WO2015042748A1 - Dehydrin protein dh7 from cotton, and coding gene and use thereof - Google Patents

Abstract

Disclosed are a dehydrin protein GhDH7 from cotton, a coding gene thereof, and a use thereof in breeding a transgenic plant with improved drought tolerance.

Description

 FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a dehydrin protein derived from cotton and a gene encoding the same, and a drought tolerance thereof Increased use in transgenic plants. Background technique

 Stresses such as temperature, salt and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to agricultural production and the natural environment. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters, which is the bottleneck of agricultural development in many regions. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350-40 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 spring droughts frequently reach 10 years.

 Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The use of conventional breeding methods to improve the drought resistance of crops is limited by the long cycle and lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.

When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. 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 will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION The present inventors cloned a coding gene of a dehydrin protein (designated herein as "H7)) of cotton using SSH (Suppression Subtractive Hybridization) in combination with RACE (rapid amplification of cDNA ends), and determined its DNA sequence. And found that after introducing it into plants for over-expression, it can significantly improve the drought tolerance of transgenic plants, and these The shape can be stably inherited.

 A first aspect of the invention provides a gene encoding a dehydrin protein/) H7 of cotton (herein designated GhDH7, the sequence of which is SEQ ID NO: 2.

 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 35S-G)H7-2300 vector shown in Fig. 2.

 The 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 present invention provides a method for improving drought 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 causing the gene Expression; 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 a plant 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 Tissue; 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 drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is Arabidopsis thaliana.

 The seventh aspect of the present invention provides the gene-encoded protein according to the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector C35S-G)H7-2300) of G)H7 (Fig. la-lb).

 Figure 2 is a plasmid map of the plant expression vector (35S-G) H7-2300 of GhDH7.

 Figure 3 shows the results of drought tolerance simulation experiments of G)H7 T1 transgenic Arabidopsis plants (in the figure, T1G1) and non-transgenic Arabidopsis plants (in the figure, CK) as controls. (Fig. 3a is an Arabidopsis plant that grows normally for 20 days; Fig. 3b shows an Arabidopsis plant that has been treated for 14 days after normal growth for 14 days).

Figure 4 is a graph showing the results of protein expression verification at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants. M is DNA Ladder Marker (DL2000, TakaRa), 1-4 is a drought-tolerant transgenic Arabidopsis T1 plant, 5-8 is a non-transgenic Arabidopsis control, and 9-14 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant. (In order: T1G1, T1G2, T1G3, T1G4, T1G5, T1G6). BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

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

 The specific method is:

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

(1) Test materials:

冀棉14 (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded onto sterilized vermiculite at 16 °C, photoperiod 16 hours light / 8 hours dark (light intensity 2000 - 3000 Lx), 1/2 MS liquid medium per week (containing 9.39 mM KN0 ₃ , 0.625 mM KH ₂ P0 ₄ , 10.3 mM NH ₄ N0 ₃ , 0.75 mM MgSO ₄ , 1.5 mM CaCl ₂ , 50 μΜ ΚΙ, 100 μΜ Η ₃ ΒΟ ₃ , 100 M MnS0 ₄ , 30 μΜ ZnS0 ₄ , 1 μΜ Na ₂ Mo0 ₄ , 0.1 μΜ CoCl ₂ , 100 μΜ Na ₂ EDTA, 100 μΜ FeS0 ₄ ) once. When the seedlings were cultured for about 1 month, they were used for experiments.

 (2) Material handling:

 The above test seedlings were divided into two groups, each with 4 pots and 1 pot per pot. The first group was a control group, which was cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and was normally watered. The second group was the drought treatment group, cultured at 25 °C, photoperiod of 16 hours light/8 hours darkness, stopped watering, and treated for 10 days. After the treatment, the leaves of the top two groups of the seedlings were cut in time. After the nitrogen was rapidly frozen, it was stored in a -70 ° C refrigerator.

