTACTTCA CTGGAAACAC AAAAGATGAC AAGAATATAT TCGAGGCAAG

1141 ACAGCATGAT H GAACTCAGTC GTGGAGATAA TTTCAGCAGC AATTACCATC

 GTGGTdTTG CA TTCCAGA AGGTCT Cn^ TTGGCAGTGA CCCTAACTTT

 E

 1261 ATGAAGCA^ TGATGGCTGA TCATGCAATG GTCAGAAAAC TTTCAGCTTG

 1321 CA CAATCTG CACAGATA G L ACAGGTACCC TTACTTTAAA TGAAATGAAG

 ] '--

1381 GTTATGGAGT GAAAGAATTA ATGGGCAGTA AA AGCACCT 44 _ L

 AATGTCCATA AGTTACTGCA GCTCTCAACA (HU GTAC T '1 ATAAG

1501 GATCAGTTCC TGAAATTTCT GGTAGTC cCAA CTGAGAAAGC

 1561 CAGATTTGGG GATGAAGCTA GATGACCCAA AGCAAAACTA

 1621 CAAGTAGAGG CCTTCAATTC AGAGAAGAAG AGGAGTGGAG TTCTGATAAG GAGGAAGAGT

1681 GAGAGTGGCG GGTGCAGTGG AAGGGTGCTG CTTGGCCATG

1741 TGCTCACAAT Al'l'ATGATAG AAGTGGTGTA EH AGAAAGAGTA

18 01 GAGATGGGGA AAGTTATTGA GGATATGGCA GCCAA AGCT

 1861 CACACACAAA ATCCAAAAGA CAATGAACGA GTTTTGCAAG AAAGTGGACT O O CA CTTGCTA

 O

 1921 GGGCTGGTAG GTTTGAAAGA CCCATGTCGT GAAAAGCAG AGAAGCT GC

 H

 1981 ATAAATGCTG GAGTAAACAT CAAGATTATA ACTGGAGACA ATATC TCAC AGCAAA LGGCT

 E E

 2 041 ATAGCAACTG AATGTGGCA TTTGCAACCC AATGAGGATT TGAGGGAAGC TGTAATAGAA

2101 GGTGTACAGT TCCGAAATTA CTCACCAGAA GAAAG-AATGG CTAAGATCAA CAAAATTTGT

2161 GTAATGGCCA GATCCTCACC TTTTGACAAA CTTCTGATGG TACAGTGTC AAAGCAGAAT

2221 GGTCATG TG TAGCAGTCAC TGGGGACGGC ACCAATGA G CACCAGCTTT AAAGGAAGCA

2281 GATATTGGAC TTCGATGGG AATCCAAGGA ACAGAAGTTG CCAAGGA ^AG CTCAGACATA

 ATCATTT GG ATGATAACTT TACTTCTGTG GTGACTGT T TAAGG GGGG TAGGTGTGTC

 24 01 TTCAATA3⁄4CA TTCAGA^ATT CATTCAGTTT CAGCTTACAG TGAATATAGC AGCACTAGTC

2461 ATCAACTTCA TTGCTGCAGT ATCTTCTGGT G^A3⁄4TCCCAT TGACAGCAGT CCAACTATTG

 TGGGTGA3⁄4CT TAATA3⁄4TGGA GCTTTAGCTT TGGCCACTGA GCGACCTACC

2581 AATGATCTCA TGACAAAGCC ACCTGTGGGT CGATCCAAGC CCCTCATATC CAACATCATG

2641 TGGAGGA3⁄4CC TCATTGCTCA AGCCTTGTAT CAGGTTGCAG TTCTGCTAAC CCTTCAGTTC

2701 AGGGGAAAAT TTATCTTTGA TGTGGATA^A AAGGTCAATA ACACACTTAT TTTCAACACT

2761 TTTGTCCTCT GCC^AGTGTT TA3⁄4TGAGTTC ΑΆ.Τ G C C AG AA AACTTGA^AA GA3⁄4GAATATA

2821 TTCCAGGGAC TACACAAGA , CAAACTCTTT CTAGGGATCA TTGCCAT^AC CATAATTCTT

2881 CAAGTGGTAA TGGTGGAATT CTTGAAGAGG TTTGCT^ACA GA3⁄4CTGGGGG

3⁄4ATGGGGTA TTGTCCTGGC CACTTGGCTG TGGATTCCAG Example 3 Construction of a plant expression vector for the GhATPase-2 gene

 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The 35S promoter and Tnos terminator were selected as promoters and terminators of the G/L4 Pa-2 gene, respectively. The construction flow chart is shown in Figure 1.

