WO2014026311A1

WO2014026311A1 - Cotton protein kinase and encoded gene and use thereof

Info

Publication number: WO2014026311A1
Application number: PCT/CN2012/079997
Authority: WO
Inventors: 崔洪志; 梁丽; 王建胜; 何云蔚
Original assignee: 创世纪转基因技术有限公司
Priority date: 2012-08-13
Filing date: 2012-08-13
Publication date: 2014-02-20
Also published as: CN103748224A; CN103748224B

Abstract

Provided are a polypeptide sequence and encoded gene of the protein kinase GPK1 originating from cotton. Also provided are the uses of the protein kinase GPK1 and the encoded gene thereof in the cultivation of a transgenic plant with improved drought tolerance.

Description

A cotton protein kinase and its coding gene and application

FIELD OF THE INVENTION The present invention relates to protein kinases and their coding genes and applications, and more particularly to a cotton-derived cotton protein kinase GPK1 and its encoding gene, and its use in breeding transgenic plants with improved drought tolerance. BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, and cause great losses to crop yield. The impact of drought on crop yield is It takes the first place in natural adversity, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas. According to statistics, the world's dry and semi-dry areas account for 34% of the land area; China's dry and semi-arid areas account for 52% of the country's land area, and the annual area is 20 to 2.7 million hectares. About 3 billion cubic meters, due to lack of water, less than 350 to 4 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and the spring drought frequently reaches 10 years and nine times. .

Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques. It is especially difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promoted the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification systems and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions. In recent years, studies on the ability of plants to improve stress tolerance through transgenic techniques, as well as studies on stress-tolerant crops, xerophytes and halophytes have yielded significant results for stress-related genes and signal transduction. The system has a further understanding (Liu Q.1998. Two transcrip tion facto rs, DREB1 and DREB2, w ith an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature - responsive gene exp ression Plant, 10: 1391-1406; KAN GJY.2002. A rabid op sis basic leucine zipper p ro teins that mediate stress 2responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABE H. 2003.A rabid op sis AtMYC2 (bHLH) and AtMYB2(MYB) Function as transcrip tional activato rs in abscisic acid signaling. Plant Cell, 15: 63-78. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies on the function and expression regulation of stress-responsive genes are dominant, but the link between stress-resistance-related signaling pathways and the mechanism of the entire signaling network system remains to be further studied. SUMMARY OF THE INVENTION The present inventors cloned a DNA sequence encoding a gene of a protein kinase (designated herein as GPK1) of cotton using a combination of SSH and RACE. It was found that the introduction of transgenic plants significantly improved the drought tolerance of transgenic plants, and these traits were stably inherited.

The first aspect of the present invention provides a gene encoding a protein kinase GPK1 of cotton, the sequence of which is SEQ ID NO: l o

A second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GhGPK1-2300 carrier shown in Fig. 2.

The third aspect of the present invention provides a recombinant cell comprising the nucleotide sequence 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 present invention provides a method for improving drought tolerance of a plant, comprising: introducing the nucleotide sequence 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 The gene is expressed; preferably, the plant is tobacco.

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

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 drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.

A seventh aspect of the present invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as shown in SEQ ID NO: 1. DRAWINGS Figure 1 is a construction flow of a plant expression vector (rd29A-GhGPK1-2300) of GhGPK1.

Figure 2 is a plasmid map of the plant expression vector (rd29A-GhGPK1-2300) of GhGPK1.

Figure 3 shows the drought tolerance simulation results of GhGPK1 T1 transgenic tobacco plants (in the figure, T1R1; right, T1R3) and non-transgenic tobacco plants (left, CK) as controls.

Figure 4 shows the results of protein expression verification at the transcriptional level of transgenic T1 tobacco plants and non-transgenic control plants. detailed description

The invention is further illustrated by the following non-limiting examples. Example 1. Cotton SSH library construction under drought stress:

The specific method is:

A subtractive library was constructed by inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit. The mRNA of the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a driver. The specific steps are as follows:

(1) Test materials:

冀棉14 (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded on vermiculite vermiculite, cultured at 25 ° C, photoperiod 16 h / 8 h, poured 1 per week /2MS medium (9·39 mM KN03, 0. 625 mM KH2P04, 10. 3 mM NH4N03, 0.75 mM MgS04, 1. 5 mM CaC12, 50 μ Μ KI , 100 μ Μ H3B03, 100 μ Μ MnS04, 30 μ M ZnS04, 1 μ Μ Na2Mo04, 0. Ι μ Μ CoC12, 100 μ M Na2EDTA, 100 μ Μ FeS04) once. It was used for experiments when the seedlings were as high as 25_30 cm.

