WO2015024143A1 - Zinc finger protein zat10-1 from cotton, and coding gene and uses thereof - Google Patents

Abstract

The present invention relates to a plant protein, a coding gene and uses thereof, and in particular, to a zinc finger protein ZAT10-1 from cotton, a coding gene thereof, and uses thereof in breeding a transgenic plant with improved salt tolerance.

Description

 Cotton zinc finger protein ZAT10-1 and its coding gene and application

FIELD OF THE INVENTION The present invention relates to plant proteins and their encoding genes and applications, and more particularly to a cotton-derived zinc finger protein ZAT10-1 and its encoding gene, and its use in the cultivation of transgenic plants having 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 improvement of plant resistance to salt and alkali, drought-tolerant ability and the selection of 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} 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, which has made breakthroughs in the research in this field ( Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53: 1247-1273; Zhang ZL. 2011. Arabidopsis Floral Initiator SKB1 Confers High Salt Tolerance by Regulating Transcription and Pre- mRNA Splicing through Altering Histone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cel l, 23: 396-411).

Higher plant cells can sense changes in physicochemical parameters in the external environment through various pathways, thereby transforming extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions. The transcription factor is activated, and the activated transcription factor acts on the functional gene to initiate 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 factors for plant salt tolerance have not been found; the molecular mechanism of plant salt tolerance is not well understood. SUMMARY OF THE INVENTION The present inventors cloned a zinc finger protein (designated herein as ZAT10-1) encoding gene of Gossypiim herbaceim D of African cotton using SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends). And its DNA sequence was determined. Furthermore, it was found that after introduction into the recipient plant and expression thereof, the salt tolerance of the recipient plants was significantly improved, and these traits were stably inherited.

 The first aspect of the invention provides a gene encoding a zinc finger protein ZAT10-1 of cotton (designated herein as

GhZA T10-l), 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 An expression control sequence for the expression vector is operably linked; preferably, the vector is the 35S-GhZAT10-l-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 present 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 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 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. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a construction flow of a plant expression vector (35S-GhZAT10-l-2300) of a gene (Fig. la-lb).

 Figure 2 is a plasmid map of the plant expression vector of the gene (35S-GhZAT10-l-2300). Figure 3 shows the results of salt tolerance simulation experiments of transgenic 1\ transgenic Arabidopsis plants (right panel, TVV3-6) and non-transgenic Arabidopsis plants (left panel, CK).

 Figure 4 is a verification result of molecular detection of genes at the transcriptional level in 1\ generation transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. The upper and lower partial bands shown in the figure belong to Π and tac - respectively as internal parameters. 1_4 is a salt-tolerant non-transgenic control Arabidopsis plant, 5-12 is a salt-tolerant T1 transgenic Arabidopsis plant (in the order of Ί\Α3-1, Ί\Α3-6, Ί\Α3-8 and Ί \Α3_16 four salt-tolerant strains, 2 strains per strain), 13 is 35S-GhZAT10-l-2300 plasmid PCR positive control, 14-17 is transgenic salt-tolerant Arabidopsis plants (subordinate to 2 strains) 2 strains were tested, and 18 was a blank control without template. The results showed that the transcription level of GhZATW-l in salt-tolerant transgenic Arabidopsis thaliana plants was higher in the salt-tolerant transgenic Arabidopsis thaliana plants, and the transcription level of the salt-tolerant transgenic Arabidopsis plants was very high. low. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.

 The unrecognized restriction endonucleases mentioned in the examples below were purchased from New England Biolabs.

