CN105143246A - Cysteine protease cysp1-2 of cotton and coding gene, and use thereof - Google Patents

Abstract

Provided in the present invention are a cysteine protease cysp1-2 derived from cotton and a coding gene thereof, and the use of the gene in the cultivation of transgenic plants having improved drought tolerance.

Description

Cysteine protease cysp1-2 of cotton and coding gene, and use thereof

One cotton cysteine proteinase cyspl-2 and its encoding gene and applied technical field

The present invention relates to cysteine proteinase and its encoding gene and application, the more particularly to one cysteine proteinase cyspl-2 and its encoding gene from cotton, and its application in the genetically modified plants that drought tolerance is improved are cultivated.

Technical background

Abiotic stress, such as arid, salt marsh, extreme temperature, chemical contamination and oxygen injury can cause serious harm to growing for plant, extreme loss is caused to crop yield, wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, it is endangered equivalent to other disaster sums, be many areas be agricultural development bottleneck.According to statistics, the dry early, semiarid zone in the world accounts for the 34% of land area;China is dry early, semiarid zone accounts for the 52% of area, year area suffered from drought up to 200 270 ten thousand hectares, the national billion cubic meter of the annual water shortage in irrigation district about 300 receives 350 400 hundred million kilograms of grain because of water shortage less；Particularly China relationship and primary grain producing such as North China, northeast and northwest, is the area of China's water shortage most serious, and spring drought frequently reaches 10 years nine chances.

Because the resistance to coercive of plant belongs to quantitative character mostly, existing available germ plasm resource is deficient, and the difficulty for improveing stress tolerance in plants using traditional breeding method is quite big, cultivates really resistance to stress kind just particularly difficult.In recent years, with to plant stress-resistance molecular mechanism research deepen continuously and Protocols in Molecular Biology fast development, degeneration-resistant research is deep into molecular level from physiological level, promotes the development of plant stress-resistance genetic engineering.Corresponding responsing reaction can be produced when plant is being forced, to reduce or eliminate the harm brought to plant.This responsing reaction of plant is one and is related to polygenes, multi signal approach, the complex process of polygenes product.These genes and its expression product can be divided into 3 classes：（1) gene and product of signal cascade amplification system and transcription control are participated in；（2) gene and its expression product directly worked to protection biomembrane and protein；(3) protein related with transhipment to the intake of water and ion.In recent years, research of the plant to stress-tolerance ability is improved by transgenic technology, and significant achievement is all achieved to the research for coercing crops, xerophyte and halophytes with tolerance, stress-related genes and signal transduction system there has also been and further understand（Liu Q.1998. Two transcription factors, DREB1 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. ) .

Cysteine proteinase is used as the important protease family of a class, the various physiology courses of wide participation plant.Many studies have shown that cysteine proteinase mRNA can accumulate under environment-stress such as low temperature, arid and condition of salt stress, it is responsible for the degraded of impaired or albuminate under adverse environmental factor, and peptide fragment is provided or free amino acid for the synthesis of new albumen.But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.In the research in terms of the function and expression regulation of degeneration-resistant response gene important basis is provided by the research that network system is transmitted in the contact between the signaling pathways related to plant stress-resistance and whole signal.

The content of the invention

The present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned a cysteine proteinase for cotton（Be named as cyspl-2 herein) encoding gene.And find to be conducted into after recipient plant, the drought tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a cysteine proteinase cyspl-2 for cotton encoding gene（GhcyspI-2 is named as herein), its sequence is SEQ ID N0:2.

Second aspect of the present invention provides a kind of recombinant expression carrier, and it contains the gene described in first aspect present invention, and it is inserted into a kind of expression vector by the gene and obtained, it is preferable that the expression vector is pCAMBIA2300;And the nucleotide sequence of the gene is operably connected with the expression control sequence of the recombinant expression carrier；Preferably, the recombinant expression carrier is the rd29A-Ghcysp 1-2-2300 carriers shown in accompanying drawing 2.

Third aspect present invention provides a kind of recombinant cell, and it contains the recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention；Preferably, the recombinant cell is restructuring agrobatcerium cell.

Fourth aspect present invention provides a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：Effectively producing plant Under the conditions of cultivate containing the gene described in first aspect present invention, the plant of the recombinant expression carrier described in second aspect of the present invention or plant tissue；Preferably, the plant is tobacco.