(2) Material handling:

The test seedlings were divided into two groups, each with 4 pots and 1 pot per pot. The first group was the control group, cultured at 25 ° C, lighted, and normally watered. The second group was drought treatment group, 25 °C, light culture, stop watering, treatment for 10 days. After treatment, cut the leaves of the top 1/3 of the two groups of seedlings in time, and quickly freeze with liquid nitrogen at -70 °C. Store in the refrigerator.

(3) Total RNA extraction:

The cotton leaves of the control and drought-treated groups were taken 0.5 g, and the total RNA of cotton was extracted with a plant RNA extraction kit (invitrogen). Determined by HITACHI's UV spectrophotometer U-2001 Absorbance values of total RNA at 260 nm and 280 nm, 0D260/0D280 ratio of 1. 8-2· 0, indicating high total RNA purity, total RNA integrity was detected by 1.0% agarose gel electrophoresis, The brightness of the 28S band is approximately twice that of the 18S band, indicating good RNA integrity. The mRNA was isolated using Qiagen's Oligotex mRNA purification assay (^ (purification of polyA+ RNA from total RNA).

(4) Inhibition subtractive hybridization:

In order to increase the availability of the Expressed sequence tag (EST) (unigene), the gene is not cleavable and the obtained sequence is in the untranslated region. The laboratory uses Rsal, Hael ll to digest the double-stranded cDNA (according to the protocol described in the subtractive kit, obtained by reverse transcription of mRNA), and performs two sets of suppression subtraction. Other steps and methods are PCR-selected by Clontech. The method shown in the TM cDNA Subtraction Kit was used for suppression subtractive hybridization, and finally the second PCR product of the two groups of forward subtractive hybridization cDNA fragments was combined.

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

The second PCR product of the forward subtracted hybrid cDNA fragment (QIAquick PCR Purification Kit, purchased from Qiagen) was ligated to the pGEM-T Easy (purchased from Promega kit) vector according to the procedure of the pGEM_T Easy kit. The specific steps are as follows: The following components were sequentially added using a 200 μl PCR tube: 2nd PCR product of purified forward subtractive hybridization cDNA fragment 3 μ 1, Τ4 ligase buffer 5 μ 1, pGEM-T Easy vector 1 μ 1, Τ4 DNA The ligase 1 μ ΐ was ligated overnight at 4 °C. 10 μL of the ligation reaction product was added to 100 μL of competent E. coli JMI09 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 μL of LB medium (1 % Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. In a 37°C water bath, cultured at 225 r/min for 30 min, 200 μL of bacterial solution was planted in 50 LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) of μ g/mL ampicillin was incubated at 37 ° C for 18 h. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 360 white colonies (number: Gh-D001 to Gh-D360). All white clones were picked in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 g/mL ampicillin, cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C. . The nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the broth, and 292 positive clones were obtained. All positive clones were sent to Yingjieji (Shanghai). ) Trading Co., Ltd. sequencing

(6) cDNA sequencing analysis of differential clones:

After removing the DNA sequencing results from the vector and the ambiguous sequence and the redundant cDNA, a total of 180 samples were obtained.

EST (unigene). It was found by BlastN that 102 unigenes have homologous sequences in GenBank (more than 50% protein homology), 33 EST functions are unknown or hypothetical proteins, and another 45 have not obtained homologous It is speculated that it may be a shorter sequence in the untranslated region at the 3 ', 5 ' end. Example 2 Cloning of cotton protein kinase gene encoding gene GhGPK1

After the cloned Gh-D193 was detached from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence belonged to the mitogen-activated protein kinase. The full-length gene encoded by the clone Gh-D193 was named here. For GhGPKl, the corresponding protein was named GPK1.

SEQ ID No: 3

1 ACACTAAACA ACTTTTGTTG GGACTGGAAT ATCTTCACAA AAATAGAATC GTGCATAGAG

61 ACATCAAGGG AGCAAACATT CTTGTGGATA ACAAGGGTTG TATCAAACTT GCAGACTTTG

121 GTGCATCCAA AAAAGTTGTC GAGTTGGCTA CAATAAATGG AGCCAAGTCA ATGAAGGGAA 181 CTCCTTATTG GATGGCTCCT GAAGTTATTC TCCAAACTGG GCATAGCTTC TCTGCCGATA 241 TTTGGAGTGT TGGCTGT

Cloning of the full-length gene of GhGPK1

Based on the Gh-D193 gene fragment that has been obtained, two specific primers were designed as the 5'-end specific primer for 3' RACE.