 (3) Total RNA extraction:

The cotton leaves of the control and drought-treated groups were each 0.1 g, and the total RNA of cotton leaves was extracted with a plant RNA extraction kit (purchased from Invitrogen). The absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The ratio of OD ₂₆₀ / OD ₂₈₀ was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose gel was used. Gel electrophoresis detected the integrity of total RNA. The brightness of the 28S band was about twice that of the 18 S band, indicating that the integrity of RN A was good. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purification of poly A+ RNA from total RNA, purified polyA+ RNA from total RNA). (4) Suppression of subtractive hybridization:

Suppression Subtractive Hybridization performed by PCR-select ^TM cDNA Clontech's method shown Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and P 2 g Driver cDNA as starting materials. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 h 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 two products of the first forward subtractive hybridization were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain a rich set.

 In order to increase the availability of the Expressed sequence tag (EST) (unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps. The cDNA was digested with Driver cDNA and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications. Finally, the second inhibitory PCR products of the two groups of forward subtractive hybridization cDNA fragments were combined.

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

 The second PCR product of the above combined positive subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega) -T Easy vector ligation, the specific steps are as follows: The following components are sequentially added to a 200 μΐ PCR tube: the second inhibitory PCR product of the purified combined positive subtractive hybridization cDNA fragment 3 μΚ2χΤ4 DNA ligase buffer 5 Μ1, pGEM-T Easy vector 1 μ1, Τ4 DNA ligase 1 μΐ, and ligated overnight at 4 °C. Then 10 μL of the ligation reaction product was added to 100 μL of competent E. coli JM109 (purchased from TAKARA) and mixed, ice bath for 30 min, heat shock at 42 ° C for 60 s, ice bath for 2 min, and 250 LB liquid. The medium (containing 1% tryptone (purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Chinese medicine) was placed in a 37 ° C shaker. The culture was shaken at 225 r/min for 30 min, and the obtained bacterial solution was the subtracted library bacterial solution. Add glycerin to a final concentration of 20% (V/V) and store at -80 °C for later use.

200 μL of the subtractive library solution was applied to 50 g/mL ampicillin (purchased from Beijing Bayerdi), 40 g/mL X-gal (5-bromo-4-chloro-3-indol-β -D-galactoside), 24 g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) (X-gal and IPTG are purchased from TAKARA) on LB (ibid.) on solid culture plates Incubate at 37 ° C for 18 hours. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 198 white colonies (number: Gh-B001 to Gh-B198). The picked white colonies were inoculated separately into LB liquid medium containing 50 g/mL ampicillin in a 96-well cell culture plate (CORNING), and cultured overnight at 37 ° C, and then glycerol was added thereto. The final concentration of glycerol is 20% (volume ratio) and stored at -80 ° C for later use. The cultured colony clones were subjected to nested PCR (primer Primer 1 and Primer 2R, PCR-selectTM cDNA Subtraction Kit from Clontech) for PCR amplification, and 190 positive clones were obtained, and then all were positive. The clone was sent to Yingjie 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 135 ESTs (Unigene;) were obtained. After analysis, there were 21 contigs with 114 single sequences. BlastN found that 48 of the ESTs (Unigene) have homologous sequences in GenBank, 32 ESTs are unknown or hypothetical proteins, and 34 have not obtained homologous matches, presumably at the 3' or 5' end. A shorter sequence of regions. Example 2 Cloning of cotton dehydrin protein-encoding gene GhDH7

 After the cloned Gh-B76 was detached from the redundant DNA, the sequence was SEQ ID NO: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the dehydrin protein. The full-length coding gene corresponding to the clone Gh-B76 was named G). H7, its corresponding protein is named £)H7.

SEQ ID NO: 3

 1 GAAGGTGAAG GCGGAGAGAA GAAGAAGAAG AAGAAAAACG AGAAGGGAAA GAAAGAACAG 61 GACAGTGCAG TCCCAGTGGA GAAGTGCGAT GAGGCAGCAA CAGTTCACCA CTCAGAGACG 121 CCGGAAAAGA AGGGTTTCAT GGATAAGATC AAAGACAAAC TCCCAGGACA GCATAAGAAA 181 GATGAAGAGG ACACCACCCA ACCACCAGCT GCTGCTGCCC CAACTGAGAA CGACCACCAT 241 GAAGGAGAGA CAAAAGAGAA GAAGGGATTT TTGGAGAAAA TCAAGGAGAA AATTCCTGGT 301 TACCACTCCA AAAGAGAGGA CGAGAAGGAA AAAGAGACCA CTGCTCCCCA TTAA

Cloning of the full-length coding gene of DH7

 Sequence analysis according to SEQ ID NO: 3 which has been obtained: SEQ ID NO: 3 is the 3 '-end sequence of the coding gene G ) H7 . Based on the sequence of SEQ ID NO: 3 which has been obtained, the following three specific primers were designed as specific primers for reverse transcription primers and 5 'RACE.

