WO2014172840A1 - Cysteine protease cysp1-1 of cotton and coding gene, and use thereof - Google Patents

Abstract

The present invention relates to a cysteine protease cysp1-1 derived from cotton and a coding gene thereof, and the use thereof in the cultivation of transgenic plants having improved drought tolerance.

Description

 A cotton cysteine protease cyspl-1 and its coding gene and application

FIELD OF THE INVENTION The present invention relates to cysteine proteases and their encoding genes and applications, and in particular to a cotton-derived cysteine protease cyspl-1 and a gene encoding the same, and in the cultivation of transgenic plants with improved drought tolerance Applications. BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, and cause great losses to crop yield. The impact of drought on crop yield is It takes the first place in natural adversity, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas. According to statistics, the world's dry and semi-arid areas account for 34% of the land area; China's dry and semi-arid areas account for 52% of the country's land area, the annual drought area is 22.7 million hectares, and the national irrigation district lacks about 300 water per year. Million cubic meters, due to lack of water, less than 355 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.

Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques, and it is particularly difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promoted the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification systems and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions. In recent years, studies on the ability of plants to improve stress tolerance through transgenic techniques, as well as studies on stress-tolerant crops, xerophytes and halophytes have yielded significant results for stress-related genes and signal transduction. The system has a further understanding (Liu Q.1998. Two transcription factors, DREB 1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis . Plant Cell, 10: 1391-1406; KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABEH.2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78.

 As a family of important proteases, cysteine proteases are widely involved in various physiological processes in plants. Numerous studies have shown that cysteine protease mRNA accumulates under environmental stress such as hypothermia, drought and salt stress, which is responsible for the degradation of damaged or denatured proteins under adverse conditions, providing peptides or free amino acids for the synthesis of new proteins. 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 for a cysteine protease of cotton (designated herein as cyspl-1) by combining SSH (suppression subtractive hybridization) with RACE (rapid amplification of cDNA ends). It was also found that when introduced into recipient plants, the drought tolerance of transgenic plants was significantly improved, and these traits were stably inherited.

 The first aspect of the present invention provides a gene encoding a cysteine protease cyspl-1 of cotton (herein named Ghcyspl-1) having the sequence of 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, preferably, the expression vector is pCAMBIA2300; And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is the rd29A-Ghcyspl-1 - 2300 vector shown in Figure 2.

 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.

 A fourth aspect of the 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 tobacco.

A fifth aspect of the invention provides a method for preparing a transgenic plant, comprising: efficiently producing a plant The plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention is cultured under conditions; preferably, the plant is tobacco.

 A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.

 A seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as set forth in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (rd29 A-Ghcy sp 1-1-2300) of Ghcysp 1 (Fig. la-lb).

 Figure 2 is a plasmid map of the plant expression vector (rd29A-Ghcysp l-l-2300;) of (3⁄4ς)ψ-1.

Figure 3 shows the results of drought tolerance simulation experiments of (3⁄4ς) ψ-1 transgenic tobacco T _Q plants (T _Q A _{5 ;} right, ToAn) and non-transgenic tobacco plants (left, CK) as controls.

Figure 4 shows the results of molecular level detection of the transcription level of the G/zqy^-1 gene in T _Q transgenic tobacco plants and non-transgenic control plants by reverse transcription PCR. Μ is the marker DNA Ladder Marker (DL2000), 1-5 is a non-transgenic control tobacco plant, 6-18 is a transgenic tobacco To plant with significant drought tolerance, 19-24 transgenic tobacco T _Q with insignificant drought tolerance Generation plants. DETAILED DESCRIPTION OF THE EMBODIMENTS

 The invention is further illustrated by the following non-limiting examples.