Gene, the recombinant expression carrier described in second aspect of the present invention or the recombinant cell described in third aspect present invention that sixth aspect present invention is provided described in first aspect present invention are used to improve drought resistance in plants and the purposes for plant breeding；Preferably, the plant is tobacco.

Seventh aspect present invention provides the amino acid sequence of the gene code as described in first aspect present invention, such as SEQ ID NO:Shown in 1.Brief description of the drawings

Fig. 1 is Ghcyspl-2 plant expression vector Crd29A-Ghcyspl-2-2300) structure flow（Scheme la-lb).

Fig. 2 is GhcyspI-2 plant expression vector (rd29A-Ghcyspl-2-2300;) plasmid figure.

Fig. 3 is () ψ -2 transgene tobaccos T_QFor plant（In figure, T_QB_{6 ;}The right side, T_QB₁₃) and be used as control non-transgenic tobacco plants（Figure is left, CK) drought-enduring simulated experiment result.

Fig. 4 is to T using reverse transcription PCR_QFor in transgenic tobacco plant and non-transgenic reference plant

The transcriptional level of G/zqy^ -2 genes carries out the result of molecular level detection.Μ is Marker, and 1-4 is non-transgenic control tobacco, and 5-18 is the significant transgene tobacco T of drought-enduring effect_QFor plant, 19-23 is the inapparent transgene tobacco T of drought-enduring effect_QFor plant.Embodiment

The present invention is further described with reference to non-limiting example.

The restriction enzyme mentioned in example below is purchased from New England Biolabs companies.

Cotton SSH library constructions under embodiment 1, drought stress：

Specific method is：

According to the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kit specifications builds subtracted library by Subtractive hybridization method.The mRNA of the cotton seedling leaf of Osmotic treatment is used as sample using in growth course in experimentation（), tester the mRNA using untreated cotton seedling leaf is used as control（driver).Comprise the following steps that：

(1) material to be tested： (National Cotton mid-term storehouse obtains unit Cotton research institute, Unified number to Ji cotton 14：ZM-30270) it is seeded on the vermiculite of sterilizing, is cultivated under 25 °C, 16 hours photoperiods illumination/8 hour dark condition, 1/2MS culture mediums are poured weekly（9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM H₄N0₃, 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50μΜ ΚΙ, 100 μΜ Η₃Β0₃, 100 M MnSO₄, 30 μ Μ ZnS0₄, 1 μ Μ Na₂Mo0₄, 0.1 μΜ CoCl₂, 100 μΜ Na₂EDTA, 100 M FeSO₄) once.It is used to test when seedling plant height reaches 25-30cm.

(2) material process：

2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.First group is control group, in 25 °C, illumination cultivation, is just commonly using 1/2MS and is pouring；Second group is Osmotic treatment group, 25 °C, illumination (6 hours illumination/8 hour dark（The Lx of light intensity 3000-4000) culture, stop pouring, the blade of timely two groups of seedling apicals 1/3 of clip after 10 days, after liquid nitrogen quick freeze, preserved in-70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and the cotton leaf 0.5g of Osmotic treatment group are taken respectively, and the total serum IgE of cotton is extracted with plant RNA extracts kit (invitrogen).Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, OD260/OD280 ratios are 1.8-2.0, show that total serum IgE purity is higher, the integrality of total serum IgE is detected with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.Use the Oligotex mRNA purification kits of Qiagen companies（PolyA+ RNA are purified from total serum IgE) separation mRNA.

(4) Subtractive hybridization：

In order to have increased access to EST（Expressed sequence tag, EST) (unigene) validity, it is to avoid gene is without restriction enzyme site and obtained sequence in non-translational region.

This laboratory is according to the scheme described in subtractive kit, by two groups of mRNA respectively with primer

01igo (dT) 18 (TTTTTTTTTTTTTTTTTT) carries out reverse transcription, and by gained cDNA Rsal,

Haelll is digested, do two groups of suppression subtractives, the method of other steps and method as shown in the PCR-select cDNA Subtraction Kit kits of Clontech companies carries out Subtractive hybridization, finally merges second of PCR primer that two groups of positive subtractives hybridize cDNA fragments.