GH-B76GSP1: SEQ ID NO: 4:

 ATGGTGGTCGTTCTCAGTTG GH-B76GSP2 : SEQ ID NO: 5:

 TGGGTGGTGTCCTCTTCATC GH-B76GSP3: SEQ ID NO:6:

 GAAGGTGAAGGCGGAGAGAA

The kit comes with universal primers: AAP : SEQ ID NO: 7:

 GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP : SEQ ID NO: 8:

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

The Ends kit was purchased from Invitrogen).

 GH-B76GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and the mRNA extracted from cotton leaves of the drought-treated group was used as a template for reverse transcription to obtain a cDNA template, and then added according to the steps in the above 5' RACE kit instructions. Poly C j , the first round of PCR amplification using the tailed product as a template, the primer used is SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (provided by the kit, I is hypoxanthine modified) a, c, g or t), the specific steps are as follows:

 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 10 min.

 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 using SEQ ID NO: 5 and the general primer SEQ ID NO: 8. The specific steps are as follows:

 50 μΐ PCR reaction system: 5 μΐ Ι Ο Χ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 μΐ Ex Taq, 10 μΜ primer SEQ ID NO: 5 and P SEQ ID NO: 8 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min. The strip of about 450 bp in the second PCR product was recovered (the Gel Extraction Kit was purchased from OMEGA), and it was ligated into the pGEM-T Easy Vector vector, and then converted to JM109 (the specific method is the same as above), and 6 randomly selected. The white colonies were inoculated separately in an LB liquid medium containing 50 μl of ampicillin, and cultured overnight at 37 ° C, and then glycerin was added to a final concentration of glycerol of 20% by volume, and stored at -80 ° C until use. The primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above). 6 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. Sequencing, a 5' end sequence of the cDNA of the gene was obtained. The resulting 5 'RACE product clone 06 was sequenced to obtain the sequence of SEQ ID NO: 9:

1 GGGGGGGGGG ATGGCTGAGG AGCATACCAA GGTTGGTGGT GAGGAAGCAG TGGAGAGCAA

61 GGAGCGAGGG ATGTTTGATT TCTTGGGGAA GAAAGAAGAT GAGAAGCCTC AACCTCAAGA 121 GGAGGTGGTC GCCACCCAGT TTGAGAAAGT CAAGATAGAA GAGGAACATA AGGAAGATGA

181 GAAGAAACAC AGTCTCCTGG ACAAGCTTCA CCTATCCAAT AGCAGCTCCA GCTCTTCTAG

241 TGACGAGGAA GAAGGTGAAG GCGGAGAGAA GAAGAAGAAG AAGAAAAACG AGAAGGGAAA

301 GAAAGAACAG GACAGTGCAG TCCCAGTGGA GAAGTGCGAT GAGGCAGCAA CAGTTCACCA

361 CTCAGAGACG CCGGAAAAGA AGGGTTTCAT GGATAAGATC AAAGACAAAC TCCCAGGACA

421 GCATAAGAAA GATGAAGAGG ACACCACCCA The sequence obtained by 5 ' RACE SEQ ID NO: 9, was spliced with the obtained sequence SEQ ID NO: 3 to obtain SEQ ID NO: 10:

 1 GGGGGGGGGG ATGGCTGAGG AGCATACCAA GGTTGGTGGT GAGGAAGCAG TGGAGAGCAA

61 GGAGCGAGGG ATGTTTGATT TCTTGGGGAA GAAAGAAGAT GAGAAGCCTC AACCTCAAGA

121 GGAGGTGGTC GCCACCCAGT TTGAGAAAGT CAAGATAGAA GAGGAACATA AGGAAGATGA

181 GAAGAAACAC AGTCTCCTGG ACAAGCTTCA CCTATCCAAT AGCAGCTCCA GCTCTTCTAG

241 TGACGAGGAA GAAGGTGAAG GCGGAGAGAA GAAGAAGAAG AAGAAAAACG AGAAGGGAAA

301 GAAAGAACAG GACAGTGCAG TCCCAGTGGA GAAGTGCGAT GAGGCAGCAA CAGTTCACCA

361 CTCAGAGACG CCGGAAAAGA AGGGTTTCAT GGATAAGATC AAAGACAAAC TCCCAGGACA

421 GCATAAGAAA GATGAAGAGG ACACCACCCA ACCACCAGCT GCTGCTGCCC CAACTGAGAA

481 CGACCACCAT GAAGGAGAGA CAAAAGAGAA GAAGGGATTT TTGGAGAAAA TCAAGGAGAA

541 AATTCCTGGT TACCACTCCA AAAGAGAGGA CGAGAAGGAA AAAGAGACCA CTGCTCCCCA

601 TTAA According to the sequence analysis of SEQ ID NO: 10, SEQ ID NO: 10 is the full length sequence of GhDH7. A pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:

GhDHTF: SEQ ID NO: 1 1:

 ATGGCTGAGGAGCATACCAAG

GhDH7R: SEQ ID NO: 12:

 TTAATGGGGAGCAGTGGTCTC AP : SEQ ID NO: 13:

 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT The G) full-length coding sequence of G7 was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.

 The RNA of the drought-treated cotton was extracted as a template, the primer SEQ ID NO: 13 was used as the reverse transcription primer, the cotton cDNA was obtained by reverse transcription, and then the PfuUltra II Fusion HS DNA Polymerase of stratagene was used, and the cDNA of the cotton obtained above was used as a template. Perform a PCR reaction. 50 μΐ PCR reaction system: 5 μΐ 10 X PfuUltra II reaction Buffer 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primers SEQ ID NO: 1 1 and SEQ ID NO: 12 2.0 μ1, and 37.5 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 51 °C for 25 s, extension at 72 °C for 30 min), extension at 72 °C for 5 min.

PCR amplification product plus A tail: PCR product hydration to 400μ1, first extracting with chloroform to remove protein, suck The supernatant was added to a solution of 40 μl of 3 M sodium acetate solution, and 2 volumes of absolute ethanol were added thereto, allowed to stand at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μΐ Ι Ο Χ Εχ Buffer 0.5 μΐ 5 mM dATP and P 1.0 μΐ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 600 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector (to obtain G) H7-pGEM plasmid), and then transformed into JM109, and 8 white colonies were randomly picked and inoculated to contain 50. G/mL ampicillin was cultured in LB liquid medium, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C until use. The primers SEQ ID NO: 1 1 and SEQ ID NO: 12 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, sequence. Is SEQ ID NO: 2, the amino acid sequence of the encoded protein is SEQ ID NO: 1