 Example 1. Cotton SSH library construction under salt stress:

 The specific method is:

 The subtraction subtraction library was constructed by the method of inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit. The mRNA of the leaves of the cotton seedlings treated with salt was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control. The 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 onto a sterilized vermiculite substrate, cultured at 25 ° C, light dark cycle 16 h / 8 h, each 1/2MS liquid medium (containing 9.39 mM KN0 ₃ , 0. 625 mM KH ₂ P0 ₄ , 10. 3 mM N N0 ₃ , 0.75 mM MgS0 ₄ , 1. 5 mM CaCl ₂ , 50 μ M KI, 100 μ Μ H ₃ B0 ₃ , 100 μ Μ MnS0 ₄ , 30 μ M ZnS0 ₄ , 1 μ M N3⁄4Mo0 ₄ , 0. 1 μ Μ CoCl ₂ , 100 μ M N3⁄4 EDTA, 100 μM 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 is the processing group, 25. C. Incubate under light, place in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treat for 6 hours. After the treatment, the leaves of the two groups of seedlings should be cut out in time and quickly frozen with liquid nitrogen. Store in a -70 ° C refrigerator.

 (3) Total RNA extraction:

The cotton leaves of the control and the salt-treated group were each 0.5 g, and the total RNA of the cotton leaves was extracted with the plant RNA extraction kit (Invitrogen). Absorbance values of total RNA at 260 nm and 280 nm, 0D ₂₆ , were determined using a HITACHI UV spectrophotometer U-2001. /0D 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 The integrity of the RNA is good. The mRNA was isolated using Qiagen's Oligotex mRNA purification assay [JC (Purification of poly A+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

 Suppression subtractive hybridization was performed as indicated by Clontech's PCR-selectTM cDNA Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain a 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 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 products of the two first subtractive hybridizations 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 enrichment.

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

The second PCR product of the combined positive subtractive hybridization cDNA fragment (using QIAquick PCR) according to the method described in the product manual for the pGEM-T Easy kit (purchased from Promega) Purification Kit purification, purchased from Qiagen) and pGEM_T Easy vector, the specific steps are as follows: The following components were sequentially added using a 200 μΐ PCR tube: The second PCR product of the purified positive subtractive hybridization cDNA fragment 3 μ 1, 2 Χ Τ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μ ΐ, ligated overnight at 4 °C. Take 10 ligation reaction products, add 100 μL of E. coli JM109 competent cells (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, then add 250 μL LB medium (including 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, and 200 μL of culture solution after shaking culture was taken. Planted in 50 μg/mL ampicillin (purchased from Beijing Bayer), 40 μg/mL X-gal ( 5-bromo-4chloro_3_吲哚-β _D_galactoside), 24 μ g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) (X-gal and IPTG were purchased from TAKARA) on LB (ibid.) solid culture plates, 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 300 white colonies (number: Gh-S2-001 to Gh-S2-300). All white colonies were inoculated separately in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μg/mL ampicillin, and cultured overnight at 37 °C with glycerol to a final concentration of 20% at -80 °C. Save spare. The nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to verify the PCR amplification of the bacteria, and 231 positive clones were obtained. All positive clones were sent to Yingjiejieji. (Shanghai) Trading Co., Ltd. sequencing

 (6) cDNA sequencing analysis of differential clones:

 After removing the DNA sequencing results of the above 231 differential clones and removing the vector and the ambiguous sequence and the redundant cDNA, a total of 203 effective ESTs (Unigene) were obtained. Example 2 Cloning of the African cotton zinc finger protein gene GhZA TW-1

 After the sequencing result of the clone Gh-S2-123 was removed, the sequence was SEQ ID NO: 3. The full-length coding gene corresponding to SEQ ID NO: 3 was named GhZA TW-l, and the corresponding protein was named as ZAT10-1.

SEQ ID NO: 3

 1 ACGGTTTGTA ACAAGGGATT TCCATCTCAT CAAGCTTTAG GAGGACACAA GGCCAGTCAC

61 AGGAAACTCG TTGTTGGAGC TGGAGATCAC CTCACCATCA CCACCGCCGC CACCAAGACC

121 ACTGCTGATG TCTCCGCTGT TGCTGCCACC ACAGTCACGC CGCCTAAAAA TTTACCTCTC 181 ATGATTAATC GAGTTGGTAA CAAGAATCAC GTGTGTTCAA TTTGTAACAA GACATTCTCT 241 TCAGGTCAAG CTTTAGGAGG GCATAAGCGA TGCCACTACG AAGCTGGCAG CAATAACAAC