(5) structure of cDNA subtracted libraries and preliminary screening, clone and identification

The positive subtractive of merging is hybridized to second of PCR primer of cDNA fragments（QIAquick PCR Purification Kit are purified, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega) carrier, according to method shown in pGEM-T Easy reagent kit product specifications, comprises the following steps that：Following ingredients are sequentially added with 200 μ PCR pipes：The positive subtractive of purifying hybridizes 4 ligase buffer solution of second of PCR primer 3 μ 1, Τ 5 μ 1, pGEM-T Easy carriers 1 μ 1, the μ of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.Then 10 μ coupled reaction products are taken, are added in 100 μ competence e. coli jm109s (being purchased from TAKARA), the min of ice bath 30, the s of heat shock 60, the min of ice bath 2 separately add 250 μ L LB fluid nutrient mediums（Contain 1% tryptone（Tryptone, purchased from OXOID), 0.5% yeast extract（Yeast Extract, purchased from OXOID) standing grain P l% NaCl (be purchased from traditional Chinese medicines））After be placed in 37 °C of shaking tables, with the min of 225 r/min shaken cultivations 30,200 bacterium solutions are then therefrom taken to plant in containing 50 g/mL ampicillins, the g/mL IPTG of 40 g/mL X-gaK 24 (X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4）With IPTG (isopropyl-β-D- Thiogalactopyranosides）Purchased from TAKARA) LB is (ibid）On solid culture flat board, 37 °C of 18 h of cultivation.Count diameter in culture plate>1 mm clear white and blue colonies number, random 300 white colonies of picking (numbering：Gh-A001 to Gh-A300).By the white colony of institute's picking be inoculated in 96 porocyte culture plates (CORNING) (glycerol adding is to (the volume ratio of final glycerol concentration 20% after the LB fluid nutrient mediums of ^g/mL ampicillins, 37 °C of overnight incubations containing 5）, saved backup in -80 °C.To the colony clone cultivated with the standing grain P Primer 2R of nest-type PRC primer Primer 1 (the PCR-selectTM cDNA Subtraction Kit kits from Clontech companies）Bacterium solution PCR amplification checkings are carried out, 222 positive colonies is obtained, Invitrogen is being sent to all positive colonies（Shanghai）Trade Co., Ltd is sequenced

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 145 ESTs are obtained（Expressed sequence tag, EST) (unigene).Find that wherein 92 unigene have homologous sequence in GenBank through BlastN（Albumen homology more than 50%）, 37 EST Unknown Functions or for assume albumen, separately there are 47 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3 ', 5 ' end non-translational regions.

The cotton cysteine proteinase encoding gene Ghcyspl-2 of embodiment 2 clone

One of effective clone that embodiment 1 is obtained Gh-A112 sequencing result removes after redundant DNA, and sequence is SEQ ID NO:3, sequence analysis shows the amino acid sequence and known cysteine of the coding of the sequence Protease has compared with high homology, and the clone Gh-A112 full-length genes encoded are named as（) ψ -2, corresponding albumen is named as cyspl-2.

