WO2014205595A1

WO2014205595A1 - Atp hydrolase atpase-1 derived from cotton, and coding gene and use thereof

Info

Publication number: WO2014205595A1
Application number: PCT/CN2013/000748
Authority: WO
Inventors: 王建胜; 何云蔚; 梁远金; 陈淼
Original assignee: 创世纪转基因技术有限公司
Priority date: 2013-06-24
Filing date: 2013-06-24
Publication date: 2014-12-31
Also published as: CN105073984B; CN105073984A

Abstract

Provided are a plant protein, and a coding gene and a use thereof, particularly, an ATP hydrolase derived from cotton, a coding gene thereof, and a use thereof in cultivating a transgenic plant with improved salt tolerance.

Description

Cotton ATP hydrolase ATPase-1 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-1 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 of an ATP hydrolase (designated herein as ATPase-1) 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 were significantly improved in salt tolerance by transgenic technology and introduced into plants, and these traits were stably inherited.

The first aspect of the present invention provides an ATP hydrolase ATPase-1 encoding gene (designated herein as GhATPase-1) 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-1-2300 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-l-2300) of the G/zATPsae-1 gene (Fig. la-lb).

Figure 2 is a plasmid map of the plant expression vector (35S-GhATPase-l-2300) of the G/zATPase-1 gene.

Fig. 3 shows the salt tolerance test results of the T1 generation Arabidopsis thaliana plants transgenic with GhATPase-1, T lh2 showed significant salt tolerance, and the results of T lh8 and T lhlO were similar thereto, and are not shown here.

Figure 4 shows the results of molecular level detection of the transcription level of GhATPase-1 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 (Tlh2, Tlh8, TlhlO, respectively), 9 is a plasmid PCR positive control (35S-GhATPase-l-2300) Plasmids, 10-13 are non-transgenic control Arabidopsis plants.

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, and placed at a final concentration of 200 mM. Treatment in 1/2 MS liquid medium of NaCl for 6 hours. After the treatment, the roots of the two seedlings were cut out in time, quickly frozen with liquid nitrogen, and 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 g/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 stored at -80 °C for later use. The nested PCR primers Primer 1 and Primer 2R (PCR-select ^TM cDNA Subtraction Kit from Clontech) were used to perform PCR amplification verification on the cultured bacteria, and 231 positive clones were verified, and then all were Positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.

(6) cDNA sequencing analysis of differential clones:

After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 203 Expressed sequence tags (ESTs) (Unigene) were obtained. Example 2 Cotton ATP hydrolase gene GhATPase-1 clone

After removing the redundant DNA from the clone of the colony Gh-S2-117 in the identified cotton SSH library, the sequence was SEQ ID No: 3, and 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-1, and its corresponding protein was named ATPase-l o.

SEQ ID No: 3:

1 ACACTGGA3⁄4A GCAGCTGCAG AAATAATCGT AGCCATGTGC TCAGACTATT ATGAGAGCAA 61 TGGGC GCATA AC GTCCATGG ATGAAGACCA AAGGAC CAGA ATTGAAACAA TAATCCAAAG

121 TATGC CCGCT AC TAGCTTAC GATGCATTGC TTTTC CTCAT AAC CAAGTCT CACA3⁄4AAACA

181 CATGGAATC T GTTGATCATA GCGAAAAGAC ACATCA3⁄4AGA ATA3⁄4AACi3⁄4AG ATGC TCTAAC

2, 4 . C T T G C T AG G G ATAGTTGGTT TGAAGG AT C C AT GT CG C C C G G T G T 'C A.3⁄4 A A A.AG C T GT GG A

301 AGCTTGTAAA TCTGCAGGGG TGGACATCAA AATGATTACT GGAGACAATA TTTTCACAGC 361 A^AAGCTATA GCTGCAGAAT GTGGAATTCT AGGAGCAGAT TACL3⁄4ATGAAG AAAGTGGACA

421 AGCTATAGAA GGTATTG, 3⁄4AT TTCGAAATTA CACACCTGAA GAGAGAATGG AGAAGATCGG

481 GAAGATCAAG GTGATGGCAA GG CCTCTCC ATTTGACAAG CTTCTGATGG T

Cloning of the full-length coding gene of GhATPase-1 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-1 GSP 1: SEQ ID No: 4:

GCTGCAGAAT GTGGAATTCT AG

GhATPase-1 GSP2: SEQ ID No: 5:

GTGATGGCAA GGTCCTCTCC AT

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 and the universal primer AUAP for PCR amplification, 4 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the cDNA of the gene was obtained. 3 ' end.