301 AGCGGCAGCG ATGGTGCTAA GTTACCGAGC CAAAGTCAAC GTGACTTTGA CTTAAATTTG

361 CCTGCTGGAC CGCAGGAACT AAGT

 Cloning of the full-length coding gene of GhZA TlO-1

 Based on the sequence of SEQ ID NO: 3 that has been obtained, two specific primers were designed as the 5'-end specific primer for 3' RACE: Gh ZAT10-1_1 GSP1: SEQ ID NO: 4:

 GGTCAAGCTTTAGGAGGGCAT

 Gh ZAT10-1_2 GSP2: SEQ ID NO: 5:

 CGATGGTGCTAAGTTACCGAG

 Reagents come with universal primers:

 AP: SEQ ID NO: 6:

 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT

AUAP: SEQ ID NO: 7:

 GGCCACGCGTCGACTAGTAC

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

 Using SEQ ID NO: 4 and 3' primer SEQ ID NO: 7 (the AUAP primer provided by the kit), reverse transcription of SEQ ID NO: 6 primer (AP primer derived from the kit) and cotton mRNA. The cDNA was used as a template for the first round of PCR amplification. Specific steps are as follows:

 50 μ 1 PCR reaction system: 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 mRNA reverse transcribed cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO : 4 and P SEQ ID NO: 7 each of 2.0 μ 1, and 35 μl 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 53 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.

 The obtained PCR product was diluted 50 times with double distilled water and 2.0 μl was used as a template, and SEQ ID NO:

5 and 3' primers SEQ ID NO: 7 for the second round of PCR amplification, the specific steps are as follows:

50 μ 1 PCR reaction system: 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq, 10 μΜ primers SEQ ID NO: 5 and SEQ ID NO: 7 each of 2.0 μ 1, and 35 μ 1 of double distilled water. PCR reaction conditions: 94 ° C pre-denaturation 5 Min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min. The second PCR product recovery fragment (Gel Extraction Kit was purchased from OMEGA) was ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (the same method as above), and 8 white colonies were randomly picked up to contain 50 g/mL ampicillin. Incubate in LB liquid medium, incubate at 37 °C overnight, add glycerol to a final concentration of 20%, and store at -80 °C until use. SEQ ID NO: 5 and 3' primer SEQ ID NO: 7 were used for PCR amplification of bacterial cells, and 6 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Co., Ltd. for sequencing and sequencing, and 3' cDNA of the gene was obtained. end.

 The resulting 3' RACE product clone was sequenced to obtain a sequence spliced with SEQ ID NO: 3 to obtain ID NO: 8:

 ACGGTTTGTA ACAAGGGATT TCCATCTCAT CAAGCTTTAG GAGGACACAA GGCCAGTCAC AGGAAACTCG TTGTTGGAGC TGGAGATCAC CTCACCATCA CCACCGCCGC CACCAAGACC ACTGCTGATG TCTCCGCTGT TGCTGCCACC ACAGTCACGC CGCCTAAAAA TTTACCTCTC ATGATTAATC GAGTTGGTAA CAAGAATCAC GTGTGTTCAA TTTGTAACAA GACATTCTCT TCAGGTCAAG CTTTAGGAGG GCATAAGCGA TGCCACTACG AAGCTGGCAG CAATAACAAC AGCGGCAGCG ATGGTGCTAA GTTACCGAGC CAAAGTCAAC GTGACTTTGA CTTAAATTTG CCTGCTGGAC CGCAGGAACT AAGTACTCCC GACGTTAACC AAAGAGATAA ATATTCCACC GACGATGAAG TGGTGGAGAA TCCTTGGCCG CCGGCAAAGC GCGTCGGCTG CCACGTTTGT TGAAAGTCGT AGTAAAATAA GGATTAGATT CAGATTAGGC GAAT

 Based on the sequence of SEQ ID NO: 3 that has been obtained, three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.