 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:

The subtractive library was constructed by the method of inhibition subtractive hybridization according to the method shown in the PCR-selectTM cDNA Subtraction Kit kit of Clontech. During the experiment, the mRNA of cotton seedling leaves treated with drought during the growth period was used as a tester, and the mRNA of untreated cotton seedling leaves 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, cultured at 25 °C, photoperiod of 16 hours light/8 hours dark, each 1/2MS medium (9.39 mM KNO ₃ , 0.625 mM KH ₂ PO 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 ΒΟ ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1 μΜ Να ₂ Μο0 ₄ , O. ^M CoCl ₂ , ΙΟΟμΜ Na ₂ EDTA, 100 M FeSO ₄ )-time. It was used for experiments when the seedlings were as high as 25-30 cm.

 (2) Material handling:

 The test seedlings were divided into two groups, each with 4 pots and 1 pot per pot. The first group was the control group, cultured at 25 °C, light culture, normal 1/2MS watering; the second group was drought treatment group, cultured at 25 °C, photoperiod 16 hours light/8 hours dark, stop watering After 10 days, the leaves of the top 1/3 of the seedlings of the two groups were cut out in time, quickly frozen with liquid nitrogen, and stored in a -70 °C refrigerator.

 (3) Total RNA extraction:

 Cotton leaves of 0.5 g in the control and drought-treated groups were taken, and total RNA of cotton was extracted using the plant RNA extraction kit (Invitrogen). The absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD260/OD280 ratio was 1.8-2.0, indicating that the total RNA was of high purity and was detected by 1.0% agarose gel electrophoresis. The integrity of the total RNA, the 28S band is about twice as bright as the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

In order to increase the availability of the Expressed Sequence Tag (EST) (unigene), the gene is not cleavable and the obtained sequence is in the untranslated region. Subtractive laboratory according to the protocol described in the kit, and the other ij by two points at the bow I mRNA was 01igo (dT) ₁₈ (TTTTTTTTTTTTTTTTTT) reverse transcription, the resulting cDNA was digested with Rsal, Haelll, making two groups suppression subtractive other steps and methods by PCR-select ^TM cDNA Clontech's subtraction kit kit shown a second method of suppression subtractive hybridization of PCR products, and finally merge the two forward subtractive hybridization cDNA fragments.

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

The second PCR product of the combined positive subtractive hybridization cDNA fragment (QIAquick PCR) Purification Kit purified, purchased from Qiagen) and pGEM-T Easy (purchased from Promega kit) vector, according to the method shown in the pGEM-T Easy kit product manual, the specific steps are as follows: Add the following ingredients in sequence using a 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment was 3 μl, Τ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μ1, T4 DNA ligase 1 μl, 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), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 μL LB liquid medium (containing 1 % Tryptone (purchased from OXOID), 0.5% Yeast Extract (purchased from OXOID) and P 1% NaCl (purchased from Sinopharm) were placed in a 37 ° C shaker at 225 r/min. Incubate for 30 min, then take 200 μ of bacterial solution from 50 g/mL ampicillin, 40 igl L X-gaK 24 g/mL IPTG (X-gal ( 5-bromo-4-chloro-3-indole- β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactopyranoside) were purchased from TAKARA) on LB (ibid.) solid culture plates and incubated at 37 ° C for 18 h. Count the number of clear white and blue colonies with a diameter > lmm in the culture plate and randomly pick 300 white colonies (number: Gh-C001 to Gh-C300). The picked white colonies were inoculated into LB liquid medium containing 50 g/mL ampicillin in 96-well cell culture plate (CORNING), and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of glycerol of 20% by volume. , and keep it at -80 °C. The cultured colony clones were verified by PCR PCR using nested PCR primers Primer 1 and Primer 2R (PCR-selectTM cDNA Subtraction Kit from Clontech) to obtain 243 positive clones, and all positive clones were sent. Yingjie Jieji (Shanghai) Trading Co., Ltd. was sequenced.

