WO2015024147A1 - Zinc finger protein zpt5-5 from cotton, and coding gene and uses thereof - Google Patents

Abstract

Provided are a zinc finger protein ZPT5-5 and a coding gene thereof, and uses of thereof in breeding a transgenic plant with improved salt tolerance.

Description

 Cotton zinc finger protein ZPT5-5 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 ZPT5-5 and its encoding gene, and its use in the cultivation of transgenic plants having improved salt tolerance. Technical Background Salt stress is one of the most important abiotic stress hazards in the world's agricultural production. Salinized soil is usually dominated by sodium, calcium or magnesium salts, which is the main factor affecting plant growth and causing food and economic crops to reduce production. 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 ZPT5-5) of African cotton ossypi herbaceum L. by SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends). The gene, and its DNA sequence was determined. It was also found that when introduced into a recipient plant and expressed, the salt tolerance of the recipient plants was significantly improved, and these traits were stably inherited.

 The first aspect of the present invention provides a gene encoding a zinc finger protein ZPT5-5 of cotton (designated herein as

GhZPT5-5), 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-GhZPT5-5-2300 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-GhZPT5-5-2300) of a gene (Fig. la-lb).

 Figure 2 is a plasmid map of the plant expression vector of the gene (35S-GhZPT5-5-2300). Figure 3 shows the results of salt tolerance simulation experiments of transgenic Arabidopsis plants (right panel, T\F7-8) and non-transgenic Arabidopsis plants (left panel, CK) as controls.

 Figure 4 is a verification result of molecular detection at the transcriptional level in 1\ generation transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. Μ is DL2000 DNA Ladder Marker, 1_4 is a non-tolerant non-transgenic control Arabidopsis plant, 5_12 is a salt-tolerant T1 transgenic Arabidopsis plant (in order to belong to T\F7-2, T\F7-5, T\) F7_8 and T\F7_13 four salt-tolerant strains, each strain

2 strains), 13 is a 35S-GhZPT5-5-2300 plasmid PCR positive control, and 14 is a blank water control. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.

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

Biolabs.

Example 1. Cotton SSH library construction under salt stress:

 The specific method is:

 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, incubated 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 N3⁄4EDTA, 100 μ M FeS0 ₄ ) once. It was used for experiments when the seedlings were as long as 25-30 cm.

 (2) Material handling:

 The test seedlings were divided into 2 groups of 4 plants each. The first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium. The second group 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 group and the salt treatment group were respectively taken 0.5 g, and the plant RNA extraction kit was used.

(Invitrogen) Extracts total RNA from cotton leaves. 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. mRNA was isolated using the Qiagen Oligotex mRNA Purification Kit (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 forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM_T were prepared according to the method described in the product manual of the pGEM-T Easy kit (purchased from Promega). Easy carrier connection, specific steps such as Bottom: The following components were sequentially added using a 200 μΐ PCR tube: 2nd PCR product of purified positive subtractive hybridization cDNA fragment 3 μ 1, Τ4 ligase buffer 5 μ 1 , pGEM-T Easy vector 1 μ 1, Τ4 DNA ligase 1 μ ΐ and ligated overnight at 4 °C. 10 L of ligation reaction product was added to 100 E. coli JM109 competent cells (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, then 250 μL LB medium (containing 1%) Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. It was placed in a 37°C water bath, shaken at 225 r/min for 30 min, and 200 bacteria cultured in shaking culture was planted in the 50 g/mL ampicillin (purchased from Beijing Bayerdi), 40 μg/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), 24 μg/mL IPTG (Iso-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. Nested PCR primer Primer 1 and P Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) was used to carry out PCR amplification and validation, and 231 positive clones were obtained. All positive clones were sent to the UK. Base (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

 Sequencing results of the clone Gh-S2-090 After the redundant DNA was removed, the sequence was SEQ ID NO: 3. Here, the full-length coding gene corresponding to SEQ ID NO: 3 was named 63⁄4Z/ 5-5, and the corresponding protein was named. For ΖΡΤ5-5.