 GhZATIO- 1_1 GSP1 : SEQ ID NO: 9:

 GTGACTGTGGTGGCAGCAACAG

 GhZATIO- 1_2 GSP2: SEQ ID NO: 10:

 GGTGATGGTGAGGTGATCTCCAG

 GhZATIO- 1_3 GSP: SEQ ID NO: 11:

 CCTAAAGCTTGATGAGATGGAAATC

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

Using SEQ ID NO: 10 and the universal primer AAP (provided with the kit), reverse transcription of cotton mRNA The cDNA (reverse transcription primer SEQ ID NO: 9) was used as a template for the first 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 mRNA reverse transcribed cDNA, 1. 0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primers SEQ ID NO: 10 and AAP each 2. 0 μ 1, and 35 μ 1 Double steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min), 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: 11 and the universal primer AUAP (provided by the kit), 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 M Primers SEQ ID NO: 11 and AUAP were each 2.0 μl, and 35 μl 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 V for 2 min), extension at 72 °C for 10 min. The second round of PCR product was recovered (purchased from OMEGA using the Gel Extraction Kit), ligated into the pGEM_T Easy vector, transformed into JM109 competent cells (specific method as above), and the transformed bacterial solution was coated in 50 Screening was performed on LB solid medium of yg/mL ampicillin, 40 μg/mL X_gal, 24 μg/mL IPTG. Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 μg/mL ampicillin, and cultured overnight at 37 °C, glycerol was added to a final concentration of 20%, and stored at -80 °C until use. Using SEQ ID NO: 11 and the universal primer AUAP for PCR amplification (the reaction system and reaction conditions are the same as above), three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the cDNA of the gene was obtained. 5' end.

 The sequence obtained by cloning the obtained 5' RACE product was spliced with the sequence of SEQ ID NO: 8, and SEQ ID NO: 12 was obtained.

 1 ACTATAATAG CCCACACCAC ACTTCACTCT CCAAAGGACG ACTTTTATGA TCAGATTCAG

61 ACGTGCCAAT ATTGGTGAGA GCAGAGTATT GCAATTTCTT TTCACCCTTC TTTCTCTCCA 121 CTTATCAACC ACTTTCCTTC CTACTTCCTC CACTCTTCCA TATAAAAATC AATCCTTCCT 181 TCTTCACTGA AGCACTCACA TAACAAGAGA AGAATCATGG CTCTTGACAC ATTGAATTCC 241 CCAACTTCAG CTCCTCCACT GCTGGACCGT GATGATATAG TCGACCTCCA CTGTGTTGAG 301 CCCTGGACCA AGAGGAAAAG AACCAAACGA CCCAGATCCG AAAACCCACC AACCGAAGAA

361 GAATACTTAG CTCTTTGCCT TCTCATGCTG GCTCAGGGAA CAACTACCAG AACCATTCCC 421 TCAGCCTCGG CTGCTTCCAG AACCTCCCTC AACCTTTACA AGTGTACGGT TTGTAACAAG 481 GGATTTCCAT CTCATCAAGC TTTAGGAGGA CACAAGGCCA GTCACAGGAA ACTCGTTGTT

541 GGAGCTGGAG ATCACCTCAC CATCACCACC GCCGCCACCA AGACCACTGC TGATGTCTCC

601 GCTGTTGCTG CCACCACAGT CACGCCGCCT AAAAATTTAC CTCTCATGAT TAATCGAGTT

661 GGTAACAAGA ATCACGTGTG TTCAATTTGT AACAAGACAT TCTCTTCAGG TCAAGCTTTA

721 GGAGGGCATA AGCGATGCCA CTACGAAGCT GGCAGCAATA ACAACAGCGG CAGCGATGGT

781 GCTAAGTTAC CGAGCCAAAG TCAACGTGAC TTTGACTTAA ATTTGCCTGC TGGACCGCAG

841 GAACTAAGTA CTCCCGACGT TAACCAAAGA GATAAATATT CCACCGACGA TGAAGTGGTG

901 GAGAATCCTT GGCCGCCGGC AAAGCGCGTC GGCTGCCACG TTTGTTGAAA GTCGTAGTAA

961 AATAAGGATT AGATTCAGAT TAGGCGAAT A pair of primers were designed according to the sequence of SEQ ID NO: 12 as follows:

 GhZA T10-l F: SEQ ID NO: 13:

 ATGGCTCTTG ACACATTGAA TTC

 GhZA T10-l R: SEQ ID NO: 14:

 TCAACAAACG TGGCAGCC

 The GhZA TW-1 full-length coding sequence was cloned by SEQ ID NO: 13 and SEQ ID NO: 14. The 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 HS DNA polymerase, 10 μ Μ primer SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 μl, and 30 μl 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 2 min), extension at 72 °C for 10 min.