SEQ ID NO:3

1 GCTTTTTCAA CTATTGCTGC AGTGGAAGGG ATAAACAAAA TCATTACAGG CGACCTCATA

61 GTTCTCTCAG AGCAAGAATT AGTGGATTGT GATACATCCT ACAATGAAGG ATGCAATGGT

121 GGCCTTATGG ATTATGCCTT TGAATTCATC ATTAAAAATG GTGGCATTGA CACTGAAGAA

181 GATTACCCTT ATTCCAATCA TAATGGCAGA TGTGACACCT ATAGGAAGAA TGCCAAGGTT

241 GTTTCAATTG AAGCTTATGA AAATGTTCCG GAAAACGACG AGGGTGCATT GAAAAAGGCA

301 GTCTCAAATC AACCGGTTAG TGTTGCTATC GAAGCCGGAG GCAGGGCTTT CCAATTATAT

361 CAATCTGGTA TATTTGATGG TCAATGTGGC ACACAATTAG ACCATGGTGT GACTATTGTT

421 GGATATGGTA CTGAAAATGG TAAAGACTAT TGGATTGTAA GGAATTCATG GGGTGATAAT

481 TGGGGAGAGG CAGGCTATGT CCGGATGGAA CGAAACGTGG TCGATACCAA GACCGGTAAA

541 TGCGGAATTG CAATGGAAGC CTCTTATCCT ATCAAAACGG GACGAAATCC ACCTAATCCC

601 GGTCCTTCTC CGCCGTCTCC CGTAAAGCCT CCTTCTGTTT GTGATAATTA TTATAGTTGT

661 CCCGAGAGCA ATACTTGTTG TTGCGTCTTC GAGCAGTATG GCTACTGCCT CGCGTGGGGA

721 TGTTGCTCGA TTGAGGCTGC TACTTGTTGT GAAGACAGCT ATAGCTGCTG TCCACATGAC

781 TACCCTGTTT GCAACATAAA TGAAGGAACT TGCTTGATGA GCAAGGATAA TCCGTTGGGA

841 GTGAAGGCGA TGAAGCGGAC TCCGGCTAAA CCGTTCCGGG GAGATGGAAG TGTAGTCGGA

901 AAAAGCAGGG CTTAAATCGA AAACCGACAT GTATGATGGT AAAGCAGCTG TGAACAAACA

961 GGTTGCTGTT AATTTCTATT GTCCTCTGTA TTTCATTTCA TTATTTATTA ATGGCTTCTA

1021 AGAATTTGAT TACTTGTCAA CATGTAATGT TGTTTATACT TTCTTCGTAT TAATTATTAA

1081 ATTTAGGAA

The clone of Ghcyspl-2 full-length genes

Ghcyspl-2 genetic fragments (the SEQ ID NO obtained:3), there is terminator codon TAA, it is only necessary to be 5 ' RACE.

According to the Ghcyspl-2 genetic fragments obtained（SEQ ID NO:3) three specific primers, are designed, reverse transcription primer and 5'RACE 3 ' end primers are used as.

Ghcyspl-2 GSP 1： SEQ ID NO: 4：

GGTTGATTTGAGACTGCCTTTT

Ghcyspl-2 GSP2： SEQ ID NO: 5：

AAACAACCTTGGCATTCTTCCT

Ghcyspl-2 GSP3： SEQ ID NO: 6：

AAGGGTAATCTTCTTCAGTGTC

Experimental procedure is operated by kit specification（5'RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）. With SEQ ID NO:5 (kit is carried with 5' universal primers AAP）, cDNA (the reverse transcription primer SEQ ID NO for the mRNA reverse transcriptions extracted with Osmotic treatment group cotton leaf:4) first round is carried out for template

PCR is expanded, and is comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems： 5 μΐ ΙΟ Εχ

Buffer, 3 μ 2.5 mM the μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 5 and AAP, and 35 μ distilled water.PCR reaction conditions：

94 °C of pre-degenerations 5 min, 94 denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:6 carry out second with 3' ends primer AUAP takes turns PCR amplifications, comprises the following steps that：

50 μ PCR reaction systems：The first round PCR primer of 5 μ Ι Ο Ε χ Buffer, 3 μ 2.5 mM dNTP, 2.0 μ dilution, l .O l Ex Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 6 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 V are denatured 30 s, 58 °C of 30 s of annealing, and 72V extends 2 min, after 33 circulations, 72 °C of 10 min of extension.It is about 1000bp bands that second of PCR primer, which reclaims fragment,（Gel Extraction Kit are purchased from OMEGA) it is connected to pGEM-T Easy carriers, it is transformed into JM109 (specific method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 g/mL ampicillins, and glycerol adding to final glycerol concentration is after 37 °C of overnight incubations

20% (volume ratio）, -80 °C save backup. SEQ ID NO:6 carry out bacterium solution PCR amplifications with 3' ends primer AUAP（Reaction system and reaction condition are ibid）, 3 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of gene 5' ends.

After the 5'RACE product clonings sequencing of gained, splice with clone Gh-A112 sequencing results, obtain

SEQ ID NO: 17：

1 ACTCATACAT CACTACACTT TTTTCCACCC CACACATTCA AAATCCTCTA TATGGATTCA

61 CAACCATCAA CAATGGCGAT TCTCTTGCTG ATGATGTTCA CTTTATCATC AGCTTTCGAC

121 ATGTCAATCG TATCCTACGA TAAATCCCAT CCCGACCGGT CGATGTCTAG TTGGAGAACC

181 TTCGACGAGG TTATGGCAAT GTATGAGGAC TGGCTTGTAA AACATGGCAA AGTTTACAAT 241 GGTTTAGGAG AGAAAGAGAA AAGGTTCCAG ATTTTCAAAG ATAATCTCAG GTTCATCGAT

301 GAACATAACT CGGAGGAGAC ACATAGTTTC AAGCTTGGGT TGAACCAGTT CGCCGACTTG

361 ACCAACGAGG AGTACCGTTT TACTTACTTG GGTGTTAAGA AACCTAATAA GAAGGTTTCC

421 AAGAGGAGTG ATCGTTACGT GCAGCTCCTC GGCCAGGCGG CCTTGCCGGA TTCTGTTGAT

481 TGGAGGACAA AAGGTGCTGT GGCTCCGGTT AAAGATCAAG GTTCTTGTGG GAGTTGCTGG 541 GCTTTTTCAA CTATTGCTGC AGTGGAAGGG ATAAACAAAA TCATTACAGG CGACCTCATA