 (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 167 Expressed sequence tags (EST) (unigene) were obtained. It was found by BlastN that 82 unigenes have homologous sequences in GenBank (more than 50% protein homology), 23 EST functions are unknown or hypothetical proteins, and another 35 have not obtained homologous matches, presumably at 3 ' , a shorter sequence of 5 'end untranslated regions. Example 2 Cloning of a cotton cysteine protease encoding gene (3⁄4ς)-1

After the sequencing result of Gh-C192, which is one of the effective clones obtained in Example 1, was removed, the sequence was SEQ ID NO: 3, and sequence analysis indicated that the encoded amino acid sequence of the sequence was related to known cysteine. The acid protease sequence has high homology. In this paper, the full-length gene encoded by the clone Gh-C192 is named Ghcyspl-1, and the corresponding protein is named cysp 1-1.

 SEQ ID NO: 3

 1 AAACTTGGGT TGAACAAGTT CGCTGATTTA ACTAACCAGG AGTACCGTTC CATGTTTTTG 61 GGTACAAAGA GTGACCCTAA ACGTCGAGTC ATGAAGTCCA AAAACCCCAG CCAACGCTAT 121 GCTTCCCGCG CCGGCGACAG GTTGCCGGAA TCTTTTGACT GGAGAGATCA TGGAGCCGTT 181 ACTCCAGTGA AGGATCAAGG GCGTTGCGGA AGTTGCTGGG CATTTTCAAC GATTGAAGCT 241 GTTGAAGGCA TAAATAAAAT CGCCACCGGC GAACTAATCT CTTTGTCAGA GCAAGAGCTA 301 GTAGATTGTG ACCGATCCTA CGACGCTGGT TGCGATGGAG GCCTAATGGA TTATGCCTTC 361 CAATTCATTA TTGACAACGG TGGCATTGAC TCTGAACAAG ACTATCCTTA CCTTGGTGCT 421 GATAATAACC AATGCGATCC AACGAGGAAG AATGCTAAGG TTGTCAGCAT TGATGGGTAC 481 GAGGATGTTG TTCAATATGA TGAGAAGGCA TTGAAGAAGG CTGTATCACA TCAACCTGTG 541 AGTGTCGCCA TTGAAGCTAG TGGCAGAGCT TTCCAACTCT ACGAATCGGG AGTTTTCAGC 601 GGTGAATGCG GGTCAGCATT AGACCACGGC GTGATTGTCA TCGGATATGG CATGGATGAG 661 AACGGTCAGG AATATTGGAC AGTGAGGAAC TCATGGGGCA GCGGTTGGGG TGAAGATGGA 721 TACATAAGGA TGGAGCGTAA CGTTGATGAC CGTGCTGGCA AGTGTGGCAT TGCGATGGAG 781 GCTTCATATC CTGTTAAGAA TGGGACAAAC ATCATCAAAC CTTACTGGAC TAATGAAGAC 841 ACTCAGAAAA TTAGCAGT GC CTGAGAGTTA AAGATGTGTG CCAGCTGCTG TTTTGGGCCA 901 TGGGAGAAGA GGTGTGCCAT GAGTTA

 Cloning of the full-length gene of Ghcyspl-1

 The Ghcyspl-I gene fragment (SEQ ID NO: 3) that has been obtained already has a stop codon TGA, and only 5 ' RACE is required.

 Based on the G/zc ^ -1 gene fragment (SEQ ID NO: 3) which has been obtained, three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.