PCR amplification product plus A tail: PCR product plus 2.5 times the volume of absolute ethanol, placed at _20 °C for 10 minutes, centrifuged, supernatant, dried, dissolved in 21 μl of double distilled water. Add 2. 5 μ 1 ΙΟ Χ Εχ Buffer, 0. 5 μ 1 5 mM dATP, 2. 5 μ 1 ΙΟ Χ Εχ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 900 bp was recovered (using the Omega recovery kit) and ligated into the pGEM T-easy vector (GhZA TW+pG was obtained, and the resulting plasmid was transformed into JM109 competent cells (method supra) The transformed bacterial solution was applied to LB solid medium containing 50 μg/mL ampicillin, 40 g/mL X-gal, and 24 g/mL IPTG for screening. 10 white colonies were randomly picked. Inoculated in LB liquid medium containing 50 μg/mL ampicillin, cultured overnight at 37 °C Glycerol is brought to a final concentration of 20% and stored at -80 °C until use. SEQ ID NO: 13 and SEQ ID NO: 14 were tested for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ. ID NO: 2, the amino acid sequence of the encoded ZAT1O-1 protein is shown in SEQ ID NO: 1.

 Amino acid sequence of ZAT10-1 protein: SEQ ID NO : 1

 1 ALDTLNSPT SAPPLLDRDD

 2 1 IVDLHCVEPW TKRKRTKRPR

 4 1 SENPPTEEEY LALCLL LAQ

 61 GTTTRTIPSA SAASRTSLNL

 81 YKCTVCNKGF PSHQALGGHK

 101 ASHRKLWGA GDHLTITTAA

 12 1 TKTTADVSAV AATTVTPPK

 14 1 LPL I RVGN K HVCS ICNK

 161 TFSSGQALGG HKRCHYEAGS

 181 NNNSGSDGAK LPSQSQRDFD

 2 01 LNLPAGPQEL STPDWQRDK

 22 1 YSTDDEWEN PWPPAKRVGC

 24 1 HVC*

 Nucleotide sequence of the GhZA T10-1 encoding gene: SEQ ID NO: 2

 1 ATGGCTCTTG ACACATTGAA TTCCCCAACT TCAGCTCCTC CACTGCTGGA CCGTGATGAT

61 ATAGTCGACC TCCACTGTGT TGAGCCCTGG ACCAAGAGGA AAAGAACCAA ACGACCCAGA

121 TCCGAAAACC CACCAACCGA AGAAGAATAC TTAGCTCTTT GCCTTCTCAT GCTGGCTCAG

181 GGAACAACTA CCAGAACCAT TCCCTCAGCC TCGGCTGCTT CCAGAACCTC CCTCAACCTT

241 TACAAGTGTA CGGTTTGTAA CAAGGGATTT CCATCTCATC AAGCTTTAGG AGGACACAAG

301 GCCAGTCACA GGAAACTCGT TGTTGGAGCT GGAGATCACC TC AC CATC AC CACCGCCGCC

361 ACCAAGACCA CTGCTGATGT CTCCGCTGTT GCTGCCACCA CAGTCACGCC GCCTAAAAAT

421 TTACCTCTCA TGATTAATCG AGTTGGTAAC AAGAATCACG TGTGTTCAAT TTGTAACAAG

481 ACATTCTCTT CAGGTCAAGC TTTAGGAGGG CATAAGCGAT GCCACTACGA AGCTGGCAGC

541 AATAACAACA GCGGCAGCGA TGGTGCTAAG TTACCGAGCC AAAGTCAACG TGACTTTGAC

601 TTAAATTTGC CTGCTGGACC GCAGGAACTA AGTACTCCCG ACGTTAACCA AAGAGATAAA 661 TATTCCACCG ACGATGAAGT GGTGGAGAAT CCTTGGCCGC CGGCAAAGCG CGTCGGCTGC 721 CACGTTTGTT GA Example 3 Construction of Gene Plant Expression Vector

 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 constitutive promoter 35S containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the gene, respectively. The construction process is shown in Figure 1.