Based on the GhATPase-1 gene fragment that has been obtained, the following three specific primers were designed as the 3'-end specific primer for 5' RACE. GhATPase-1 GSP3 : SEQ ID No: 6:

CAAACCAACT ATCCCTAGCAAG

GhATPase-1 GSP4: SEQ ID No: 7:

TCTGCTCCTT TGGTCTTCAT CC

GhATPase-1 GSP5 : SEQ ID No: 8:

GCACATGGCT ACGATTATTT CTG Experimental procedure according to the kit instructions ( 5 ' RACE System for Rapid Amplification of cDNA

The Ends kit was 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 using SEQ ID NO: 8 and the 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: 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.

Recover the second round of PCR product with a fragment of approximately 1800 bp (Gel Extraction Kit was purchased from

OMEGA), and ligated it into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the same method as above), and applied the transformed bacterial solution to 50 μlmL ampicillin, 40 ^ glmL Screening was performed on LB solid medium of X-gaK 24 g/mL IPTG. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 μ _§ /ιη1 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. spare. Bacterial PCR using SEQ ID NO: 8 and primer AUAP Amplification verification (reaction system and reaction conditions are the same as above), 6 positive clones were obtained, and 4 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the 5' end of the cDNA of the gene was obtained. After the obtained 5 'RACE product clone was sequenced, it was spliced with the above 3' RACE product sequencing result and the SEQ ID No: 3 sequence to obtain (3⁄4 2 3⁄4 -7 full-length cDNA sequence SEQ ID No: 9:

1 AAAGCACAAA ACAGACTTCC AATTCAAGCA CTTCTTCCAA CTGGTTAAAG AAACCCAGCA

61 ACAACCATGT CTAGCAGTGA CGGTAATCAA ATTTACGATT GCGGTACATT ACTTTTCAAA

121 GTCACCACCT CTGGCTTCAC CACCGCTCAA AAGCGTTGGA GGATTGCCTA TGCATCGATA

181 TATTCTGTGC GGGTAATGCT TTCTCTTGCC AAGGAGATAA TATCGAAGAG AGGTATTGAA

241 CAACCCTCGA TCATCTCGGA TTTGCATCCT TACGTTGCGC TAGATGTTGA ACCCTCGAGC

301 TCTCCGCATT GGGGTGAAAA ATTTTCATCT TCCTCCTTTG CTCCGAAAAT TGATCGGAAG

361 AGACTTGTGG AGACCGTGAA AGAAAAGGAC TTGGTTTCTC TTCACCAGCT TGGAGGAGTC

421 GAAGGCATTG CCGCTGCTCT TGGAACAAAT CCTGAAAAGG GAATCCGAGA TGATGATCGA

481 GACGTTGTGA AAAGACAGGA GATGTTTGGT ACCAATACTT ACCACAAGCC ACCTCCCAAA

541 GGGTTACTGT ATTTTGTCCT GGATGCTTTC AAGGACACCA CAATTCTTAT CCTCCTGGTC

601 TGTGCTGCTC TTTCTCTTGG TTTTGGTATC AAAGAGCATG GTGCAGCAGA GGGTTGGTAT

661 GAAGGCGGAA GTATTTTCGT TGCCGTCTTT CTTGTGATTG TTGTCTCTGC ACTCAGTAAC

721 TTCAGACAGG AGACTCAATT TGACAAGCTA TCAAAAATAA GTAACAATAT CAAAGTCGAA

781 GTTGTTAGAG GTGGCCGCCG TCGACAAGTA TCTATTTTTG ATCTTGTTGT TGGAGATGTT

841 GTCTTCCTGA AGATTGGAGA TCAAATTCCA GCAGATGGGC TGTTCTTGGA CGGTTATTCA

901 CTGCAAGTGG ATGAATCGAG CATGACAGGA GAAAGTGACC ATATGGAAGT AGATGCCACC

961 AGGAACCCGT TCCTTTTTTC TGGTTCGAAA GTGGCAGATG GTTATGGTCA AATGCTTGTA

1021 GCATCCGTGG GAATGGATAC TACATGGGGA GAAATGATGA GCTCGATAAC CAGTGATAAA

1081 AACGAAAGAA CTCCACTACA AGAACGACTT GACAGACTTA CCTCTTCTAT CGGAAAGGTA

1141 GGTCTTGCGG TTGCCTTTCT AGTCCTTGTA GTCTTATTAA TTCGTTATTT CACTGGGAAT

1201 ACAGAAGATG ACAATGGGAA CACGGAGTAT ATTGGTAGCA AGACCAGCGT AGATGATATC

1261 TTAAATGCTG TTGTTCGTAT AGTTTCGGCT GCAGTAACTA TTGTGGTAGT TGCAATTCCG

1321 GAAGGTCTAC CATTGGCTGT CACTCTTACA CTTGCTTACT CAATGAAAAG AATGATGGCT

1381 GATCAGGCAA TGGTTAGAAA ACTTTCAGCT TGTGAAACGA TGGGCTCAGC TACCATCATT

1441 TGTACTGACA AAACTGGAAC CTTGACACTA AACCAGATGA AGGTAACCCA GTTTTGGCTC

1501 GGCCAAGAGT CCATCAAGGA AGATCATTCC AACATCATTG ACCACGCTGT TCTTGAATTA

1561 TTCTACCAAG GTGTTGTTTT GAACACAACT GGTAGTGTTT GCAAACCCGT CTCCGGATCC

1621 CTACCTGAAT TTTGTGGCAG TCCAACAGAA AAGGCGATTC TTTCTTGGGC TGTCTTAGGT

1681 TTGGATCTGG ATATGGAAAA ATTGAAGCAA AAATACAGCA TTCTCCATGT TGAAACTTTC

1741 AACTCGGAGA AAAAGAGAAG TGGGGTTTCA GTAAGGAGAA AAACAGATGA AACTCTTCAT

1801 GTACACTGGA AAGGAGCTGC AGAAATAATC GTAGCCATGT GCTCAGACTA TTATGAGAGC

1861 AATGGGGGCA TAAGGTCCAT GGATGAAGAC CAAAGGAGCA GAATTGAAAC AATAATCCAA

1921 AGTATGGCCG CTAGTAGCTT ACGATGCATT GCTTTTGCTC ATAAGCAAGT CTCACAAAAA

1981 GAGATGGAAT GTGTTGATGA TAGCGAAAAG AC AC AT C AAA GAATAAAAGA AGATGGTCTA

2041 ACCTTGCTAG GGATAGTTGG TTTGAAGGAT CCATGTCGCC CAGGTGTCAA AAAAGCTGTG

2101 GAAGCTTGTA AATCTGCAGG GGTGGACATC AAAATGATTA CTGGAGACAA TATTTTCACA

2161 GCAAAAGCTA TAGCTGCAGA ATGTGGAATT CTAGGAGCAG ATTACAATGA AGAAAGTGGA 2221 CAAGCTATAG AAGGTATTGA ATTTCGAAAT TACACACCTG AAGAGAGAAT GGAGAAGATC