 Amino acid sequence of DH7 protein: SEQ ID NO: 1

MAEEHTKVGG EEAVESKERG FDFLGKKED EKPQPQEEW ATQFEKVKIE EEHKEDEKKH

 61 SLLDKLHLSN SSSSSSSDEE EGEGGEKKKK KKKEKGKKEQ DSAVPVEKCD EAATVHHSET

121 PEKKGF DKI KDKLPGQHKK DEEDTTQPPA AAAPTENDHH EGETKEKKGF LEKIKEKI PG

181 YHSKTEDEKE KETTAPH

 G) nucleotide sequence of the H7-encoding gene: SEQ ID NO: 2

 1 ATGGCTGAGG AGCATACCAA GGTTGGTGGT GAGGAAGCAG TGGAGAGCAA GGAGCGAGGG

61 ATGTTTGATT TCTTGGGGAA GAAAGAAGAT GAGAAGCCTC AACCTCAAGA GGAGGTGGTC

121 GCCACCCAGT TTGAGAAAGT CAAGATAGAA GAGGAACATA AGGAAGATGA GAAGAAACAC

181 AGTCTCCTGG ACAAGCTTCA CCTATCCAAT AGCAGCTCCA GCTCTTCTAG TGACGAGGAA

241 GAAGGTGAAG GCGGAGAGAA GAAGAAGAAG AAGAAAAAGG AGAAGGGAAA GAAAGAACAG

301 GACAGTGCAG TCCCAGTGGA GAAGTGCGAT GAGGCAGCAA CAGTTCACCA CTCAGAGACG

361 CCGGAAAAGA AGGGTTTCAT GGATAAGATC AAAGACAAAC TCCCAGGACA GCATAAGAAA

421 GATGAAGAGG ACACCACCCA ACCACCAGCT GCTGCTGCCC CAACTGAGAA CGACCACCAT

481 GAAGGAGAGA CAAAAGAGAA GAAGGGATTT TTGGAGAAAA TCAAGGAGAA AATTCCTGGT

541 TACCACTCCA AAACAGAGGA CGAGAAGGAA AAAGAGACCA CTGCTCCCCA TTAA Example 3 G) Construction of H7 gene plant 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 Pnos promoter was used to replace the CaMV35S promoter containing the double enhancer in the ΝΡΤΠ gene to reduce the expression of prion protein in plants. . The 35S promoter and the terminator Tnos were inserted upstream of the Pnos promoter as the promoter and terminator of the GhDH7 gene, respectively, and the GhDH7 gene was between the 35S promoter and the Tnos terminator.

 Pnos were amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 14 and SEQ ID NO: 15, using TaKaRa's PrimeSTAR HS DNA polymerase.

50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 μl, And 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (denaturation at 94 ° C for 30 s, annealing at 56 ° C for 30 s, extension at 72 ° C for 30 s), extension at 72 ° C for 10 min. The resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.

SEQ ID NO: 14:

 GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15 :

 ATCCAGATCTAGATCCGGTGCAGATTATTTG The primers SEQ ID NO: 16 and SEQ ID NO: 17 were used to amplify Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. The resulting PCR product was digested with Sacl, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2.

SEQ ID NO: 16:

 AAGG^GCJCGAATTTCCCCGATCGTTCAAA

 SEQ ID NO: 17:

 TCKGAA rrCCC AGTGAATTCCCGATCT AGT A The 35S promoter was amplified using the primers SEQ ID NO: 18 and SEQ ID NO: 19 using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μΙ ΡΟ Reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 primer 8 £0 10 ^^0: 18 and 8 £ 0 10 ^^0: 19 each of 2.0 ^, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. After digestion with HindIII and Pstl, the obtained PCR product was ligated to the same (preferably as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3.

SEQ ID NO: 18:

 ACT^GCrJATGGTGGAGCACGACACTCT

SEQ ID NO: 19: TGACJGC^GAGAGATAGATTTGTAGAGAGAGAC GhDH7 (template was the positive GhDH7-pGEM plasmid obtained in Example 2) was amplified using primers SEQ ID NO: 20 and SEQ ID NO: 21, using strafine's PfuUltra II Fusion HS DNA Polymerase. 50 μl ΡΟ Reaction system: 5 μΐ lOxPfuUltra II reaction Buffer, 0.5 μl 25 mM dNTP, 2.0 μΐ GhDH7-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primers SEQ ID NO: 20 and SEQ ID NO: 21 each of 2.0 μl, and 37.5 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 58 °C for 25 s, extension at 72 °C for 30 s), extension at 72 °C for 5 min. The resulting PCR product was ligated by Pstl and Sacl (connection method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-G)H7-2300.

 SEQ ID NO: 20:

 AACTGCAG ATGGCTGAGGAGCATACCAAG SEQ ID NO: 21:

AAGGAGCTC TTAATGGGGAGCAGTGGTCTC Example 4 35S-GhDH7-2300 expression vector for transformation of Agrobacterium

Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was plated on LB solid medium containing 50 g/ml rifampicin and 50 g/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 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 streptomycin, and cultured overnight (about 12-16 hours) to OD ₆ at 28 °C. _{The K)} value is 0.4, and a seed bacterial liquid is formed. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, and cultured at 28 ° C for 2 to 2.5 hours with shaking. To OD ₆ . . =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; resuspend the cells by adding a certain amount of ice-cold 10% glycerol (volume), centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; Cold 10% glycerol (volume) was washed 3-4 times repeatedly; the bacterial pellet was resuspended by adding an appropriate amount of ice-cold 10% glycerol (volume) to prepare LBA4404 competent cells, which were dispensed at 40 μM/tube. Store at -70 ° C for later use.

Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μΐ of the positive 35S-G)H7-2300 plasmid obtained in Example 3 was added to 40 μΐ of competent cells, and the mixture was mixed and ice bathed for about 10 min. Transfer the mixture of competent cells and 35S-GhDH7-2300 plasmid DNA to a ice-cold electric shock cup (purchased from bio-rad) with a pipette, tap to bring the suspension to the bottom of the electric shock cup, be careful not to have bubble. Place the electric shock cup on the slide of the electric shock room, push The slide places the electric shock cup to the base electrode of the shock chamber. Using a 0.1 cm size electric shock cup, the MicroPulser (purchased from bio-rad) program is set to "Agr" and the shock is applied once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently mix the competent cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 hour with shaking. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ _§ /ιη1 rifampicin, 50 μ _§ /ιη1 streptomycin and 50 μ _§ /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use.

 Example 5 Agrobacterium-mediated transformation was used to obtain transgenic Arabidopsis thaliana

Plants to be transformed: Arabidopsis seeds (Columbia type, Arabidopsis thaliana Bioresource Center, Ohio State University) Seeded in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 hours light Sprouting in an 8 hour dark incubator. After 7-10 days, transplanted into a plastic crucible with a diameter of 7.5 cm containing peat soil and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 ° C, 16 hours light / 8 hours dark Growing in the incubator. 1/2MS medium (9.39 mM KN0 ₃ , 0.625 mM KH ₂ P0 ₄ , 10.3 mM H ₄ N0 ₃ , 0.75 mM MgS0 ₄ , 1.5 mM CaCl ₂ , 50 μΜ ΚΙ, 100 μΜ Η ₃钵 per 前 before transplanting. ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1 μΜ Na ₂ Mo0 ₄ , 0.1 M CoCl ₂ , 100 μΜ Να ₂ ΕϋΤΑ, 100 M FeSO ₄ ) 40 ml, and the soil moisture is replenished in time after transplanting. The nutrient solution is properly watered during the growth period. Every 3-4 weeks (or longer) as needed. In order to obtain more flower buds on each plant, when the first inflorescence of most plants is formed, the first inflorescence is cut off, and the apical dominance is removed, prompting the simultaneous emergence of multiple secondary inflorescences. Dip is prepared when most inflorescences are about 1-10 cm high (about 4-8 days after the first inflorescence is cut).

Culture of Agrobacterium: After removing the bacterial solution of the Agrobacterium-positive transformed clones preserved in Example 4, a single Agrobacterium colony was picked and inoculated into 10 mL of sterile LB liquid medium (containing 75 mg/L of rifampicin, 100 mg/L streptomycin and 100 mg/L kanamycin were shaken overnight at 250 °C/min at 28 °C. Then inoculate the obtained bacterial solution in a volume ratio of 1% to 2% to 200 mL of sterile LB liquid medium (containing 75 mg/L rifampicin, 100 mg/L streptomycin, and 100 mg/L Kana). In the case of chloramphenicol, at 250 °C at 28 °C, the concentration of Agrobacterium reached 00 ₆₍₎₍₎ = 1.8, then at 4 °. Centrifuge at 3000 r/min for 15 min, discard the supernatant and use the dip medium (the dip medium is 5.0% (w/v) sucrose and 0.05% (500 μΙ7ί) of Silwet L in 1/2MS medium. -77) Resuspend Agrobacterium and suspend to OD ₆ QQ about 0.80.

Inflorescence Dip: The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that the above ground tissues are completely immersed in the Agrobacterium suspension for 3-5 s and gently agitated. There should be a liquid film on the plant after infiltration. Dip-infected plants are placed in plastic trays, covered with clean plastic or cling film to moisturize, and then placed in low light or dark places overnight. Take care to prevent direct sunlight from directing the plant. Remove the cover approximately 12-24 hours after processing. The plants are cultured normally, and the plants are further grown for 3-5 weeks until the pods are browned and dried. The seeds were harvested and the seeds were dried and stored in a centrifuge tube at 4 °C.