 Primer SEQ ID NO: 15 and SEQ ID NO: 16 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121, 1· 0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 15 and SEQ ID NO: 16 each of 2.0 μ 1, and 31 μl of double distilled water. PCR reaction conditions: pre-variability 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 f coR I, Bgl II and ligated into pCAMBIA2300 (purchased from Promega, T4 ligase cassette) to obtain pCAMBIA2300_1.

 SEQ ID NO: 15:

 GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 16:

 ATCCAGATCTAGATCCGGTGCAGATTATTTG

 Primers SEQ ID NO: 17 and SEQ ID NO: 18 were used to amplify Tnos using the pBI121 plasmid as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 1 5XPS Buffer, 3 μ 1 2· 5 mM dNTP, 1.0 μ 1 ρΒΙ121, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μΜ primer SEQ ID NO: 17 and P SEQ ID NO: 18 each of 2.0 μ 1, and 31 μ 1 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 Sac I, EcoR I and ligated into pCAMBIA2300-1 to obtain pCAMBIA2300_2.

SEQ ID NO: 17: AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 18:

 TCAGAATTCCGCAGACGCTGCACTTGT

 The CaMV 35S promoter was amplified using the primers SEQ ID NO: 19 and SEQ ID NO: 20 using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μ 1 PCR reaction system: 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 pCAMBIA 2300 plasmid DNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μΜ primer SEQ ID NO: 19 and SEQ ID NO: 20 each of 2.0 μ 1, and 31 μl 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 obtained PCR product was ligated by the digestion of ^ ζ 'κ! III, Pst I (the ligation method is the same as above) to pCAMBIA2300-2 to obtain pCAMBIA2300_3.

 SEQ ID NO: 19:

 ACTAAGCTTATGGTGGAGCACGACACTCTC

SEQ ID NO: 20:

 TGACTGCAGAGAGATAGATTTGTAGAGAGAGACTGGTG

 Primers SEQ ID NO: 21 and P SEQ ID NO: 22 ψ GhZATlO-1 (template is the positive GhZAT1O+G plasmid obtained in Example 2), using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 GhZAT10-l-pGEM, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 21 and SEQ ID NO: 22 each of 2.0 μl and 31 μl 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 2 min), extension at 72 °C for 10 min. The resulting PCR product was ligated by / 3⁄4ί I, Sac I (ligation as described above) to pCAMBIA2300-3, and the plant expression vector 35S-GhZAT10-l-2300 (Fig. 2) was obtained.

 SEQ ID NO: 21:

 TGACTGCAGATGGCTCTTGACACATTGAATTC SEQ ID NO: 22:

AAGGAGCTCTCAACAAACGTGGCAGCC Example 4 Transformation of Agrobacterium by 35S-GhZAT10-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. Pick a single colony and inoculate 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin. Incubate overnight at 28 °C (about 12-16 h) to 0D600 value. 0. 4, forming a seed bacterial liquid. 5 ml of 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 with shaking 2 2. 5 h to 0D 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; 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; 10% with ice pre-cooling The glycerin was repeatedly washed 3-4 times; the bacterial pellet was resuspended by adding an appropriate amount of ice-cold 10% glycerol to prepare LBA4404 competent cells. It was then dispensed in 40 μl/tube and stored at _70 °C for later use.