 Ghcyspl-1 GSP 1: SEQ ID NO: 4:

 GTGGCGATTTTATTTATG

 Ghcyspl-1 GSP2: SEQ ID NO: 5:

 GAGTAACGGCTCCATGATC

 Ghcyspl-1 GSP3: SEQ ID NO: 6:

 GACGTTTAGGGTCACTCTTTG

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

Using the SEQ ID NO: 5 and 5' universal primer AAP (provided with the kit), the reverse-transcribed cDNA extracted from cotton leaves of the drought-treated group (reverse transcription primer SEQ ID NO: 4) was used as a template for the first round. PCR amplification, the specific steps are as follows: Ex Taq purchased from TAKARA, 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, 10 μΜ primers SEQ ID NO: 5 and AAP 2.0 μl each, and 35 μΐ double distilled water. PCR reaction conditions:

Pre-denaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The obtained PCR product was diluted 50-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: 6 and the 3' primer AUAP. 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 diluted first round PCR product, l .O l Ex Taq, 10 μΜ primers SEQ ID NO: 6 and AUAP each 2.0 μ1 , and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 94 V denaturation for 30 s, annealing at 58 °C for 30 s, 72V extension for 2 min, 33 cycles, and extension at 72 °C for 10 min. The second PCR product was recovered from a fragment of about 600 bp (Gel Extraction Kit from OMEGA), ligated to pGEM-T Easy vector, transformed into JM109 (specific method as above), and randomly picked 10 white colonies containing 50 g/mL. The ampicillin was cultured in LB liquid medium, and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of glycerol of 20% by volume, and stored at -80 ° C until use. Using SEQ ID NO: 6 and the 3' primer AUAP for bacterial PCR amplification (reaction system and reaction conditions as above), three positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. The 5' end of the cDNA.

 After the obtained 5' RACE product was cloned and sequenced, it was spliced with the cloned Gh-C192 sequencing result to obtain

 SEQ ID NO: 17:

 1 TCCTTTCTCT TCACCAAACC ACTCCCTCCA ATAACCATAA CAACCCTTGT CATTTCTCCC

61 ATGGCAACCA CAGTAACACC CATTTCCACC CTCTTTTTCC TCTTCTTCAC CTTATCCTGG 121 GCAACCAGCT TCAGCCAAAA TCACTGGAGG AGCGATGATG AAGTGATGAG CTTGTACCAA 181 GCTTGGCTCA TTAAACATGG CAAACAATAC AACGGTATAG GGGAGGAAGA AAACCGGTTC 241 GATATCTTTA AGGATAACTT GAAATTTATC GACCAACATA ACTCCAAAAA CACCACATAC 301 AAACTTGGGT TGAACAAGTT CGCTGATTTA ACTAACCAGG AGTACCGTTC CATGTTTTTG 361 GGTACAAAGA GTGACCCTAA ACGTCGAGTC ATGAAGTCCA AAAACCCCAG CCAACGCTAT 421 GCTTCCCGCG CCGGCGACAG GTTGCCGGAA TCTTTTGACT GGAGAGATCA TGGAGCCGTT 481 ACTCCAGTGA AGGATCAAGG GCGTTGCGGA AGTTGCTGGG CATTTTCAAC GATTGAAGCT 541 GTTGAAGGCA TAAATAAAAT CGCCACCGGC GAACTAATCT CTTTGTCAGA GCAAGAGCTA 601 GTAGATTGTG ACCGATCCTA CGACGCTGGT TGCGATGGAG GCCTAATGGA TTATGCCTTC 661 CAATTCATTA TTGACAACGG TGGCATTGAC TCTGAACAAG ACTATCCTTA CCTTGGTGCT 721 GATAATAACC AATGCGATCC AACGAGGAAG AATGCTAAGG TTGTCAGCAT TGATGGGTAC 781 GAGGATGTTG TTCAATATGA TGAGAAGGCA TTGAAGAAGG CTGTATCACA TCAACCTGTG 841 AGTGTCGCCA TTGAAGCTAG TGGCAGAGCT TTCCAACTCT ACGAATCGGG AGTTTTCAGC 901 GGTGAATGCG GGTCAG CATT AGACCACGGC GTGATTGTCA TCGGATATGG CATGGATGAG 961 AACGGTCAGG AATATTGGAC AGTGAGGAAC TCATGGGGCA GCGGTTGGGG TGAAGATGGA 1021 TACATAAGGA TGGAGCGTAA CGTTGATGAC CGTGCTGGCA AGTGTGGCAT TGCGATGGAG 1081 GCTTCATATC CTGTTAAGAA TGGGACAAAC ATCATCAAAC CTTACTGGAC TAATGAAGAC 1141 ACTCAGAAAA TTAGCAGTGC CTGAGAGTTA AAGATGTGTG CCAGCTGCTG TTTTGGGCCA 1201 TGGGAGAAGA GGTGTGCCAT GAGTTA