 Using primers SEQ ID NO: 12 and SEQ ID NO: 13, Pnos was amplified using the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 l PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 plasmid, 1.0 μ ΐ PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 12 and SEQ ID NO : 13 each of 2.0 μ ΐ and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with EcoRI, Bglll, and ligated into pCAMBIA2300 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.

 SEQ ID NO: 12

 GCACGAATTC ggcgggaaac gacaatctga

 SEQ ID NO: 13

 ATCCAGATCTAGATCCGGTGCAGATTATTTG

 Tnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 using the pBI121 plasmid as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μ 1 PCR reaction system: 10 μ ΐ 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 1.0 μ 1 pBI121 plasmid, 1.0 μ 1 Prime STAR, 10 μ Μ primers SEQ ID NO: 14 and SEQ ID NO: 15 2.0 μl and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by Kpnl and EcoRI (Promega T4 ligase kit) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.

 SEQ ID NO: 14:

 AAGGGTACCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 15:

TCAGAATTCCCAGTGAATTCCCGATCTAGTA The 35S promoter was amplified using the primers SEQ ID NO: 16 and SEQ ID NO: 17 using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase using TaKaRa. 50 μ 1 PCR reaction system: 10 μ 1 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 pCAMBIA 2300 plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 16 and P SEQ ID NO: 17 each of 2.0 μΐ and 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by HindIII, Sail digestion (ligation method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.

 SEQIDNO: 16:

 ACTAAGCTTTAGAGCAGCTTGCCAACATGGTG

SEQIDNO: 17:

 TGAGTCGACAGAGATAGATTTGTAGAGAGAGACT Amplifies the full-length sequence of the G/^ Pa^-2 encoding gene with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template is the positive GhATPase-2-pGEM plasmid obtained in Example 2), using PrimeSTAR of TaKaRa HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μΐ 5XPS Buffer 3 μ 1 2.5 mM dNTP 1.0 μ 1 GhATPase-2-pGEM plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 18 and SEQ ID NO: 19 2.0 μ 1 and 31 μ 1 double distilled water. PCR reaction conditions: Pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 3 minutes), extension at 72 ° C for 10 minutes. The obtained PCR product was ligated by Sall and Smal (connection method as above) to pCAMBIA2300-3, and the plant expression vector 35S-GhATPase-2-2300 was obtained after verification (Fig. 2).

SEQIDNO: 18

 ACTGTCGACATGTCGTTAAGACACCGCAACA SEQIDNO: 19

 ACTCCCGGGTC ATGCTGGA ATCC ACTTAACTA

Example 4 Transformation of Agrobacterium by 35S-GhATPase-2-2300 Expression Vector

Agrobacterium tumefaciens LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin Incubate at 28 ° C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin, and cultured overnight (about 12-16 hours) to OD ₆ by shaking at 28 °C. . A value of 0.4 forms a seed broth. 5 ml of culture-activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin, and cultured at 28 °C. -2.5 hours to OD _6QQ = 0.8. The ice bath solution was shaken for 10 minutes every 3 minutes to allow the bacteria to enter a dormant state uniformly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; resuspend the cells by adding 1 ml of ice pre-cooled 10% (by volume) glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; 10% (by volume) of the pre-cooled glycerin was washed 3-4 times; then the bacterial pellet was resuspended by adding an appropriate amount of ice-cold 10% (by volume) glycerol to prepare LBA4404 competent cells to 40 μM/tube. Dispense and store at -70 ° C for later use.