 SEQ ID NO: 3

 1 GATGTTATTC GTCGGTCTTC CTCTTCGTCG TCACCGCCTC CGCCGCCGCC TGCTTTGAAG

61 TTGTCTTACA AGTGTAGTGT TTGTGACAAG GCGTTCCCTT CTTATCAAGC TTTGGGCGGT 121 CATAAAGCCA GCCACCGCAA ACCCCTTTCC GCCGACGCCG CTACCACCAC CGCCGCCGTC

181 AACGTCGATA ACCCATCAAC AACCAGCACC GCCACCACCA TCACCAGCAG CGGTAGGCTT 241 CACGAGTGTT CCATCTGCCA CAAGAGTTTC CCTACGGGCC AAGCCTTGGG TGGTCATAAA 301 CGCTGCCACT ACGAAGGTGG CAACAACAAC AACAAAAATA ACAACAACAG CGGTAGCGTT 361 AGCGTTAGCG GGGTTACGTC TTCGGATGGG GGCGCGTTGA GCCACAACCA CCGTGCAGTC 421 GACTTTGACT TTGACCTCAA CTTGCCAGCC TTGCCGGAGT TCAGTCAAAT GT Cloning of full-length coding gene

 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:

 GhZPT5-5 GSP1: SEQ ID NO: 4:

 CGAGTGTTCCATCTGCCACAAGAG

 GhZPT5-5 GSP2: SEQ ID NO: 5:

 CGCTGCCACTACGAAGGTG

 Reagents come with universal primers:

 AP: SEQ ID NO: 6:

 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTT 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-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 3' primer SEQ ID NO: 7, 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 The first round of PCR product diluted by μ 1 , 1. 0 μ 1 Ex Taq, 10 μ Μ of primers SEQ ID NO: 5 and SEQ ID NO: 7 each of 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 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 (specifically the same 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 'end primer SEQ ID NO: 7 were subjected to bacterial cell PCR amplification, and 6 positive clones were obtained, which were sent to Yingji 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 SEQ ID NO: 8:

 1 GATGTTATTC GTCGGTCTTC CTCTTCGTCG TCACCGCCTC CGCCGCCGCC TGCTTTGAAG

61 TTGTCTTACA AGTGTAGTGT TTGTGACAAG GCGTTCCCTT CTTATCAAGC TTTGGGCGGT

121 CATAAAGCCA GCCACCGCAA ACCCCTTTCC GCCGACGCCG CTACCACCAC CGCCGCCGTC

181 AACGTCGATA ACCCATCAAC AACCAGCACC GCCACCACCA TCACCAGCAG CGGTAGGCTT

241 CACGAGTGTT CCATCTGCCA CAAGAGTTTC CCTACGGGCC AAGCCTTGGG TGGTCATAAA

301 CGCTGCCACT ACGAAGGTGG CAACAACAAC AACAAAAATA ACAACAACAG CGGTAGCGTT

361 AGCGTTAGCG GGGTTACGTC TTCGGATGGG GGCGCGTTGA GCCACAACCA CCGTGCAGTC

421 GACTTTGACT TTGACCTCAA CTTGCCAGCC TTGCCGGAGT TCAGTCAAAT GTACCCAGAT

481 GAAGAAGAGG TCCAAAGCCC ATTGCCGACC AAGAAACCAC GTTTCTTGAT CGCCAAGAAA

541 GAGAAACTGG ATTCTTCATT AGCATAAGAT TTAGAATTAA ATTGAAAAAT TCAGATAACC

601 TTGTTTTTAG TTTTCAATAT TTTTTCTGGG ATTTGTATTT TGTTTT

 Based on the sequence of SEQ ID NO: 8 that has been obtained, three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.

 GhZPT5-5 GSP1 : SEQ ID NO : 9:

 GCAGATGGAACACTCGTGAAG

 GhZPT5-5 GSP2 : SEQ ID NO : 10:

 GGAACGCCTTGTCACAAAC

GhZPT5-5 GSP3 : SEQ ID NO : 11: GGAAGACCGACGAATAACATC

 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: 10 and the universal primer AAP (provided with the kit), and the cDNA obtained by reverse transcription of cotton mRNA (reverse transcription primer SEQ ID NO: 9) was used as a template. 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 μ 1 mRNA reverse transcribed cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 10 and AAP each with 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 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 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 ΙΟΧΕχ Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq, 10 μM primer SEQ ID NO: 6 and P AUAP 2.0 μ 1, and 35 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 72V for 2 min), extension at 72 °C for 10 min. A second round of PCR product band (purchased from OMEGA using the Gel Extraction Kit) was recovered and ligated into the pGEM_T Easy vector, which was then transformed into JM109 competent cells (specifically as above), and the transformed bacterial solution was Screening was performed on LB solid medium containing 50 μg/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 g/mL ampicillin, and cultured overnight at 37 ° C, glycerin was added to a final concentration of 20%, and stored at -80 ° C for 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 Yingji 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 GCATTGAATT GACTTGGAAG CAGTCATATG AAATCATAGT CTCCACCTCA TGAGAGCATC