601 GTTCTCTCAG AGCAAGAATT AGTGGATTGT GATACATCCT ACAATGAAGG ATGCAATGGT

661 GGCCTTATGG ATTATGCCTT TGAATTCATC ATTAAAAATG GTGGCATTGA CACTGAAGAA 721 GATTACCCTT ATTCCAATCA TAATGGCAGA TGTGACACCT ATAGGAAGAA TGCCAAGGTT

781 GTTTCAATTG AAGCTTATGA AAATGTTCCG GAAAACGACG AGGGTGCATT GAAAAAGGCA

841 GTCTCAAATC AACCGGTTAG TGTTGCTATC GAAGCCGGAG GCAGGGCTTT CCAATTATAT

901 CAATCTGGTA TATTTGATGG TCAATGTGGC ACACAATTAG ACCATGGTGT GACTATTGTT 961 GGATATGGTA CTGAAAATGG TAAAGACTAT TGGATTGTAA GGAATTCATG GGGTGATAAT

1021 TGGGGAGAGG CAGGCTATGT CCGGATGGAA CGAAACGTGG TCGATACCAA GACCGGTAAA

1081 TGCGGAATTG CAATGGAAGC CTCTTATCCT ATCAAAACGG GACGAAATCC ACCTAATCCC

1141 GGTCCTTCTC CGCCGTCTCC CGTAAAGCCT CCTTCTGTTT GTGATAATTA TTATAGTTGT

1201 CCCGAGAGCA ATACTTGTTG TTGCGTCTTC GAGCAGTATG GCTACTGCCT CGCGTGGGGA 1261 TGTTGCTCGA TTGAGGCTGC TACTTGTTGT GAAGACAGCT ATAGCTGCTG TCCACATGAC

1321 TACCCTGTTT GCAACATAAA TGAAGGAACT TGCTTGATGA GCAAGGATAA TCCGTTGGGA

1381 GTGAAGGCGA TGAAGCGGAC TCCGGCTAAA CCGTTCCGGG GAGATGGAAG TGTAGTCGGA

1441 AAAAGCAGGG CTTAAATCGA AAACCGACAT GTATGATGGT AAAGCAGCTG TGAACAAACA

1501 GGTTGCTGTT AATTTCTATT GTCCTCTGTA TTTCATTTCA TTATTTATTA ATGGCTTCTA 1561 AGAATTTGAT TACTTGTCAA CATGTAATGT TGTTTATACT TTCTTCGTAT TAATTATTAA

1621 ATTTAGGAA

According to SEQ ID NO:17 sequences Design pair of primers are as follows：

GhcyspJ-2F: SEQ ID NO: 7：

ATGGCGATTCTCTTGCTGAT

GhcyspJ-2R: SEQ ID NO: 8：

TTAAGCCCTGCTTTTTCCGA

Pass through SEQ ID NO:7 and SEQ ID NO:8 clone GhcyspI-2 total lengths.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, the cDNA for the mRNA reverse transcriptions extracted using Osmotic treatment group cotton leaf enters performing PCR reaction as template.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR, 10 μ Μ primer

SEQ ID NO:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：

94 °C of pre-degenerations 5 min, 94 denaturation 30 s, 54 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.

Pcr amplification product adds A tails：PCR primer adds the absolute ethyl alcohol of 2.5 times of volumes, and -20 °C are placed 10 minutes, centrifugation, supernatant is removed, is dried, is dissolved with 21 μ distilled waters, add 2.5 μ Ι Ο χ Ε χ Buffer, 0.5 μ 15 ι η Μ dATP, 2.5 μ Ι Ο χ Ε χ Taq.Reaction condition：70 °C are reacted 30 minutes.It will obtain about

1400 bp DNA fragmentation is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers, conversion JM109 (method is ibid), random 10 white colonies of picking are in containing 50 g/mL ampicillins

Cultivated in LB fluid nutrient mediums, glycerol adding is to (the volume ratio of final glycerol concentration 20% after 37 °C of overnight incubations）, - 80 °C save backup. SEQ ID NO:7 and SEQ ID NO:8 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 4 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO: 2.

According to the nucleotide sequence, it is known that the cyspl-2 protein amino acid sequences of the gene code such as SEQ ID Ν Ο:Shown in 1.