 Transformation of Agrobacterium: Thaw competent cells on ice, add 1 μl of the positive 35S-GhAZF2-l-2300 plasmid obtained in Example 3 to 40 μl of competent cells, mix and heat for about 10 min. . The mixture of the competent cells and the 35S-GhAZF2-l-2300 plasmid after the ice bath was transferred to an ice-cold 0.1 cm-sized electric shock cup (purchased from bio-rad) using a micropipette. Knock the suspension to the bottom, taking care not to have air bubbles. The electric shock cup is placed on the slide of the electric shock chamber, and the slide is pushed to place the electric shock cup to the base electrode of the electric shock chamber. Set the program for MicroPul ser (purchased from bio-rad) to "Agr" and shock once. Immediately remove the electric shock cup and add 1 ml of pre-warmed LB liquid medium at 28 °C. Quickly and gently mix the cells with a micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. 100~200 μl of the 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 Arabidopsis Planting for Transformation

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, seeding 20-30 Arabidopsis 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 h black Culture in dark/12 h light, 1/2 MS liquid medium was watered every 7 days. After 30 days of culture, 4-5 plants were retained per pot, and the photoperiod was adjusted to 8 h dark/16 h light culture. After most of the plants are bolted, the entire main moss is cut off at the base of the inflorescence, and the apical dominance is obtained. After about 1 week, 4-6 new side mosses grow in the axillary buds, and the flower buds form part of the flower buds and partially flower or When 1-2 pods are formed, they can be used for transformation. Example 6 Arabidopsis flower leaching transformation

The bacterial solution of the Agrobacterium LBA4404-transformed positive clone containing the 35S-GhAZF2-l-2300 plasmid obtained in Example 4 was inoculated to an LB liquid medium containing 50 μg/ml kanamycin overnight, and the next morning. The cells were inoculated at 1:50 into a new LB medium (1 L) containing 50 μg/ml kanamycin and cultured for about 8 hours to Agrobacterium liquid 0D ₆ . . 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 corresponding volume of the infiltration medium (1/2 MS; 5% sucrose; adjust to pH 5.7 with K0H; and contain 200 μM/L Si lwet L_77), making 0D ₆ . . Around 0.8. The upper part of Arabidopsis thaliana was gently and spirally immersed in the Agrobacterium-containing dyeing medium, and gently shaken clockwise for about 2 minutes, covered with a transparent plastic cover to maintain humidity, and placed in a greenhouse overnight. After 24 hours, the plastic transparent cover was removed and the water was poured through. 2-3 weeks after soaking, 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. 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 1/2 MS solid screening medium containing 50 μg/ml kanamycin (a maximum of 1500 seeds were plated in a 150 mm diameter plate), and vernalized at 4 °C. day. Then, the cells were cultured for 7-10 days at a constant temperature of 22 ° C, an illumination intensity of 3500-4000 lx, and an illumination period of 8 h dark/16 h. Seeds that can germinate and grow normally are transgenic seeds. After the germination of the transgenic seeds on a 1/2 MS solid plate containing 50 μg/ml kanamycin for 2 weeks, the positive plants capable of growing were transferred to the soil for further cultivation, and 1-2 leaves were cut per plant. The DNA was extracted as a template, and PCR detection was carried out using primers SEQ ID NO: 13 and SEQ ID NO: 14. The negative plants were removed by PCR, and the seeds of the positive plants were collected and labeled as T. A3-1 to T. A3-20 and save. Example 8 Overgrowth of GhZA TW-1 transgenic Arabidopsis thaliana 1\ generation plants The selection of water-absorbing soil with good soil moisture and soft soil was combined with nutrient soil (1:1) as the soil for Arabidopsis thaliana planting. Will T. A3-1 to T. A3-20 was planted in 2 pots per transformation line, 2 pots were planted in non-transgenic control Arabidopsis, and 20-30 seeds were seeded per pot. After sowing, the film is covered with a film to provide a moist environment for plant growth. The temperature was 22 ° C, the light intensity was 3500-4000 lx, the photoperiod was 12 h dark / 12 h light culture, and 1/2 MS liquid medium was watered every 7 days. After 25 days of culture, the leaves of the transgenic Arabidopsis lines were excised and their genomic DNA was extracted as a template, and PCR detection was carried out using primers SEQ ID NO: 13 and SEQ ID NO: 14. PCR-negative plants were removed, and 7-8 positive seedlings were retained. After 10 days of culture, 4-5 transgenic Arabidopsis thaliana or non-transgenic control Arabidopsis thaliana seedlings with uniform size were kept in each pot for salt tolerance experiments. Example 9 Salt tolerance test of overexpressing GhZA TW-1 transgenic Arabidopsis thaliana 1\ generation plants