Transgenic seed screening: formulated with 1/4 MS (4.695 mM KN0 ₃ , 0.3125 mM KH ₂ P0 ₄ , 5.15 mM H4NO3, 0.375 mM MgS0 ₄ , 0.75 mM CaCl ₂ , 25 μΜ ΚΙ, 50 μΜ Η ₃ ΒΟ ₃ , 50 M MnSO ₄ , 15 M ZnS0 ₄ , 0.5 μΜ Να ₂ Μο0 ₄ , 0.05 M CoCl ₂ , 50 μΜ Na ₂ EDTA, 50 M FeSO4) A large amount of aqueous solution of the element, add 0.8% agar powder, and heat it in a microwave oven until the agar is completely dissolved. Cool to about 50 ° C, add the required amount of 50 mg of kanamycin, shake well, pour 25 mL into each dish, set the bench to cool and solidify, then seed. Pour the weighed seeds on a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the container at 4 °C for 72 hours, then move to Germinating in a 23 ° C, 16 hour light / 8 hour dark incubator, regular seed germination and seedling growth, and timely transplanting the resistant seedlings into the nutrient soil. After transplanting, the soil moisture is added to the water in time. The nutrient solution is properly watered during the growth period. Take 0.1 g of Arabidopsis leaves grown for 20 days, extract DNA, and amplify GhDH7 with SEQ ID NO: 11: P SEQ ID NO: 12, 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μl Εχ Τα 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 51 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 7 min, identification of PCR as positive Plants are numbered (T1G1-T1G12) and stored. Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing GhDH7. The sterilized vermiculite was soaked with 1/2 MS medium. T1G1-T1G6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 °C, 10 hours light culture/14 hours dark culture cycle, 1/2MS every 7 days, after 20 days of culture 4 seedlings of uniform size were kept in each pot for drought experiments. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana drought for 14 days (without watering), 25 °C, 10 hours light culture / 14 hours dark culture cycle. The drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, and there were 26 strains of T1G1, T1G2, T1G3, T1G4, T1G5 and T1G6. 21 out of 4) Arabidopsis thaliana survived and continued to grow, showing significant drought tolerance (see Figures 3a and 3b, using T1G1 as an example, T1G2, T1G3, T1G4, T1G5, T1G6 results and T1G1 is similar, not shown here). Example 7 Verification of G) H7 protein expression at the transcriptional level

 Control Arabidopsis thaliana plants, drought-tolerant transgenic Arabidopsis T1 plants (T1G1, T1G2, respectively)

10 strains of T1G3, T1G4, T1G5, T1G6) and drought-tolerant transgenic Arabidopsis thaliana T1 plants The leaves were each 0.05 g, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 total RNA as a template and reverse transcription primer as SEQ ID NO: 13). The relative expression of DH7 protein was detected by amplifying GhDH7 by SEQ ID NO: 11 and SEQ ID NO: 12.

 PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primer SEQ ID NO: 11 and P SEQ ID NO: 12 each 2.0 μ1, and 30 ΐ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 10 min.

 The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, TakaRa), 1-4 is a drought-tolerant transgenic Arabidopsis T1 plant, 5-8 is a non-transgenic Arabidopsis control, 9-14 is Drought-tolerant transgenic Arabidopsis thaliana T1 plants (in order: T1G1, T1G2, T1G3, T1G4, T1G5, T1G6). The size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of G)H7 (about 600 bp). The results showed that there was no GhDH7 transcription in Arabidopsis thaliana, and GhDH7 transcription was stronger in T1 generation plants of drought-tolerant transgenic Arabidopsis thaliana. The transcription of GhDH7 in the T1 generation of Arabidopsis thaliana plants was weak.

Claims

claims 1. A protein encoded by a cotton dehydrin encoding gene, whose amino acid sequence is SEQ ID NO: 1. 2. The gene encoding the protein of claim 1, whose nucleotide sequence is shown in SEQ ID NO: 2. 3. A recombinant expression vector, which contains the gene of claim 2 and the nucleotide sequence of the gene is operably linked to the expression control sequence of the expression vector. 4. The recombinant expression vector according to claim 3, which is the 35S-G)H7-2300 vector shown in Figure 2. 5. A recombinant cell containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell. 6. A method for improving plant drought tolerance, comprising: combining the gene described in claim 2 or the gene described in claim 2 The recombinant expression vector described in 3 or 4 is introduced into a plant or plant tissue and the gene is expressed; preferably, the plant is Arabidopsis thaliana. 7. A method for preparing transgenic plants, comprising: cultivating plants or plant tissues containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions that are effective for producing plants. 8. The method of claim 7, wherein the plant is Arabidopsis thaliana. 9. The use of the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for improving plant drought tolerance and for plant breeding. 10. The use of claim 9, wherein the plant is Arabidopsis thaliana.