The amino acid sequence of cyspl-2 albumen： SEQ ID NO: 1

1 MAILLLMMFT LSSAFDMS IV

21 SYDKSHPDRS MSSWRTFDEV

41 MAMYEDWLVK HGKVYNGLGE

61 KEKRFQI FKD NLRFIDEHNS

81 EETHSFKLGL NQFADLTNEE

101 YRFTYLGVKK PNKKVSKRSD

121 RYVQLLGQAA LPDSVDWRTK

141 GAVAPVKDQG SCGSCWAFST

161 IAAVEGINKI ITGDLIVLSE

181 QELVDCDTSY NEGCNGGLMD

201 YAFEFI IKNG GIDTEEDYPY

221 SNHNGRCDTY RKNAKWS IE

241 AYENVPENDE GALKKAVSNQ

261 PVSVAIEAGG RAFQLYQSGI

281 FDGQCGTQLD HGVTIVGYGT

301 ENGKDYWIVR NSWGDNWGEA

321 GYVRMERNW DTKTGKCGIA

341 MEASYPIKTG RNPPNPGPSP

361 PSPVKPPSVC DNYYSCPESN

381 TCCCVFEQYG YCLAWGCCS I

401 EAATCCEDSY SCCPHDYPVC

421 NINEGTCLMS KDNPLGVKAM

441 KRTPAKPFRG DGSWGKSRA

461 *

The nucleotide sequence of Ghcyspl-2 encoding genes： SEQ ID NO:2

1 ATGGCGATTC TCTTGCTGAT GATGTTCACT TTATCATCAG CTTTCGACAT GTCAATCGTA

61 TCCTACGATA AATCCCATCC CGACCGGTCG ATGTCTAGTT GGAGAACCTT CGACGAGGTT

121 ATGGCAATGT ATGAGGACTG GCTTGTAAAA CATGGCAAAG TTTACAATGG TTTAGGAGAG

181 AAAGAGAAAA GGTTCCAGAT TTTCAAAGAT AATCTCAGGT TCATCGATGA ACATAACTCG

241 GAGGAGACAC ATAGTTTCAA GCTTGGGTTG AACCAGTTCG CCGACTTGAC CAACGAGGAG

301 TACCGTTTTA CTTACTTGGG TGTTAAGAAA CCTAATAAGA AGGTTTCCAA GAGGAGTGAT

361 CGTTACGTGC AGCTCCTCGG CCAGGCGGCC TTGCCGGATT CTGTTGATTG GAGGACAAAA

421 GGTGCTGTGG CTCCGGTTAA AGATCAAGGT TCTTGTGGGA GTTGCTGGGC TTTTTCAACT 481 ATTGCTGCAG TGGAAGGGAT AAACAAAATC ATTACAGGCG ACCTCATAGT TCTCTCAGAG

541 CAAGAATTAG TGGATTGTGA TACATCCTAC AATGAAGGAT GCAATGGTGG CCTTATGGAT

601 TATGCCTTTG AATTCATCAT TAAAAATGGT GGCATTGACA CTGAAGAAGA TTACCCTTAT

661 TCCAATCATA ATGGCAGATG TGACACCTAT AGGAAGAATG CCAAGGTTGT TTCAATTGAA

721 GCTTATGAAA ATGTTCCGGA AAACGACGAG GGTGCATTGA AAAAGGCAGT CTCAAATCAA

781 CCGGTTAGTG TTGCTATCGA AGCCGGAGGC AGGGCTTTCC AATTATATCA ATCTGGTATA

841 TTTGATGGTC AATGTGGCAC ACAATTAGAC CATGGTGTGA CTATTGTTGG ATATGGTACT

901 GAAAATGGTA AAGACTATTG GATTGTAAGG AATTCATGGG GTGATAATTG GGGAGAGGCA

961 GGCTATGTCC GGATGGAACG AAACGTGGTC GATACCAAGA CCGGTAAATG CGGAATTGCA

1021 ATGGAAGCCT CTTATCCTAT CAAAACGGGA CGAAATCCAC CTAATCCCGG TCCTTCTCCG

1081 CCGTCTCCCG TAAAGCCTCC TTCTGTTTGT GATAATTATT ATAGTTGTCC CGAGAGCAAT

1141 ACTTGTTGTT GCGTCTTCGA GCAGTATGGC TACTGCCTCG CGTGGGGATG TTGCTCGATT

1201 GAGGCTGCTA CTTGTTGTGA AGACAGCTAT AGCTGCTGTC CACATGACTA CCCTGTTTGC

1261 AACATAAATG AAGGAACTTG CTTGATGAGC AAGGATAATC CGTTGGGAGT GAAGGCGATG

1321 AAGCGGACTC CGGCTAAACC GTTCCGGGGA GATGGAAGTG TAGTCGGAAA AAGCAGGGCT

The Ghcyspl-2 gene plant expression vector establishments of 1381 TAA embodiments 3