 Transformation of Agrobacterium: The LBA4404 competent cells were thawed on ice, and 1 μl of the plasmid 35S-GhATPase-2-2300 obtained in Example 3 was added to 40 μl of the competent cells, and the mixture was mixed and ice bathed. About 10 minutes. The mixture of the competent cells after the ice bath and the 35S-GhATPase-2-2300 plasmid was transferred to an ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) using a micropipette, and tapped to make the suspension Arrive at the bottom of the electric shock cup, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. Set the program of MicroPulser (purchased from Bio-Rad) to "Agr" and shock once. Immediately remove the electric shock cup and add 200 μl of 预 medium pre-warmed at 28 °C. Quickly and gently mix the sensitive cells with a micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 1 hour at 225 rpm. 100-200 μM of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μg/ml rifampicin, 50 μg/ml streptomycin, 50 μg/ Ml Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Receptor Material Arabidopsis Culture

Select the vermiculite with good water absorption and soft soil to match the nutrient soil (1:1) as the soil for Arabidopsis planting. Flower pots with a diameter of 9 cm, 20-30 seeds per pot (Columbia type, from the Arabidopsis Bioresource Center, Ohio State University). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22 ° C, light intensity 3500-4000 lx, light cycle 12 hours dark, 12 hours light culture, 1/2MS watered every 7 days, after 30 days of culture, 4-5 plants per pot, the light cycle is adjusted to After 8 hours of darkness and 16 hours of light culture, after most of the plants were twitched, the entire main moss was cut off at the base of the inflorescence, and the top edge was taken. After about 1 week, it grew 4-6 in the axillary bud. A new genus of the genus, which can be used for transformation when the inflorescence forms a flower bud and partially blooms or forms 1-2 pods. Example 6 Arabidopsis flower leaching transformation:

The Agrobacterium liquid of the transformed expression vector 35S-GhATPase-2-2300 obtained in Example 4 was inoculated to an LB liquid medium containing 10-50 μ ₈ /ηι 1 kanamycin (kan), and cultured overnight. Inoculated in the morning to 1:50 in a new LB medium (1 L) containing 50 μ ₈ /ηι 1 kanamycin, and cultured for about 8 hours until the Agrobacterium liquid OD600 should be between 1.0 and 1.2. Centrifuge at 5000 rpm for 5 minutes at room temperature, discard the supernatant, and suspend the Agrobacterium pellet in the impregnation medium (1/2MS; 5% sucrose; adjust to pH 5.7 with KOH; 0.02% Silwet L-77) to make the OD600 at 0.8. about. The upper part of the Arabidopsis thaliana prepared for transformation prepared in Example 5 was slowly and spirally immersed in the Agrobacterium-containing dyeing medium, and gently swayed clockwise for about 2 minutes, and covered with a transparent plastic cover. Humidity, put in the greenhouse overnight. After 24 hours, remove the plastic transparent cover and pour through the water. After 2-3 weeks, ensure that the plants are hydrated. When the plant stops flowering and the first fruit pod matures and turns yellow, it is covered with a paper bag. When all the pods in the paper bag turn yellow, the watering is stopped. After 1-2 weeks of drying, the seeds are collected and the transformants are screened. Untransformed Arabidopsis fruit pods were taken as controls. Example 7 Screening of transgenic positive transformants from Arabidopsis thaliana