2281 GGGAAGATCA AGGTGATGGC AAGGTCCTCT CCATTTGACA AGCTTCTGAT GGTACATGAG

2341 GTGGCAAAGG AGAGCTCAGA CATTGTCATA TTGGATGATA ATTTCAGCTC AGTTGCCACA

2401 GTTTTAAGAT GGGGAAGATG TGTATATAAC AACATACAGA AATTCATTCA ATTTCAGCTT

2461 ACCGTTAATG TTGCTGCTCT TGTAATTAAC TTCATTGCAG CAGTTTCTGC TGGTGAGGTT

2521 CCCTTGACAG CCGTTCAATT GCTGTGGGTA AACCTCATCA TGGACACCTT AGGTGCTCTA

2581 GCCCTTGCAA CAGATAGGCC CACCAAGGAA CTAATGAAGA AGCCGCCCGT CGGTCGAACT

2641 GAGCCCCTCA TTACTAATGT CATGTGGAGG AATCTTTTAG CCCAAGCAGT ATACCAGATA

2701 GCTATTCTCT TGATCTTGCA ATTTAGAGGT GAATCTATGT TCAATGTGCC TAAGAGGGTA

2761 AAGGACACAC TGATTTTCAA TACTTTCGTT CTCTGCCAAG TTTTCAACGA ATTCAACGCA

2821 AGGAAGTTGG AGAAGCAAAA CGTCTTCCAA GGCATTCTTC AGAACAGATT GTTTCTGGGA

2881 ATCGTGGGCA TAACCATCAT TCTTCAGGTG GTTATGGTGG AATTTCTAAA GAAGTTTGCA

2941 GATACTGAGA GATTGGAACT ATGGCAGTGG GGGGTTTGTA TCTTATTCGC AGCTTTCTCA

3001 TGGCCAATTG CATGGGTTGT GAAGCTCATA CCTGTTTCGG ACAAACCCTT CTTCAGTTAT

3061 CTCAAGCGAT CAAAATCATC TTCACAATTA AATAAGTCAT CTACCACAAG AAACCTTCAA

3121 TCTGCAGGCA TGGAACTAGA AGTGCAATAA GATCATGTTC TGAAAGAACA AGGATGTAAG

3181 TTCAAAATCA CTTCACTGAC ACAAAGGACA ACATAAGGAG ATCATGATTC TAGATTTCGT

3241 ATACTTTAAG AGCTACTAAC TTATATTTAT GTATGTATAT GTAAAATGAC TTTTCTGATA

3301 AAGGCCTTGG AACCATTGGT CCCAGATTCA ATCTAACCTC ATGGAGCTTT GCTCTTATCT

3361 TTACCTAGGA TGTTTCTAGT TCCACGCCAA TTGTTAGTTG ACCCTTTAGT AATGGCTTAT

3421 TCCTTTCTAA ATCTGAAACA GGATCATATG TGCTACCTAT TGTACTTATT CTCGTAAAAA

3481 TGAAATACCC TTAATGTATC TACCTAAAAA AAAAAAAAAA AAAAA A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:

SEQ ID No: 10:

ATGTCTAGCA GTGACGGTAA TC SEQ ID No: 11:

TTATTGCACT TCTAGTTCCA TGC The G/L4 Pa-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 TaKaRa's PrimeSTAR HS DNA polymerase. 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 for 10 minutes.

PCR amplification product plus A tail: Add 2.5 volumes of absolute ethanol to the PCR product, and place 10 points at -20 °C. The clock was 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-lpGEM plasmid, and then the ligated product was transformed into E. coli JM109 competent cells (method supra) The transformed bacterial solution 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 are the same as above), three positive clones were obtained, and three 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-1 protein: SEQ ID NO: 1