GhGPKl GSP1 : SEQ ID NO : 4:

ACTTTTGTTGGGACTGGAAT AT

GhGPKl GSP2: SEQ ID NO: 5:

CAAAAATAGAATCGTGCATAGAG

The experimental procedure was carried out 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 3' primers AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template. The specific steps are as follows: Ex Taq purchased from TAKARA, 50 μ 1 PCR reaction system: 5 μ 1 lO X Ex Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 mRNA reverse transcribed cDNA, 1. 0 μ 1 Ex Taq 10 μ M primers SEQ ID NO: 4 and AUAP each 2.0 μl, and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.

The obtained PCR product was diluted 50-fold with double-distilled water, and then 2.0 μl was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and 3' primer AUAP, and the specific steps were as follows: 50 μ 1 PCR reaction System: 5 μ 1 10 X Ex Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 diluted first round PCR product, 1. 0 μ 1 Ex Taq 10 μ Μ primer SEQ ID NO : 5 And AUAP each of 2. 0 μ 1, and 35 μ 1 of the double Steamed water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The second PCR product was recovered from a fragment of approximately 1600 bp (Gel Extraction Kit from OMEGA). ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (specifically as above), randomly picked 10 white colonies containing 50 μ G/mL ampicillin was cultured in LB liquid medium, and cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID NO: 5 and the 3' primer AUAP were used for PCR amplification of the bacterial cells, and three positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained.

Based on the GhGPK1 gene fragment that has been obtained, three specific primers were designed as the 3'-end specific primer for 5' RACE.

GhGPKl GSP3: SEQ ID NO: 6:

TATCGGCAGAGAAGCTATGC

GhGPKl GSP4: SEQ ID NO: 7:

GAATAACTTCAGGAGCCATCC

GhGPKl GSP5: SEQ ID NO: 8:

TTCCCTTCATTGACTTGGCTCC

The experimental procedure was performed according to the kit instructions (5 '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: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification. The specific steps are as follows: Ex Taq was purchased from TAKARA, 50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2 · 5 mM dNTP, 2. 0 μ 1 mRNA reverse transcribed cDNA, 1. 0 μ 1 Ex Taq 10 μM of primers SEQ ID NO: 7 and AAP each of 2.0 μl, and 35 μl of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The obtained PCR product was diluted 50 times with double distilled water, and then 2.0 μl was used as a template, and the second round of PCR amplification was carried out with SEQ ID Ν0:8 and 3' primer AUAP, and the specific steps were 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 diluted first round PCR product, 1. 0 μ 1 Ex Taq 10 μ Μ primer SEQ ID NO : 8 And AUAP each of 2.0 μl, and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 mino, second PCR product recovery The fragment is approximately 800 bp (Gel Extraction Kit from OMEGA) attached to pGEM-T Easy Vector, transformed to JM109 (as described above), and randomly picked 10 white colonies in LB liquid containing 50 μg/mL ampicillin The medium was cultured, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID N0: 8 and 3 ' The primer AUAP was used for PCR amplification (reaction system and reaction conditions as above), and four positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 5' end of the cDNA of the gene was obtained.

The obtained 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result. The full-length cDNA sequence of GhGPK1 was obtained as SEQ ID NO: 19.

SEQ ID NO: 19:

1 ACCTCCAAAT CTTTAAGCAA ACGGATCTGA AGAAAGAAGA TTTTTGTACT CTAAATGCAG

61 GAGCTTGTTG GATCGGTTCG CCGGTCCTTT GTCTTTAGGT CTTCAACCTC CAGCGACGAT

121 GCCGGTGGAG GACTTGGAGG CTTTGTTGAG AAGATCGGCG CCAGCATTCG CAGATCGCGA

181 ATCGGCTTGT TCGCTAAGCC ACCTGCTCCA CCCGCTCTTC CTTCTGTTAA GAAAAGGGAC

241 GCTACGATCC GGTGGCGGAA GGGCGAGTTG ATTGGTTGTG GCGCCTTTGG TCGGGTTTAC

301 ATGGGGATGA ATCTTGACTC CGGCGAGTTA CTAGCTGTTA AACAGGTTTT GATAGCGGCA

361 AATGCTTCAA AGGAGAAAAC ACAGGCTCAT ATTAGAGAGC TCGAAGAAGA AGTGAAGCTT

421 CTACAGAATC TGTCACATCC AAACATTGTT AGATATTTGG GCACCGCAAG AGAGGACGAT

481 TCGTTGAATA TTCTATTGGA GTTTGTACCA GGTGGATCCA TTTCCTCACT TTTGGGGAAG

541 TTTGGATCTT TCCCCGAATC TGTTATAAGA ATGTACACTA AACAACTTTT GTTGGGACTG

601 GAATATCTTC ACAAAAATAG AATCGTGCAT AG AG AC AT C A AGGGAGCAAA CATTCTTGTG

661 GATAACAAGG GTTGTATCAA ACTTGCAGAC TTTGGTGCAT CCAAAAAAGT TGTCGAGTTG

721 GCTACAATAA ATGGAGCCAA GTCAATGAAG GGAACTCCTT ATTGGATGGC TCCTGAAGTT

781 ATTCTCCAAA CTGGGCATAG CTTCTCTGCC GATATTTGGA GTGTTGGCTG TACTGTGATT

841 GAGATGGCTA CTGGAAAGCC CCCTTGGAGC CAACAGTATC AGGAGGTTGC TGCTCTCTTT

901 CATATTGGGA CAACTAAATC TCATCCACCC ATCCCTGAGC ATCTCTCCCC TGAGGCTAAA

961 GACCTTTTGT TAAAATGCCT ACAGAAGGAA CCAGGACTGA GACCTAGTGC ATCAGACTTG

1021 CTTCAGCACC CTTTTGTCAC TGGGGACTAT CAGGAACCTC ATGCGGTGCT TCGTAGATCA

1081 ATTAGGGAAC CTGAAAATCT GGAGATGGCA TCTGGGGTGA ACCTGAGAAG CTCAATAAAT

1141 TCGGAGATCA GGTCAACCTG CACGGGTTTA AAGGACGTTT GTGAAATGGG TAGTGTGAGT

1201 TGCTCTACAG CATTTCTTGG GAAATTCTCA GAACCAGGAG CCTATTGGAG GGGAAGCAAT

1261 TGTGACAATA GCATGTGTGA G AT AG AT G AC AAAGATGATC TAGAATTTAA TTATTCTGTA

1321 AAGTTCTCCT CTGTTTTATC ATCTGCTGAC TTGAATAAAA GTTTCAATCC CATGTGTGAA

1381 CCCACTGAAG ACTGGCCACC CAAATTAGAT CAAAGTTCTG AGCTAAGCCG AAGTGGAGTA

1441 AACTTGTCTT TGGATGAAAC AATGGAAGCT GCTAGCACTC CTGGAATGTC TGGTAAGGAG

1501 GAGAATGGCT TCACTTTTCT CTGCGGACCT CCAACAGGTG AT GAT GAT G A AGAAGTTACA

1561 GAGTCAAAAA TTAGAGCCTT CCTGGATGAA AAGGCTCTGG AACTGAAGAA GCTGCAATCT 1621 CCTCTGTATG AACAGTTCTA CAACACATTG AATGGTAGTC TTCCACCTTC TGTTGGAACT 1681 GCAAATGGTG AAAATATTTT GAGCTTACCT CCTAAAAGTA GGTCACCTAA GTGGCTGCCT 1741 AGCAGAAGAC TCTCAGCAGT TGCTGATGCT GCCAACATGG TTAGCTCAAA GAGTCGTATG 1801 AATCATTTGT CAAATACTGC AGTTGTTCAT GACCGGACTT TACAAGAAAT TCAGCCACCG 1861 TCTGTTGAGG AATGGAAAGG GCAGGATATA ATTAGTCCGA GCATGAGCTT TTCTGAGAGA 1921 CAAAGGAGAT GGAAGGAAGA GCTTGACCAA GAGCTTGAGA GAAAGCGAGA GATGCTGCGG 1981 AAGACATCAT CTCCAAAGGA TAAGTTTCTA TTTGGACAAA GAGAACAAAT ACGGTCTCCA 2041 TTTCCTGGCA AGTAAATGTA GTATCCAACT TTACCTCTTT TGATGTTCCA TTGGGACTGT 2101 TCATTTCTGT ATTCTATTTT TGGTGAGAAT GGTGCCCAAT AATGTTACTC GGGCTTTATT 2161 TATGAGTGTC AGATACGAAT ATGTATTTGA TATGAGTATA TTTAATTTTT TTTCTTTTTT 2221 TTTTTTGCTT GTATAAAGTT CTTTCATATT TGTGGAAGAT ATTTGGGAGA GCCAAAAAAA 2281 AAAAAAAAAA AAA designed according GhGPKl full length cDNA a pair of primers as follows:

GhGPKF: SEQ ID NO: 9:

ATGCAGGAGCTTGTTGGATCGG

GhGPKR: SEQ ID NO: 10:

TTACTTGCCA GGAAATGGAG ACCG

The full length of GhGPK1 was cloned by SEQ ID NO: 9 and SEQ ID NO: 10.

PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 9 and SEQ ID NO: 10 each of 2·0 μ 1, and 30 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.

PCR amplification product plus A tail: PCR product plus 2.5 times absolute ethanol, _20 ° C for 10 minutes, centrifuge, remove the supernatant, air dry, dissolved in 21 μ ΐ double distilled water. Add 2. 5 μ ΐ 10 X Ex Buffer, 0. 5 μ 1 5 mM dATP, 2. 5 μ ΐ ΙΟ Χ Εχ Taq ₀ Reaction conditions: 70 ° C reaction for 30 minutes. A DNA fragment of about 1900 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector, transformed into JM109 (method as above), and 10 white colonies were randomly picked up in LB liquid containing 50 g/mL ampicillin. The medium was cultured, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at _80 ° C for use. SEQ ID NO: 9 and SEQ ID NO: 10 were subjected to bacterial cell PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the sequence was SEQ ID. NO : 2. Amino acid sequence of GPK1 protein: SEQ ID NO: 1

1 MQELVGSVRR SFVFRSSTSS

21 DDAGGGLGGF VEKIGASIRR

41 SRIGLFAKPP APPALPSVKK

61 RDATIRWRKG ELIGCGAFGR

81 VYMGMNLDSG ELLAVKGVL

101 AANASKEKTQ AHIRELEEEV

121 KLLQNLSHPN VRYLGTARE

141 DDSLNILLEF VPGGSISSLL

161 GKFGSFPESV work RMYTKGLLL

181 GLEYLHKNRI VHRDIKGANI

201 lA/DNKGCIKL ADFGASKKW

221 ELATINGAKS MKGTPYWMAP

241 EVILQTGHSF SADIWSVGCT

261 VIEMATGKPP WSQQYQEVAA

281 LFHIGTTKSH PPIPEHLSPE

301 AKDLLLKCLQ KEPGLRPSAS

321 DLLQHPFVTG DYQEPHAVLR

341 RSIREPENLE MASGVNLRSS

361 NSEIRSTCT GLKDVCEMGS

381 VSCSTAFLGK FSEPGAYWRG

401 SNCDNSMCEI DDKDDLEFNY

421 SVKFSSVLSS ADLNKSFNPM

441 CEPTEDWPPK LDQSSELSRS

461 GVNLSLDETM EAASTPGMSG

481 KEENGFTFLC GPPTGDDDEE

501 VTESKIRAFL DEKALELKKL

521 QSPLYEQFYN TLNGSLPPSV

541 GTANGEN worker: LS LPPKSRSPKW

561 LPSRRLSAVA DAANMVSSKS

581 RMNHLSNTAV VHDRTLQEIQ

601 PPSVEEWKGQ DIISPSMSFS 621 ERQRRWKEEL DQELERKREM

641 LRKTS SPKDK F LFGQREQIR

661 SPFPGK*

Nucleotide sequence of the GhGPK1 encoding gene: SEQ ID NO: 2

1 ATGCAGGAGC TTGTTGGATC GGTTCGCCGG TCCTTTGTCT TTAGGTCTTC AACCTCCAGC

61 GACGATGCCG GTGGAGGACT TGGAGGCTTT GTTGAGAAGA TCGGCGCCAG CATTCGCAGA

121 TCGCGAATCG GCTTGTTCGC TAAGCCACCT GCTCCACCCG CTCTTCCTTC TGTTAAGAAA

181 AGGGACGCTA CGATCCGGTG GCGGAAGGGC GAGTTGATTG GTTGTGGCGC CTTTGGTCGG

241 GTTTACATGG GGATGAATCT TGACTCCGGC GAG TT AC TAG CTGTTAAACA GGTTTTGATA

301 GCGGCAAATG CTTCAAAGGA GAAAACACAG GCTCATATTA GAGAGCTCGA AGAAGAAGTG

361 AAGCTTCTAC AGAATCTGTC ACATCCAAAC ATTGTTAGAT ATTTGGGCAC CGCAAGAGAG

421 GACGATTCGT TGAATATTCT ATTGGAGTTT GTACCAGGTG GATCCATTTC CTCACTTTTG