Seed disinfection: first soak for 10 minutes with 70% ethanol, occasionally suspend the seeds; then wash with sterile water four times, and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on the surface of 1/2MS solid screening medium containing 50 μ ₈ /ηι 1 kanamycin for 2 days (a 150 mm diameter plate was sown for up to 1500 seeds), 4 The solution was vernalized for 2 days, and then cultured for 7-10 days under the conditions of a constant temperature of 22 ° C, an illumination intensity of 3500-4000 lx, and a photoperiod of 12 hours of darkness/12 hours of light. . After germination of the transgenic seeds on the screening medium (MS+kan) plate for 2 weeks, the plants capable of germination and normal growth were transferred to the soil for further cultivation. 1-2 leaves of each plant capable of normal growth on the screening medium were extracted, and the DNA was extracted as a template, and PCR detection was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 (reaction system and conditions are the same as above) The plants that were negative by PCR were removed, and the seeds of the plants positive for PCR were collected and labeled as T0il-T0i21. Example 8 Transgenic Arabidopsis thaliana T1 plants overexpressing GhATPase-2 were selected for water uptake, and the soily soft vermiculite was combined with nutrient soil (1:1) as the Arabidopsis planting soil. Each transformant of T0il-T0i21 and the non-transgenic control Arabidopsis seeds were each sown in 2 pots, and 20-30 seeds were seeded per pot. After sowing, the film is covered with a film to provide a moist environment for plant growth. The temperature was 22 ° C, the light intensity was 3500-4000 lx, the photoperiod was 12 hours dark, 12 hours light culture, and 1/2 MS was watered every 7 days. After 25 days of culture, 1-2 leaf extracts were extracted from each transgenic Arabidopsis thaliana as a template, and PCR was detected using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and conditions are the same as above), and plants negative for PCR detection were removed. 7-8 PCR-positive seedlings were retained in each pot. After 10 days of continuous culture, 5-7 transgenic Arabidopsis seedlings or control Arabidopsis thaliana seedlings with uniform size were kept in each pot for salt tolerance experiments. Example 9 Salt tolerance test of transgenic Arabidopsis thaliana T1 plants overexpressing GhATPase-2 The transgenic Arabidopsis thaliana and the control Arabidopsis thaliana each retained a potted plant in Example 8 and were normally treated with 1/2 MS, respectively. One pot of plants was 1/2MS in 150 mM NaCl, and the results were observed after incubation at a constant temperature of 22 ° C, light intensity of 3500-4000 lx, and 12 hours of light culture/12 hours of darkness: T1 generation transgenic plants (TO transgenic plants) The salt tolerance of the plants grown from the seeds of the plants showed that the T1 transgenic plants Tli5, Tlil3, and Τ1Ϊ16 showed significant salt tolerance (see Figure 3, Tli5, Tlil3, T1U6 results and Similar, not shown here). Example 10 Validation of GhATPase-2 Gene Expression at Transcriptional Level

Eight T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (one of the above-mentioned Tli5, Tlil3, and Tlil6 three salt-tolerant strains), and the control plants in Example 9 were randomly selected from 4 plants. The leaves (0.05 mM) were treated with salt (150 mM NaCl) for 14 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA obtained at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template, reverse transcription primer SEQ ID NO: 11). The GhATPase-2 fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: CTGAATTGAA GGCCTCTACT TG), and its transcription was examined. PrimeSTAR HS DNA polymerization using TaKaRa The enzyme was subjected to a PCR reaction using the cDNA obtained by the above reverse transcription as a template. 50 lPCR reaction system: 10 μ 1 5XPS Buffer, 3 μ 12.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 20 each 2.0 μ1, and 30 μ 1 double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 32 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes. The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a salt-tolerant T1 transgenic Arabidopsis plant, and 10-13 is a non-transgenic control Arabidopsis plants, 9 is a plasmid PCR positive control (35S-GhATPase-2-2300 plasmid). The size of the band shown is identical to the size of the positive control (approximately 1600 bp). The results showed that there was significant transcription of G/^1⁄4^-2 in the salt-tolerant T1 transgenic Arabidopsis plants, and no GhATP-2 transcription in the non-transgenic control Arabidopsis plants.

Claims

Claim 1. An ATP hydrolase of cotton having the sequence SEQ ID NO: 1.  2. A gene encoding the ATP hydrolase of claim 1, the sequence of which is SEQ ID NO: 2.  A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and wherein the nucleotide sequence of the gene and the expression control sequence of the expression vector are operably Preferably, the expression vector is pCAMBIA2300.  4. The vector of claim 3 which is the 35 S-ThATPase-2-2300 vector shown in Figure 2. A recombinant cell comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.  A method for improving salt tolerance of a plant, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, The plant is Arabidopsis.  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.  8. The method of claim 7, wherein the plant is Arabidopsis thaliana.  9. The gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for use in improving plant salt tolerance and for use in plant breeding.  10. The use of claim 9, wherein the plant is Arabidopsis thaliana.