1 SSSDGNQIY DCGTLLFKVT

21 TSGFTTAQKR WRIAYASIYS

41 VRV LSLAKE IISKRGIEQP

61 SIISDLHPYV ALDVEPSSSP

81 HWGEKFSSSS FAPKIDRKRL

101 VETVKEKDLV SLHQLGGVEG

121 IAAALGTNPE KGIRDDDRDV

141 VKRQE FGTN TYHKPPPKGL

161 LYFVLDAFKD TTILILLVCA

181 ALSLGFGIKE HGAAEGWYEG

201 GSIFVAVFLV IVVSALSNFR

221 QETQFDKLSK IS Li IKVEVV

241 RGGRRRQVSI FDLVVGDVVF

261 LKIGDQIPAD GLFLDGYSLQ

281 VDESS TGES DH EVDATRN

301 PFLFSGSKVA DGYGQ LVAS

321 VG DTTWGE SSITSDKNE

341 RTPLQERLDR LTSSIGKVGL

361 AVAFLVLVVL LIRYFTGNTE

381 DDNGNTEYIG SKTSVDDILN

401 AVVRIVSAAV TIVVVAIPEG

421 LPLAVTLTLA YS KR ADQ

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661 CAGGAGACTC AATTTGACA , ATA3⁄4GTAACA A ATCAAAGT CGAAGTTGTT

721 AGAGGTGGCC GCCGTCGACA AGTATCTATT TTTGATCTTG TTGTTGGAGA TGTTGTCTTC

781 CTGAAGATTG GAGATCA^AT TCCAGCAGAT GGGCTGTTCT TGGACGGTTA TTCACTGCAA

841 GTGGATGAAT CGAGCATGAC AGGAGAAAGT GACCATATGG AAGTAGATGC CACCAGGAAC

901 CCGTTCCTTT TTTCTGGTTC GAAAGTGGCA GATGGTTATG GTCAAATGCT TGTAGCATCC

961 GTGGGAATGG ATACTACATG GGGAGAAATG ATGAGCTCGA TAACCAGTGA

AGAACTCCAC TAC3⁄4AGA3⁄4CG ACTTGACAGA CTTACCTCTT CTATCGGAAA GGTAGGTCTT

1 081 GCGGTTGCCT TTCTAGTCCT TGTAGTCTTA TTAATTCGTT ATTTCACTGG GAATACAGA

1141 GATGACAATG GGAACACGGA GTATATTGGT AGCAAGACCA GCGTAGATGA TATCTTAAAT

GCTGTTGTTC GTATAGTTTC GGCTGCAGTA ACTATTGTGG TAGTTG 3⁄4AT TCCGGAAGGT

1261 CTACCATTGG CTGTCACTCT TACACTTGCT TACTCAATGA AAAGAATGAT GGCTGATCAG

1321 GCAATGGTTA GA AACTTTC AGCTTGTGAA ACGATGGGCT CAGCTACCAT CATTTGTACT

1381 GACAAAACTG GAACCTTGAC ACTA^ACCAG ATGAAGGTAA CCCAGTTTTG GCTCGGCCAA

144 1 GAGTCCATCA AGGAAGATCA TTC 3⁄4ACATC ATTGACCACG CTGTTCTTGA ATTATTCTAC

1501 CAAGGTG'TTG TTTTGAACAC AAGTGG AGT GTTTGCAAAC CCGTCTCCGG ATGCCTACCT

1561 GAA TTTGTG GCAGTCCAAG AGAAAAGGCG ATTGTTTCTT GGGCTGTCT AGGTTTGGAT

1621 CTGGATA GG AAAAA'TTGAA AGCATTCTGC ATGTTGA/iAC TT CAACTCG

1681 GAGAA/iAAGA GAAGTGGGG TTCAGTi AGG AGAAA ACAG A'TGAAACTCT TCATGTAGAC

1741 TGGAAAGGAG CTGCAGA^\ AA CGTAGCC ATG GCTCAG ACTATTATGA GAGCAATGGG

18 01 GGCATAAGGT GC/iTGGATGA AGACCAAAGG AGCAGAAT G AAACAATAA'T CCAAAGTATG

18 61 GL_U CTAGTA GCTTAGGATG CA TGCTTTT GCTCATAAGC AAGTCTCACA AAAAGAGATG

^ Q 21 GAA GTG TG ATGATAGCGA AAAGACACAT CAAAGAATAA AAGAAG— TGG TCTAACC TG

1981 CTAGGGA AG TTGGT TGAA GGATCCATGT CGCCCAGG G TCA ^AAAAGC TG GGAAGCT

2 041 TGTAAATCTG CAGGGGTGGA CATCAAAATG ATTACTGGAG ACAATATTT CACAGCAAAA

2101 GCTATAGCTG CAGAA GTGG AATTCTAGGA GCAGATTACA ATGAAGAAAG TGGACAAGCT

2161 ATAGAAGGTA TGAATTTCG AAATTACACA CCTGAAGAGA GAATGGAGAA GATCGGGAAG

2221 ATCAAGGTGA TGGCAAGGTC CTCTCCATTT GACAAGCT C TGATGGTACA TGAGGTGGCA

2281 AAGGAGAGCT CAGACATTG CATATTGGAT GATAATTTCA GCTCAGTTGC CACAGTT TA

AGATGGGGAA GATGTGTATA TAACAACATA CAGAAATTCA TTCAA TTCA GC TACCGTT

24 01 AATGTTGCTG CTCTTGTAA TAACTTCATT GCAGCAGTTT C GCTGGTGA GGTTCCC TG

ACAGCCG TC AATTGCTGTG GGTAAAC TC ATCATGGACA CCTTAGGTGC TC AGCCCTT

GCAACAGATA GGCCCACCAA GGAACTAATG AAGAAGCCGC CCGTCGGTCG ^ACTGAGCCC

2581 CTCATTACTA ATGTCATGTG GAGG^ATCTT TTAGCCCAAG CAGTATACCA GATAGCTATT

2641 CTCTTGATCT TGC^ATTTAG AGGTGAATCT ATGTTC^ATG TGCCTAAGAG GGTA^AGGAC

2701 ACACTGATTT TCAATACTTT CGTTCTCTGC 3⁄4AGTTTTCA ACGAATTCA , CGCAAGGAAG

2761 TTGGAGA3⁄4GC AA^ACGTCTT CC AGGCATT CTTCAG^ACA GATTGTTTCT GGGAATCGTG

2821 GGCATAACCA TCATTCTTCA GTGGAATTTC TA^AGAAGTT TGCAGATACT

2881 AACTATGGCA TGTATCTTAT TCGCAGCTTT CTCATGGCCA

2 941 ATTGCATGGG TTGTGAAGCT CATACCTGTT TCGGACAAAC CCTTCTTCAG TTATCTCAAG

CATCTTCACA ATTA^ATAAG TCATCTACCA CAAGA ACCT TCAATCTGCA

3 061 GGCATGGAAC TAGAAGTGCA ATAA Example 3 Construction of Plant Expression Vector of GhATPase-1 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 the Tnos terminator were selected as promoters and terminators of the G/L4 Pa-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^-encoding gene with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template is the positive GhATPase-1-pGEM plasmid obtained in Example 2), using PrimeSTAR HS of TaKaRa DNA polymerase. 50 μ 1 PCR reaction system: 10 μΐ 5XPS Buffer 3 μ 1 2.5 mM dNTP 1.0 μ 1 GhATPase-1-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 resulting PCR product was ligated by Xbal and Smal digestion (ligation method) to pCAMBIA2300-3, and the plant expression vector 35S-GhATPase-l-2300 was obtained after verification (Fig. 2).