481 GGGAAGTTTG GATCTTTCCC CGAATCTGTT ATAAGAATGT ACACTAAACA ACTTTTGTTG

541 GGACTGGAAT ATCTTCACAA AAATAGAATC GTGCATAGAG ACATCAAGGG AGCAAACATT

601 CTTGTGGATA ACAAGGGTTG TATCAAACTT GCAGACTTTG GTGCATCCAA AAAAGTTGTC

661 GAGTTGGCTA CAATAAATGG AGCCAAGTCA ATGAAGGGAA CTCCTTATTG GATGGCTCCT

721 GAAGTTATTC TCCAAACTGG GCATAGCTTC TCTGCCGATA TTTGGAGTGT TGGCTGTACT

781 GTGATTGAGA TGGCTACTGG AAAGCCCCCT TGGAGCCAAC AGTATCAGGA GGTTGCTGCT

841 CTCTTTCATA TTGGGACAAC TAAATCTCAT CCACCCATCC CTGAGCATCT CTCCCCTGAG

901 GCTAAAGACC TTTTGTTAAA ATGCCTACAG AAGGAACCAG GACTGAGACC TAGTGCATCA

961 GACTTGCTTC AGCACCCTTT TGTCACTGGG GACTATCAGG AACCTCATGC GGTGCTTCGT

1021 AGATCAATTA GGGAACCTGA AAATCTGGAG ATGGCATCTG GGGTGAACCT GAGAAGCTCA

1081 ATAAATTCGG AGATCAGGTC AACCTGCACG GGTTTAAAGG ACGTTTGTGA AATGGGTAGT

1141 GTGAGTTGCT CTACAGCATT TCTTGGGAAA TTCTCAGAAC CAGGAGCCTA TTGGAGGGGA

1201 AGCAATTGTG ACAATAGCAT GTGTGAGATA GATGACAAAG AT GAT C TAG A ATTTAATTAT

1261 TCTGTAAAGT TCTCCTCTGT TTTATCATCT GCTGACTTGA ATAAAAGTTT CAATCCCATG

1321 TGTGAACCCA CTGAAGACTG GCCACCCAAA TTAGATCAAA GTTCTGAGCT AAGCCGAAGT

1381 GGAGTAAACT TGTCTTTGGA TGAAACAATG GAAGCTGCTA GCACTCCTGG AATGTCTGGT

1441 AAGGAGGAGA ATGGCTTCAC TTTTCTCTGC GGACCTCCAA CAGGTGATGA TGATGAAGAA

1501 GTTACAGAGT CAAAAATTAG AGCCTTCCTG GATGAAAAGG CTCTGGAACT GAAGAAGCTG

1561 CAATCTCCTC TGTATGAACA GTTCTACAAC ACATTGAATG GTAGTCTTCC ACCTTCTGTT

1621 GGAACTGCAA ATGGTGAAAA TATTTTGAGC TTACCTCCTA AAAGTAGGTC ACCTAAGTGG 1681 CTGCCTAGCA GAAGACTCTC AGCAGTTGCT GATGCTGCCA ACATGGTTAG CTCAAAGAGT

1741 CGTATGAATC ATTTGTCAAA TACTGCAGTT GTTCATGACC GGACTTTACA AGAAATTCAG

1801 CCACCGTCTG TTGAGGAATG GAAAGGGCAG GATATAATTA GTCCGAGCAT GAGCTTTTCT

1861 GAGAGACAAA GGAGATGGAA GGAAGAGCTT GACCAAGAGC TTGAGAGAAA GCGAGAGATG

1921 CTGCGGAAGA CATCATCTCC AAAGGATAAG TTTCTATTTG GACAAAGAGA ACAAATACGG

1981 TCTCCATTTC CTGGCAAGTA A Example 3 Construction of GhGPK1 gene expression vector

The plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the 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 inducible promoters rd29A and Tnos were selected as promoters and terminators of the GhGPK1 gene.

Pnos were amplified using the plant expression vector PBI 121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 11 and SEQ ID NO: 12, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 l PCR reaction system: 10 μ 15 XPSBuffer, 3 μ 12· 5 mM dNTP, 1· 0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μ 1, And 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, and extension at 72 °C for 10 min. pCAMBIA2300_l was obtained by EcoRI, Bglll digestion with PCAMBIA2300 (promega, T4 ligase cassette).