 A pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:

 Ghcyspl-ΓΡ: SEQ ID NO: 7:

 ATGGCAACCACAGTAACAC

 Ghcyspl-1K: SEQ ID NO: 8:

 TCAGGCACTGCTAATTTTC

 The full length of Ghcyspl-1 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8.

 PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase and cDNA from the reversed-treated mRNA extracted from cotton leaves of the drought-treated group. 50 μl ΡΟ Reaction system: 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, 33 cycles, and extension at 72 °C for 10 min.

 PCR amplification product plus A tail: PCR product plus 2.5 volumes of absolute ethanol, -20 ° C for 10 minutes, centrifuge, remove the supernatant, air dry, dissolve with 21 μΐ double distilled water, add 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μl 5 ιηΜ dATP, 2.5 μΐ ΙΟχΕχ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 1100 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector, transformed into JM109 (method as above), and 10 white colonies were randomly picked up in LB liquid containing 50 g/mL ampicillin. The medium was cultured, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of glycerol of 20% by volume, and stored at -80 ° C until use. The bacterial liquid PCR amplification was carried out with SEQ ID NO: 7 and SEQ ID NO: 8 (the reaction system and the reaction conditions were the same as above), and four positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ. ID NO: 2. According to the nucleotide sequence, the amino acid sequence of the PP2AC-2 protein encoded by the gene is shown in SEQ ID NO: 1.

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

 MATTVTPIST LFFLFFTLSW

ATSFSQNHWR SDDEVMSLYQ AWLIKHGKQY NGIGEEENRF DI FKDNLKFI DQHNSKNTTY KLGLNKFADL TNQEYRSMFL GTKSDPKRRV MKSKNPSQRY ASRAGDRLPE SFDWRDHGAV

TPVKDQGRCG SCWAFSTIEA 161 VEGINKIATG ELISLSEQEL

TPVKDQGRCG SCWAFSTIEA 161 VEGINKIATG ELISLSEQEL

 181 VDCDRSYDAG CDGGLMDYAF

 201 QFI IDNGGID SEQDYPYLGA

 221 DNNQCDPTRK NAKWS IDGY

 241 EDWQYDEKA LKKAVSHQPV

 261 SVAIEASGRA FQLYESGVFS

 281 GECGSALDHG VIVIGYGMDE

 301 NGQEYWTVRN SWGSGWGEDG

 321 YIRMERNVDD RAGKCGIAME

 341 ASYPVKNGTN I IKPYWTNED

 361 TQKISSA*

 Nucleotide sequence of GhcyspJ-1 gene: SEQ ID NO: 2

 1 ATGGCAACCA CAGTAACACC CATTTCCACC CTCTTTTTCC TCTTCTTCAC CTTATCCTGG

61 GCAACCAGCT TCAGCCAAAA TCACTGGAGG AGCGATGATG AAGTGATGAG CTTGTACCAA 121 GCTTGGCTCA TTAAACATGG CAAACAATAC AACGGTATAG GGGAGGAAGA AAACCGGTTC