SEQIDNO: 18

ACTTCTAGA ATGTCTAGC AGTGACGGTAAT C SEQIDNO: 19

ACTCCCGGGTTATTGCACTTCTAGTTCCATGC

Example 4 Transformation of Agrobacterium by 35S-GhATPase-l-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-l-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. Transfer the mixture of competent cells after ice bath and 35 S-GhATPase-1 - 2300 plasmid with a micropipette to an ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad), tap to suspend The liquid reaches 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 35S-GhATPase-1-28 expression vector 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 was 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 liquid medium and contain 5% sucrose; adjust to pH 5.7 with KOH; 0.02% Silwet L-77) , so that the OD600 is around 0.8. 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 °C was vernalized for 2 days, and then cultured for 7-10 days under the conditions of constant temperature 22 ° C, light intensity 3500-4000 lx photoperiod of 12 hours dark / 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, DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and The conditions were the same as above. The plants which were negative by PCR were removed, and the seeds of the plants positive for PCR were collected and labeled as T0hl-T0h22. Example 8 Planting of transgenic Arabidopsis thaliana T1 plants overexpressing GhATPase-1 The water-absorbing properties of the soil with good water absorption and soft soil were combined with nutrient soil (1:1) as the soil for Arabidopsis thaliana planting. Each transformant of T0hl-T0h22 and the non-transgenic control Arabidopsis seeds were each sown in 2 pots, and 20-30 seeds were sown 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 SEQ ID NO: 18 and SEQ ID NO: 19 were used as primers for PCR detection (reaction system and conditions are the same as above), and PCR detection was performed. Negative plants, 7-8 PCR-positive seedlings were retained per pot. After 10 days of continuous culture, 5-7 transgenic Arabidopsis thaliana seedlings or control Arabidopsis thaliana seedlings with uniform size were maintained in each pot. . Example 9 Salt tolerance test of transgenic Arabidopsis thaliana T1 plants overexpressing GhATPase-1 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 containing 150 mM NaCl, and the results were observed after 14 days of incubation at a constant temperature of 22 ° C, light intensity of 3500-4000 lx, and 12 hours of light culture / 12 hours of darkness. The salt tolerance of the T1 transgenic plants (plants grown from the seeds of the TO transgenic plants) showed that the T1 transgenic plants Tlh2, Tlh8 and TlhlO showed significant salt tolerance (see Figure 3, with Tlh2). For example, the results of Tlh8 and TlhlO are similar, not shown here). Example 10 Validation of G/L4 Pa-gene expression at the transcriptional level

Eight T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (one of the above-mentioned Tlh2, Tlh8, and TlhlO 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 μ _§ total RNA was used as a template and reverse transcription primer was SEQ ID NO: 11). Use primer SEQ ID

NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: TTACAAGAGC AGCAACATTA ACGGT) The G/L4 Pa^- fragment was amplified and its transcription was examined. PrimeSTAR HS with TaKaRa The DNA polymerase is subjected to a PCR reaction using the cDNA obtained by the above reverse transcription as a template. 50 μ 1 PCR reaction system: 10 yl 5XPS Buffer, 3 μ 12.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Prime STAR HSDNA polymerase, 10 μM primers SEQ ID NO: 10 and SEQ ID NO: 20 each 2.0 μ 1 , as well as

30 μΐ of 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-l-2300 plasmid). The size of the band shown is identical to the size of the positive control (approximately 2400 bp). The results showed that there was significant transcription of G/^ 1⁄4^- in the salt-tolerant T1 transgenic Arabidopsis plants, and no GhATP-1 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-1 - 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.

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.