 In Example 8, the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana were each treated without treatment, and the 1/2 MS liquid medium was normally watered; in addition, each of the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana was watered with 150 mM NaCl. 1/2 MS liquid medium, constant temperature 22 ° C, light intensity 3500-4000 lx, 12 hours light culture / 12 hours dark culture cycle, observe the experimental results after 10 days. Identification of salt tolerance of 1\ generation transgenic plants (T. plants grown from seeds of transgenic plants) showed that four strains of Ί\Α3-1, Ί\Α3-6, Ί\Α3_8 and Ί\Α3_16 showed Significant salt tolerance (see Figure 3, taking TVV3-6 as an example, the rest of the results are similar, not shown here). Example 10 Validation of ZAT10-1 protein expression at the transcriptional level

8 transgenic 1\ generation plants with good salt tolerance in Example 9 were randomly selected (in turn, the above four salt-tolerant strains of Ί\Α3-1, ΤΑ3-6, ΤΑ3-8 and T\A3_16, each 2 strains), the control plants in Example 6 were randomly selected from 4 plants, each of which was cut with 150 mM NaCl for 14 days, 0. 05 g, and total RNA was extracted with a plant RNA extraction kit (invi trogen). The absorbance values of total RNA 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 Invi trogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template and reverse transcription primer as SEQ ID NO: 14). Atactin-2 (http://www. uniprot.org/uniprot/Q96292) was amplified by primers SEQ ID NO: 23 and SEQ ID NO: 24, and the relative expression of the housekeeping gene Actin-2 protein was detected as an internal reference. The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTARHS DNA polymerase. 50 l PCR reaction system: 10 μ 15 X PS Buf f er, 3 μ 1 2· 5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 23 and SEQ ID NO: 24 each of 2.0 μl, and 30 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 32 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.

 SEQ ID NO: 23:

 GCTGATGATATTCAACCAATCGTG SEQ ID NO: 24:

 CTCTGCTGTTGTGGTGAACATG

 The relative expression of ZAT10-1 protein was detected by amplifying GhZATW-1 by the primers of SEQ ID NO: 13 and SEQ ID NO: 14.

 Using TaKaRa's PrimeSTAR HS DNA polymerase, using reverse transcribed cDNA as a template

PCR reaction. 50 yl PCR reaction system: 10 μ 15 XPS Buffer, 3 μ 12.5 mM dNTP, 2.0 μ 1 cDNA, 1· 0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 μ 1, and 30 μ 1 of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 32 cycles (denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 1 min), extension at 72 ° C for 10 min.

 The product electrophoresis results are shown in Figure 4. The upper and lower partial strips shown in the figure belong to GhZAT10-l Atactin-2, respectively, with ^ iacii?-^ as the internal reference. . 1_4 is a non-tolerant non-transgenic control Arabidopsis plant, 5-12 is a salt-tolerant T1 transgenic Arabidopsis plant (in turn, Ί\Α3-1, T\A3-6, TVV3-8, and Ί\Α3 -16 four salt-tolerant strains, 2 strains per strain), 13 is a 35S-GhZAT10-l-2300 plasmid PCR positive control, and 14-17 is a transgenic salt-tolerant Arabidopsis plant (subordinate to 2 strains) Department, 2 tests each), 18 is a blank control without template. The results showed that there was no GhZATW-1 transcription in the salt-tolerant non-transgenic control Arabidopsis plants, and the transcription level of the salt-tolerant transgenic Arabidopsis thaliana 1\ generation plants was higher. The transcription level of the salt-tolerant transgenic Arabidopsis plants was very high. low.

Claims

Claim

A zinc finger protein of cotton having the sequence SEQ ID NO: 1.

 2. A gene encoding the zinc finger protein 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 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 35S-GhZAT10-l-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.