181 GATATCTTTA AGGATAACTT GAAATTTATC GACCAACATA ACTCCAAAAA CACCACATAC

241 AAACTTGGGT TGAACAAGTT CGCTGATTTA ACTAACCAGG AGTACCGTTC CATGTTTTTG

301 GGTACAAAGA GTGACCCTAA ACGTCGAGTC ATGAAGTCCA AAAACCCCAG CCAACGCTAT

361 GCTTCCCGCG CCGGCGACAG GTTGCCGGAA TCTTTTGACT GGAGAGATCA TGGAGCCGTT 421 ACTCCAGTGA AGGATCAAGG GCGTTGCGGA AGTTGCTGGG CATTTTCAAC GATTGAAGCT

481 GTTGAAGGCA TAAATAAAAT CGCCACCGGC GAACTAATCT CTTTGTCAGA GCAAGAGCTA

541 GTAGATTGTG ACCGATCCTA CGACGCTGGT TGCGATGGAG GCCTAATGGA TTATGCCTTC

601 CAATTCATTA TTGACAACGG TGGCATTGAC TCTGAACAAG ACTATCCTTA CCTTGGTGCT

661 GATAATAACC AATGCGATCC AACGAGGAAG AATGCTAAGG TTGTCAGCAT TGATGGGTAC 721 GAGGATGTTG TTCAATATGA TGAGAAGGCA TTGAAGAAGG CTGTATCACA TCAACCTGTG

781 AGTGTCGCCA TTGAAGCTAG TGGCAGAGCT TTCCAACTCT ACGAATCGGG AGTTTTCAGC

841 GGTGAATGCG GGTCAGCATT AGACCACGGC GTGATTGTCA TCGGATATGG CATGGATGAG

901 AACGGTCAGG AATATTGGAC AGTGAGGAAC TCATGGGGCA GCGGTTGGGG TGAAGATGGA

961 TACATAAGGA TGGAGCGTAA CGTTGATGAC CGTGCTGGCA AGTGTGGCAT TGCGATGGAG 1021 GCTTCATATC CTGTTAAGAA TGGGACAAAC ATCATCAAAC CTTACTGGAC TAATGAAGAC

1081 ACTCAGAAAA TTAGCAGTGC CTGA Example 3 Construction of plant expression vector for GhcyspJ-l gene

 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter of the ΡΤΠ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The inducible promoter rd29A and the terminator Tnos were selected as promoters and terminators of the GhcyspJ-1 gene.

Using the primers SEQ ID NO: 9 and SEQ ID NO: 10, Pnos was amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and PrimeSTAR HS DNA polymerase of TaKaRa was used. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min. The resulting PCR product was digested with EcoRI, Bglll, and ligated into pCAMBIA2300 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.

 SEQ ID NO: 9:

 GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 10:

 ATCCAGATC AGATCCGGTGCAGATTATTTG SEQ ID NO: 11 and SEQ ID NO: 12 nucleotide fragments are primers, amplified with pBI121 as a template

Tnos, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 31 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 58 °C for 30 s, 72V for 30 s, 33 cycles, and 72 °C for 10 min. The resulting PCR product was digested with Sacl, EcoRI and ligated into pCAMBIA2300-1 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-2.

 SEQ ID NO: 11:

 AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 12:

 TCAGAATTCCCAGTGAATTCCCGATCTAGTA

Using SEQ ID NO: 13 and SEQ ID NO: 14 as primers, the Arabidopsis thaliana (Columbia type, purchased from the Arabidopsis Bioresource Center (www.arabidopsis.org) of the Ohio State University) was used as a template for amplification. Arabidopsis rd29A promoter (refer to Zeng J., et L. 2002, Preparation of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): 694-697 for extraction of Arabidopsis DNA ₀ ) PrimeSTAR HS DNA polymerase of TaKaRa was used. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ Arabidopsis DNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 μ1, and 31 ΐ ΐ double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 V for 30 s, annealing at 58 °C for 30 s, extension at 72V for 30 s, After 33 cycles, extend at 72 ° C for 10 min. pCAMBIA2300-3 was obtained by restriction enzyme digestion with HindIII and Pstl (connection method as above) pCAMBIA2300-2.