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoters that Ρ Τ Π genes contain double enhancers are replaced with Pnos promoters, to reduce expression of the Ρ Τ Π albumen in plant.Select inducible promoter rd29A and terminator

Tnos as Ghcyspl-2 genes promoter and terminator.

With primer SEQ ID NO:9 and SEQ ID NO:10 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS DNA polymerases.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ ρ Β Ι 121,1.0 ^ PrimeSTAR, 10 μ Μ primer SEQ ID Ν Ο:9 and SEQ ID NO:10 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of 30 s of denaturation,

56 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.By EcoRI,

The PCR primer of gained is connected to pCAMBIA2300 by kit explanation (Promega, T4 connect enzyme reagent kit) and obtains pCAMBIA2300-l by Bglll digestions.

SEQ ID NO: 9 ：

GCACGA¾TTCATACAAATGGACGAACGGAT SEQ ID NO: 10：

ATCCAGATCTAGATCCGGTGCAGATTATTTG

With primer SEQ ID NO:11 and SEQ ID NO:12 using pBI121 as template amplification Tnos, uses TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, the μ PrimeSTAR of 1.0 μ, 1 ρ Β Ι 121,1.0,10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:12 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.Illustrate that (Promega, T4 connect enzyme reagent kit) is connected to pCAMBIA2300-l and obtains pCAMBIA2300-2 by kit as PCR primer of Sacl, EcoRI digestion by obtained by.

SEQ ID NO: 11：

AAGGAGCTCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 12：

TCAGAATTCCCAGTGAATTCCCGATCTAGTA primer SEQ ID NO:13 and SEQ ID NO:14 with arabidopsis（Colombia's type, purchased from the arabidopsis Biological Resource Center of Ohio State Univ-Columbus USA（Www.arabidopsis.org)) DNA is template amplification arabidopsis rd29A promoters（With reference to Zeng J., et L. 2002, Preparation of total DNA from " recalcit rant plant taxa ", Acta Bot. Sin., 44 (6):Method in 694-697 extracts arabidopsis DNA).Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ arabidopsis DNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:13 and SEQ ID NO:14 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 denaturation 30 s, 58 °C of annealing 30 s, 72V extend 30 s, and after 33 circulations, 72V extends 10 min.It is connected to that (connection method is ibid by HindIII, Pstl digestion）PCAMBIA2300-2 obtains pCAMBIA2300-3.

SEQ ID NO: 13：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14：

TGACTGCAGTCCAAAGATTTTTTTCTTTCCAATAG SEQ ID NO:15 and SEQ ID NO:(template is that embodiment 2 is obtained to 16 amplification Ghcysp 2

), Ghcyspl-2 using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ Ghcyspl-2-pGEM, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 denaturation 30 s, 58 °C of annealing 30 s, 72V extension 2min, after 33 circulations, 72 °C of 10 min of extension.It is connected to by Sall, Sacl digestion (connection method is ibid）PCAMBIA2300-3, obtains plant expression vector rd29A- Ghcyspl-2-2300. SEQ ID NO: 15：