61 CCCCCACACT ATTTCACCCA ATACACCCTC CTCCCCTTTT CTTCGTAGTT GACCCACTTC

121 ACTTCCTTCT TAGCTACCAC CTTCACTTCC CTATATATAT ATTACCCTTT TCACTCAGTC 181 TCTCACACCA CGCCCCACCT CACAGATTAA ACCCCTCCAA AAAAGCTTAC TTTTAGCACT 241 CTGTTTTTGC TCTCAAACTC GAAGGGATTC GGAAGTATTA GATATGGCGC TTGAAGCTCT

301 GAACTCGCCG GCGACGCCTT TCACCAACAA ATACGATGAC GTGGACAACA ATTACGTCGA

361 GACATGGAAG AAAGGCAAGC GTTCGAAGCG CCAACGCGGC GACTCTCCTG CTGCTGTTGA

421 ACTTCAACCC ACCACCGAAG AAGAGTACCT CGCTCTTTGT CTCATCATGC TCGCTCGCGG

481 CTCTTCCGGT GCTGATCGTG ATGTTATTCG TCGGTCTTCC TCTTCGTCGT CACCGCCTCC

541 GCCGCCGCCT GCTTTGAAGT TGTCTTACAA GTGTAGTGTT TGTGACAAGG CGTTCCCTTC

601 TTATCAAGCT TTGGGCGGTC ATAAAGCCAG CCACCGCAAA CCCCTTTCCG CCGACGCCGC

661 TACCACCACC GCCGCCGTCA ACGTCGATAA CCCATCAACA ACCAGCACCG CCACCACCAT

721 CACCAGCAGC GGTAGGCTTC ACGAGTGTTC CATCTGCCAC AAGAGTTTCC CTACGGGCCA

781 AGCCTTGGGT GGTCATAAAC GCTGCCACTA CGAAGGTGGC AACAACAACA ACAAAAATAA

841 CAACAACAGC GGTAGCGTTA GCGTTAGCGG GGTTACGTCT TCGGATGGGG GCGCGTTGAG

901 CCACAACCAC CGTGCAGTCG ACTTTGACTT TGACCTCAAC TTGCCAGCCT TGCCGGAGTT

961 CAGTCAAATG TACCCAGATG AAGAAGAGGT CCAAAGCCCA TTGCCGACCA AGAAACCACG

1021 TTTCTTGATC GCCAAGAAAG AGAAACTGGA TTCTTCATTA GCATAAGATT TAGAATTAAA

1081 TTGAAAAATT CAGATAACCT TGTTTTTAGT TTTCAATATT TTTTCTGGGA TTTGTATTTT

1141 GTTTT

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

 GhZPT5-5 F: SEQ ID NO: 13:

 ATGGCGCTTGAAGCTCTG

 GhZPT5-5 R: SEQ ID NO: 14:

 TTATGCTAATGAAGAATCCAGTTTCTC

 The full-length coding sequence was cloned using SEQ ID NO: 13 and SEQ ID NO: 14 as primers.

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 (94 C denaturation 30 s, 58 ° C annealing 30 s, 72 C extension 2 min), 72. Extend C 10 min ₀ 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), ligated into the pGEM T-easy vector (GhZPT5+≠ was obtained), and the resulting plasmid was transformed into JM109 competent cells (method as above). The transformed bacterial solution was applied to LB solid medium containing 50 μg/mL ampicillin, 40 g/mL X-gal, 24 g/mL IPTG for screening. 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. 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 ZPT5-5 protein is shown in SEQ ID NO: 1.

 Amino acid sequence of ZPT5-5 protein: SEQ ID NO : 1

 1 MALEALNSPA TPFTNKYDDV

 21 DNNYVETWKK GKRSKRQRGD

 41 SPAAVELQPT TEEEYLALCL

 61 IMLARGSSGA DRDVIRRSSS

 81 SSSPPPPPPA LKLSYKCSVC

 101 DKAFPSYQAL GGHKASHRKP

 121 LSADAATTTA AVNVDNPSTT

 141 STATTITSSG RLHECS ICHK

 161 SFPTGQALGG HKRCHYEGGN

 181 NNNKNNNNSG SVSVSGVTSS

 201 DGGALSHNHR AVDFDFDLNL

 221 PALPEFSQMY PDEEEVQSPL

 241 PTKKPRFLIA KKEKLDSSLA

 261 * nucleotide sequence of the coding gene: SEQ ID NO: 2

ATGGCGCTTG AAGCTCTGAA CTCGCCGGCG ACGCCTTTCA CCAACAAATA CGATGACGTG 61 GACAACAATT ACGTCGAGAC ATGGAAGAAA GGCAAGCGTT CGAAGCGCCA ACGCGGCGAC