 SEQ ID NO: 13:

 ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14:

 TGACTGCAGTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO: 15 and SEQ ID NO: 16 amplify Ghcysp \ (template is Ghcyspl- obtained in Example 2, PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ Ghcyspl-l-pGEM, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 μl, and 31 μΐ double distilled water. PCR reaction conditions: 94 ° C pre- Denaturation for 5 min, 94 denaturation for 30 s, annealing at 56 °C for 30 s, 72 V for 2 min, 33 cycles, and extension at 72 °C for 10 min. Linked to PCAMBIA2300-3 by Pstl and Sacl digestion (connection method as above) pCAMBIA2300-3 Plant expression vector rd29A-Ghcyspl-l-2300.

 SEQ ID NO: 15:

 TGACTGCAGATGGCAACCACAGTAACAC SEQ ID NO: 16:

 AAGGAGCT TCAGGCACTGCTAATTTTC Example 4 rd29A-Ghcyspl-l-2300 expression vector for transformation of Agrobacterium

Agrobacterium tumefaciens LBA4404 (purchased from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin 1-2 days in advance Inoculation, culture at 28 ° C for 1 to 2 days. Pick a single colony and inoculate 5 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 streptomycin, and incubate overnight (about 12-16 hours) at 28 °C until the OD600 value 0.4, forming a seed bacterial liquid. 5 ml of culture-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 2-2.5. Hours to OD600 = 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 pre-cooled 10% glycerol (volume ratio), centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; use 10% glycerol (volume ratio) repeated washing 3-4 times; adding appropriate amount of ice pre-cooled 10% glycerol (volume ratio) resuspended Bacterial precipitation, LBA4404 competent cells were prepared, dispensed at 40 μΐ/tube, and stored at -70 °C until use.

Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μM of the rd29A-Ghcyspl-1-2300 plasmid obtained in Example 3 was added to 40 μΐ of the competent cells, and the mixture was mixed and ice bathed for about 10 min. Transfer the mixture of competent and DNA to a pre-cooled electric shock cup with a micropipette and tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. When using a 0.1cm electric shock cup, the program of the MicroPulser (purchased from bio-rad) is set to "Agr" and the electric 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 micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C. 100~200 μΐ of bacterial solution was applied to the corresponding resistant screening medium plate (LB solid medium containing 50 μ _§ /ιη1 rifampicin, 50 g/ml streptomycin, 50 μ _§ /ιη1 card Natamycin), cultured at 28 °C. Example 5 Agrobacterium-mediated leaf disc transformation was used to obtain transgenic tobacco

To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by the Institute of Tobacco, Chinese Academy of Agricultural Sciences, library number I5A00660) 30 s, washed twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 min, and wash it twice with sterile double distilled water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS ( 18.78 mM KN0 ₃ , 1.25 mM KH ₂ P0 ₄ , 20.6 mM H4NO3, 1.5 mM MgS0 ₄ , 3.0 mM CaCl ₂ , 50 μΜ , 100 μΜ Η ₃ ΒΟ ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1 μΜ Να ₂ Μο0 ₄ , 0.1 M CoCl ₂ , 100 μΜ Να ₂ ΕϋΤΑ, 100 M FeSO ₄ , 7.4 g/L agar, sucrose 30 g/L) , Prepare sterile seedlings. The leaves of sterile seedlings were cut into 5 mm×5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector rd29A-Ghcyspl-l-2300 in logarithmic growth phase for 10 min, and the bacterial liquid was sucked in the dark condition. Co-culture for 2 days (MS medium). The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin). Under light conditions (6 hours light / 8 hours dark (light intensity 3000-4000 Lx)), culture for about 45 days. After the buds grow up, they are cut and transferred to rooting medium (MS+50 mg/L kanamycin+500). The cells were cultured for about 30 days in mg/L cephalosporin. After the root system was developed, the seedlings were transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.