SEQ ID NO: 11:

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 12:

ATCCAGATCTAGATCCGGTGCAGATTATTTG

SEQ ID NO: 13 and SEQ ID NO: 14 amplify Tnos using PBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 15×PS Buffer, 3 μ 12.5 mM dNTP, 1.0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR, 10 μΜ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 μ 1, and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min by Kpnl, EcoRI digestion Connect to pCAMBIA2300-l (promega T4 ligase cassette) to obtain pCAMBIA2300-2

SEQ ID NO: 13:

AAG ^73⁄4i76GAATTTCCCCGATCGTTCAAA SEQ ID NO: 14:

TCA GAA mCCAGTGAATTCCCGATCTAGTA

SEQ ID NO: 15 and SEQ ID NO: 16 Amplification of the Arabidopsis thaliana rd29A promoter using Arabidopsis thaliana (Columbia type, available from www. arabidopsis.org) DNA as a template (see Zeng J., et L. 2002, Preparation) Of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44 (6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 Arabidopsis DNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO : 15 and SEQ ID NO: 16 each of 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min. By HindIII, Sail digestion and ligation (connection method is the same as above) pCAMBIA2300-2 obtained pCAMBIA2300_3

SEQ ID NO: 15:

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA

SEQ ID NO : 16:

TGAi7 O3⁄4CTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO: 17 and SEQ ID NO: 18 Amplified GhGPK1 (template is GhGPK1 obtained in Example 2), using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ ΐ PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 GhGPKl -pGEM, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO : 17 and SEQ ID NO: 18 each of 2.0 μl, and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The plant expression vector rd29A_GhGPKl_2300 was obtained by cleavage of Kpnl and Sail (connection method as above) PCAMBIA2300-3.

SEQ ID NO: 17:

TGA i^nATGCAGGAGCTTGTTGGATCGG

SEQ ID NO: 18:

AAG i O3⁄4CTTACTTGCCAGGAAATGGAGACCG Example 4 rd29A-GhGPKl-2300 expression vector for transformation of Agrobacterium

Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin 1-2 days in advance Single spot inoculation, culture 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 h) to 0D600 at 28 °C. The value is 0.4, and a seed bacterial liquid is formed. 5小时至0D _6。 Take 5 ml of activated bacterial solution (1: 20 ratio) inoculated in 100 ml of the same concentration of antibiotics in LB liquid medium, shaking at 2 ° C. 2 h. . =0. 8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; add a certain amount of pre-cooled 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; repeat washing with 10% glycerol3- 4 times; add the appropriate amount of ice bath pre-cooled 10% glycerol to resuspend the bacterial pellet, dispense it in 40 μM/tube, and store at -70 °C for later use.

Transformation of Agrobacterium: Thaw competent cells on ice, add 1 μl of plasmid to 40 μl of competent cells, mix and ice bath for about 10 min. Transfer the mixture of competent and DNA to the pre-cooled electric shock cup with a gun and tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) on the slide of the shock chamber and push the slide to place the electric shock cup on the base electrode of the shock chamber. When using a 0. lcm electric shock cup, the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is once. Immediately remove the electric shock cup and add LB medium pre-warmed at 28 °C. Quickly and gently spread the cells with a gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. Take 100~200 μl of the bacterial solution and plate on the corresponding resistant screening medium (LB solid medium containing 50 μg/ml rifampicin, 50 μg/ml streptomycin, 50 μg/ Ml Kanamycin), cultured at 28 °C. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation

To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by: Institute of Tobacco, Chinese Academy of Agricultural Sciences, Library No. I5A00660) 30 s, washed twice with sterile double distilled water. Then, it was immersed in 0.1% liter of mercury for 8 min, and washed twice with sterilized double distilled water to complete surface sterilization. The surface-sterilized tobacco seeds were placed in MS ( 18.78 mM KN0 ₃ , 1. 25 mM KH ₂ P0 ₄ , 20. 6 mM NH ₄ N0 ₃ , 1.5 mM MgS0 ₄ , 3. 0 mM CaCl ₂ , 50 μ M KI , 100 μ M H3BO3 , 100 M MnS0 ₄ , 30 μ M ZnS0 ₄ , 1 μ M Na ₂ Mo0 ₄ , 0.1 μM CoCl ₂ , 100 μ M N3⁄4EDTA, 100 μ Μ FeS0 ₄ , 7. 4 g/L agar, sucrose 30 g/L) was germinated under sterile conditions to prepare sterile seedlings. The leaves of the sterile seedlings were cut into 5 mm×5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhGPKl-2300 in the logarithmic growth phase for 10 min, and the bacteria were sucked up in the dark. Incubate for 2 days (MS medium). The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (face +50 mg/L kanamycin + 500 mg/L cephalosporin) After incubation for about 45 days under light conditions, the buds should be grown and transferred to rooting medium (MS+50 mg/L kanamycin + 500 mg/L cephalosporin) for about 30 days, until the root system is developed. The seedlings were then transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.