121 TCTCCTGCTG CTGTTGAACT TCAACCCACC ACCGAAGAAG AGTACCTCGC TCTTTGTCTC

181 ATCATGCTCG CTCGCGGCTC TTCCGGTGCT GATCGTGATG TTATTCGTCG GTCTTCCTCT

241 TCGTCGTCAC CGCCTCCGCC GCCGCCTGCT TTGAAGTTGT CTTACAAGTG TAGTGTTTGT

301 GACAAGGCGT TCCCTTCTTA TCAAGCTTTG GGCGGTCATA AAGCCAGCCA CCGCAAACCC

361 CTTTCCGCCG ACGCCGCTAC CACCACCGCC GCCGTCAACG TCGATAACCC ATCAACAACC

421 AGCACCGCCA CCACCATCAC CAGCAGCGGT AGGCTTCACG AGTGTTCCAT CTGCCACAAG

481 AGTTTCCCTA CGGGCCAAGC CTTGGGTGGT CATAAACGCT GCCACTACGA AGGTGGCAAC

541 AACAACAACA AAAATAACAA CAACAGCGGT AGCGTTAGCG TTAGCGGGGT TACGTCTTCG

601 GATGGGGGCG CGTTGAGCCA CAACCACCGT GCAGTCGACT TTGACTTTGA CCTCAACTTG

661 CCAGCCTTGC CGGAGTTCAG TCAAATGTAC CCAGATGAAG AAGAGGTCCA AAGCCCATTG

721 CCGACCAAGA AACCACGTTT CTTGATCGCC AAGAAAGAGA AACTGGATTC TTCATTAGCA

781 TAA 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: 9 and SEQ ID NO: 10 were used to amplify Pnos using the plant expression vector pBI 121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ ΐ 5 X PS Buffer , 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 pBI 121 , 1. 0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 9 and SEQ ID NO: 10 each of 2.0 μl, and 31 μl of double distilled water. PCR reaction conditions: pre-variation 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 ligated into pCAMBIA2300 (purchased from Promega, T4 ligase cassette) by coR I, chymase, and pCAMBIA2300_l was obtained.

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 pBI121 plasmid, 1· 0 μ 1 PrimeSTAR HS DNA polymerase, 10 μ Μ primers SEQ ID NO: 11 and SEQ ID NO: 12 each of 2.0 μ 1, and 31 μ ΐ 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 53⁄4c I, EcoR I and ligated into pCAMBIA2300-1 to obtain pCAMBIA2300_2.

 SEQ ID NO: 17:

 AAGGAGCTCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 18:

 TCAGAATTCCGCAGACGCTGCACTTGT

 Using primers SEQ ID NO: 19 and SEQ ID NO: 20 to amplify the pCAMBIA2300 plasmid as a template

CaMV 35S promoter. 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 primers SEQ ID NO: 13 and SEQ ID NO : 14 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 ligated by ^ /2dIII, /3⁄4ί 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

GhZPT5-5 (template was the positive GhZPT5-5~vG plasmid obtained in Example 2) was amplified using primers SEQ ID NO: 21 and SEQ ID NO: 22, and PrimeSTAR HS DNA polymerase of 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 GhZPT5-5-pGEM plasmid, 1. 0 μl of PrimeSTAR HS DNA polymerase, 10 μM of primers SEQ ID NO: 15 and SEQ ID NO: 16 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 V for 2 min), and extension at 72 V for 10 min. After digestion with Pst I and Sac I, the resulting PCR product was ligated (ligation method as above) to pCAMBIA2300-3, and the plant expression vector 35S-GhZPT5-5_2300 was obtained (Fig. 2).

 SEQ ID NO: 21:

 TGACTGCAGATGGCGCTTGAAGCTCTG

SEQ ID NO: 22:

 AAGGAGCTCTTATGCTAATGAAGAATCCAGTTTCTC Example 4 35S-GhZPT5-5-2300 expression vector for transformation of Agrobacterium