Take the obtained transgenic tobacco leaves and extract DNA (the same as the Arabidopsis DNA extraction method in Example 3) SEQ ID NO: 7 and SEQ ID NO: 8 (50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΐ Ex Taq, 10 μΜ primer SEQ ID NO: 9 and SEQ ID NO: 10 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 94 denaturation for 30 s, annealing at 58 ° C for 30 s, 72 ° C extension 2 min, 33 cycles, 72 °C extension for 10 min), PCR identification, preservation of positive plants for numbering ΤοΑ^ΤοΑ^ ο Example 6 Overexpression of Ghcysp l transgenic tobacco T _Q drought tolerance simulation experiment and functional identification will be extinguished The sterile vermiculite was soaked in 1/2 MS medium. T„A1-T„A20 and control tobacco tissue culture seedlings were transplanted to vermiculite, respectively. 25 °C, 10 hours light culture / 14 hours dark culture cycle, 1/2MS every 5 days, 15 days after strong seedling culture, drought stress test, transgenic tobacco, control tobacco drought for 14 days (no watering), 10 ° light culture / 14 hour dark culture cycle at 25 °C. Identification of drought resistance of T _Q transgenic plants indicated that the control plants were severely wilted, while T ₀ A ₅ , T ₀ A6, T ₀ A ₁₀ , T ₀ A _U , T ₀ A ₁₄ , T ₀ A ₁₅ , T ₀ A _Twenty-one of the 30 tobaccos in the _2Q 7 strains were able to grow normally, showing significant drought tolerance (see Figure 3, taking T ₀ A ₅ , T ₀ A _U as an example, T ₀ A6, T ₀ A ₁₀ The results of T ₀ A ₁₄ , T ₀ A ₁₅ , T ₀ A ₂ are similar to T. A ₅ , T. A _U , not shown here). Example 7 Validation of Ghcyspl-1 Gene Expression at Transcriptional Level

Control tobacco, drought-tolerant non-significant transgenic tobacco T _Q plants, drought-tolerant transgenic tobacco To plants (good growth), leaves of drought-free 14 days, 0.05 g, total RNA was extracted with 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 respective RNA concentrations. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay LIBOX Superscript III Reverse Transcriptase (.2\ag total RNA as a template, reverse transcription primer SEQ ID NO: 8). The relative expression of cyspl-1 protein was detected by amplifying G/zq-1 by SEQ ID NO: 7 and SEQ ID NO: 8. The PCR reaction was carried out using reverse-transcribed cDNA 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, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 μΐ Double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, after 29 cycles, extension at 72 °C for 10 min. The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-5 for control tobacco, 6-18 for drought-tolerant transgenic tobacco T _Q plants, 19-24 for drought-tolerant transgenic tobacco T _Q plants. The size of the band shown is consistent with the size of GhcyspJ-l (approximately 1.1 Kbp). The results showed that there was no Ghcyspl-l mRNA in the control tobacco, and the drought-tolerant transgenic tobacco T _Q- generation plants had stronger transcription of the foreign gene (3⁄4ς)ψ-1, and drought tolerance was not significant in the G/z _{C of} transgenic tobacco T _Q plants. The transcription of ^ -l is weak or not transcribed.

Claims

Claim A cysteine protease of cotton having an amino acid sequence as shown in SEQ ID NO:  2. The gene encoding the cysteine protease of claim 1, which has the nucleotide sequence of SEQ ID NO: 2.  A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and wherein the nucleotide sequence of the gene and the expression control sequence of the expression vector are operably Preferably, the expression vector is pCAMBIA2300.  4. The recombinant expression vector of claim 3 which is the rd29A-Ghcyspl-1-23 vector shown in Figure 2 of pCAMBIA2300.  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 drought 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 tobacco.  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 tobacco.  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 drought tolerance of a plant and for use in plant breeding.  10. The use of claim 9, wherein the plant is tobacco.