The obtained transgenic tobacco leaves were extracted, and DNA (the Arabidopsis thaliana DNA extraction method in Example 3) was used, and SEQ ID NO: 9: and SEQ ID NO: 10 (50 μl PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2 · 5 mM dNTP, 2. 0 μ 1 DNA, 1. 0 μ 1 Ex Taq 10 μ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2. 0 μ 1, and 35 μ 1 double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, 72 V extension for 10 min), PCR identification, positive preservation The plant is numbered T. R1_T. R20. Example 6 Drought Tolerance Simulation Experiment and Functional Identification of GhGPK1 Transgenic Tobacco T1

The sterilized vermiculite was soaked in 1/2 MS medium. T. R1-T. R20 and control tobacco seeds were sown on vermiculite, 15 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 5 days, 25 days after culture, each pot was kept 4-5 seedlings of uniform size, drought test, transgenic tobacco, control tobacco drought for 14 days (no watering), 25 ° C, 10 hours light culture / 14 hours dark culture cycle. The drought resistance of T1 transgenic plants (plants grown from seeds of TO transgenic plants) showed that the control plants were wilting, and 1\R1, 1\R3, 1\R5, 1\R8, 1\R11, 1 Twenty-five of the 34 tobacco lines of \R13 and TJ 6 were able to grow normally and showed obvious drought tolerance (see Figure 3, taking 1\R1, 1\R3 as an example, 1 R5, 1 R8, The results of 1 R11, 1 R13, TJ 6 are similar to 1 R1, 1 R3, not shown here). Example 7 Verification of GPK1 protein expression at the transcriptional level

Control tobacco, drought-tolerant transgenic tobacco T1 plants, drought-tolerant transgenic tobacco T1 plants (good growth) drought 14 days leaves 0. 05g, total RNAo extracted with plant RNA extraction kit (invitrogen) with HITACHI The UV spectrophotometer U-2001 measures the absorbance of total RNA at 260 nm and 280 nm and calculates the individual RNA concentrations. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript I I I Reverse Transcriptase (2 μg total RNA as a template, reverse transcription primer SEQ ID NO: 10). The relative expression of GPK1 protein was detected by amplifying GhGPK1 by SEQ ID NO: 9 and SEQ ID NO: 10. PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μ 1 PCR reaction system: 10 μ ΐ 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO : 9卩 SEQ ID NO: 10 each of 2.0 μl, and 30 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, after 29 cycles, extension at 72 °C for 10 min. The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-5 is control tobacco, and 6-18 is drought-tolerant transgenic tobacco T1 plant, 19-24 It is a T1 generation plant that is not tolerant to transgenic tobacco. The size of the band shown in the figure is consistent with the size of the GhGPKl-1 gene. The results showed that the control tobacco did not transcribe GhGPK1 mRNA, and the T1 generation of drought-tolerant transgenic tobacco had stronger transcription of GhGPK1, and the T1 generation of the drought-tolerant transgenic tobacco had no transcription or transcription.

Claims

Claim

A cotton protein encoding gene having a sequence of SEQ ID NO: 2.

2. A recombinant expression vector comprising the nucleotide sequence of the gene of claim 1 and operably linked to the expression control sequence of the expression vector.

3. The vector of claim 2 which is the rd29A-GhGPK1-2300 carrier shown in Figure 2.

A recombinant cell comprising the nucleotide sequence of the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.

A method for improving drought tolerance of a plant, comprising: introducing a nucleotide sequence of the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; Preferably, the plant is tobacco.

A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the nucleotide sequence of the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.

7. The method of claim 6 wherein the plant is tobacco.

The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving drought tolerance of a plant and for use in plant breeding.

9. The use of claim 8 wherein the plant is tobacco.

10. The gene-encoded protein of claim 1, as set forth in SEQ ID NO: 1.