Agrobacterium tumefaciens LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin. Incubate at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin, and cultured overnight (about 12-16 h) to 0D ₆ at 28 °C with shaking. . The 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 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 ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 ° C, collect the precipitate; pre-cool with ice 10 % glycerol was washed 3-4 times repeatedly; LBA4404 competent cells were prepared by resuspending the bacterial pellet with an appropriate amount of ice-cold 10% glycerol. 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-GhZPT5-5-2300 plasmid obtained in Example 3 to 40 μl of competent cells, mix and freeze for about 10 min. . Transfer the mixture of competent cells after ice bath and 35S-GhZPT5-5-2300 plasmid with a micropipette to an ice-cold 0. 1 cm size electric shock cup (purchased from bio-rad), tapping The suspension reaches the bottom, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber. Set the program of MicroPul ser (purchased from bio-rad) to "Agr", electric 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 screening 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

 Choose the rock with good water absorption and soft soil and the nutrient soil (1: 1) as the soil for Arabidopsis. A 9 cm diameter flowerpot, seeding 20-30 Arabidopsis seeds per pot (Columbia type, from the Arabidopsis Bioresource Center, Ohio State University, USA). 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 black / 12 h light culture, and 1/2 MS liquid medium was watered every 7 days. After 30 days of culture, 4-5 plants were kept per pot, and the light cycle 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-GhZPT5-5-2300 plasmid obtained in Example 4 was inoculated to an LB liquid medium containing 50 μg/ml kanamycin overnight, the next morning. Inoculate 1:50 into a new LB medium (1 L) containing antibiotics and culture for about 8 hours to Agrobacterium 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 dip medium (1/2 MS; 5% sucrose; adjust to pH 5.7 with K0H; and contain 200 μl/l Silwet L_77), make 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, remove the plastic transparent cover and pour the water 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: Soak for 10 minutes with 70% ethanol, and occasionally suspend the seeds; then wash them four times with sterile water 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 under the conditions of constant temperature of 22 ° C, light intensity of 3500-4000 lx, and light period of 8 h dark / 16 h. Seeds that can germinate and grow normally are genetically modified 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 surviving and continuing to grow were transferred to the soil for further cultivation, and each plant was cut 1- Two leaves, DNA was extracted as a template, and PCR was carried out using primers SEQ ID NO: 13 and P SEQ ID NO: 14. The PCR-negative plants were removed, and the seeds of the positive plants were collected and labeled as T. F7-1 to T. F7-20 and save. Example 8 Overexpression Transgenic Arabidopsis 1\Genesis Planting

 Choose the rock with good water absorption and soft soil and the nutrient soil (1: 1) as the soil for Arabidopsis planting. Will T. F2-1 to T. F2-20 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. The 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 Overexpression Salt tolerance test of 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, the experimental results were observed after 10 days. 1\ Generation of transgenic plants (T. Seeds of transgenic plants) The salt tolerance of the grown plants showed that the four lines of T\F7-2, T\F7-5, T\F7_8, T\F7_13 and T\F7_15 showed significant salt tolerance (see Figure 3). Take T\F7-8 as an example. The rest results are similar, not shown here). Example 10 Validation of ZPT5-5 protein expression at the transcriptional level

 Eight transgenic 1\ generation plants with good salt tolerance in Example 9 were randomly selected (one of the above-mentioned T\F7-2, T\F7-5, T\F7_8 and T\F7_13 four salt-tolerant lines, For each of the two strains, the control plants of Example 6 were randomly selected from 4 plants, and each of the leaves treated with 150 mM NaCl for 14 days was 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 determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template and reverse transcription primer as SEQ ID NO: 14).

 Amplification of Atactin-2 by SEQ ID NO: 23 and SEQ ID NO:

 (http://www. uniprot.org/uniprot/Q96292), detecting the relative expression of the housekeeping gene Actin-2 protein as an internal reference. PCR was carried out using TaKaRa's PrimeSTARHS DNA polymerase and reverse-transcribed cDNA as a template. 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 ZPT5-5 protein was examined by amplifying GhZPT5-5 by primers SEQ ID NO: 13 and SEQ ID NO: 14. The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 15 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 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 GhZPT5-5 and A tactin-2, respectively, with ^ iac ii ?-^ 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 (respectively belonging to the above T\F7-2, T\F7-5, T\F7-8 and T\F7_13 four salt-tolerant strains, 2 strains per strain), 13 is a 35S-GhZPT5-5-l-2300 plasmid PCR positive control, and 14-17 is a transgenic salt-tolerant Arabidopsis plant (subordinate 2) Each strain, 2 strains each), 18 was a blank control without template. The results showed that the transcription level of the salt-tolerant transgenic Arabidopsis thaliana 1\ generation plants was higher in the salt-tolerant non-transgenic control Arabidopsis plants, and the transcription level of the salt-tolerant transgenic Arabidopsis plants was very 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-GhZPT5-5-2300 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.