TGAGTCGACATGGCGATTCTCTTGCTGAT

SEQ ID NO: 16：

AAGGAGCT TTAAGCCCTGCTTTTTCCGA

The rd29A-Ghcysp 1-2-2300 expression vectors of embodiment 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competence：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1, under 28 °C shake overnight incubation (；About 12-16 hours) to OD600 values be 0.4, formed seed bacterium solution.Take the bacterium solution after 5 ml culture activation（1 :20 ratio）It is inoculated in LB fluid nutrient mediums of 100 ml containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 °C of shakes cultivate 2-2.5 hours to OD600=0.8.The min of ice bath bacterium solution 10, shakes up once every 3 min, makes bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add certain glycerine of head for precooling 10%（Volume ratio）Resuspension thalline, 4000 g centrifuge 10 min under 4 °C, collect precipitation；Use 10% glycerine（Volume ratio）Repetition is washed 3-4 times；Add 10% glycerine of appropriate ice precooling（Volume ratio）Again suspended bacterial is precipitated, that is, LBA4404 competent cells are made, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt competent cell on ice, the rd29A- Ghcysp 1-2-2300 plasmids of 1 μ embodiments 3 acquisition, the min of ice bath about 10 after mixing are added into 40 μ competent cell.Competence and DNA mixture are transferred in the electric shock of precooling cup with micropipettor, rapping makes suspension reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from bio-rad) it is put on the slideway of electroporation chamber, promote slideway to put electric shock cup to electroporation chamber base electrode.Using 0.1cm electric shock cup when, MicroPulser (be purchased from bio-rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds the LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten competent cell with micropipettor.Suspension is transferred to 1.5 ml centrifuge tube, at 28 °C, cultivated 1 hour with 225 rpm.Take 100 200 μ bacterium solution be coated with corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ_§The rifampins of/ι η 1,50 g/ml streptomysins, 50 μ_§The kanamycins of/ι η 1）, 28 °C of cultures. Embodiment 5 obtains transgene tobacco using agriculture bacillus mediated leaf disc transformation method

With 75% alcohol-pickled tobacco seed（Countries tobacco mid-term storehouse, obtains unit：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are washed twice with sterilizing distilled water.8 min are soaked with 0.1% mercuric chloride again, is washed with sterilizing distilled water twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS (18.78 mM KN0₃, 1.25 mM KH₂P0₄, 20.6 mM H4NO3,1.5 mM MgS0₄, 3.0 mM CaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 M MnSO₄, 30 M ZnSO₄, 1 μ Μ Ν α₂Μο0₄, 0.1 M CoCl₂, 100 μ Μ Ν α₂ΕϋΤΑ, 100 M FeSO₄, 7.4 g/L agar, the g/L of sucrose 30) under aseptic condition germinate, prepare aseptic seedling.Tests for sterility is taken to be cut into the leaf dish of 5 mmx5 mm sizes, with the min of During Agrobacterium leaf dish 10 of the 1-2-2300 of rd29A-Ghcysp containing expression vector in exponential phase, bacterium solution is blotted, 2 days (MS culture mediums are co-cultured under dark condition）.Blade is gone into differential medium (the MS+1 mg/L basic elements of cell division（BA)+0.1 mg/L methyl α-naphthyl acetates（NAA) the mg/L cephalosporins of+50 mg/L kanamycins+500）On, (illumination/8 hour are dark (Lx of light intensity 3000-4000) within 16 hours under illumination condition；) culture 45 days or so, cut after bud is grown up and be transferred to root media（The mg/L cephalosporins of MS+50 mg/L kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS culture mediums only added with 500 mg/L cephalosporins.

Take the transgenic tobacco leaf of acquisition, extract DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO:7 and SEQ ID NO:8 (50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, 3 μ 2.5 mM dNTP, 2.0 μ DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:9 standing grain mouthful SEQ ID NO:10 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,94 30 s of denaturation, 58 °C of 30 s of annealing, 72 °C of 2 min of extension, after 33 circulations, 72 °C of 10 min of extension), PCR identifications preserve positive plant and the overexpression GhcyspI-2 transgene tobaccos of Τ ο Β ^ Τ ο Β ^ embodiments 6 T are numbered_QDrought-enduring simulated experiment and Function Identification sterilized vermiculite is impregnated with 1/2MS culture mediums. T.A1-T.A20 and control tobacco tissue-cultured seedling are transplanted to vermiculite respectively, 25 °C, optical culture/14 hour light culture circulation in 10 hours, pour a 1/2MS for every 5 days, strong seedling culture is after 15 days, drought stress experiment is carried out, transgene tobacco, control tobacco arid (are not watered for 14 days）, 25 °C, optical culture/14 hour light culture circulation in 10 hours. T_QFor transfer-gen plant（T_QThe plant grown up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and Τ ο Β ^ T₀B₄ T。B₅、 T。B₆、 T。B₉、 T。B₁₃、 T。B₁₇、 T。B₁₉Eight strains have in totally 34 tobaccos 25 can normal growth, show significant drought tolerance（Referring to Fig. 3, with T.B₆、 T。B₁₃Exemplified by, Τ ο Β ^ T₀B₄、 T。B₅、 T。B₉、、 T。B₁₇、 T。B₁₉Result and T.B₆、 T。B₁₃It is similar, it is not shown here）.Embodiment 7 verifies Ghcyspl-2 gene expressions on transcriptional level

Control tobacco, drought-enduring not notable transgene tobacco T are taken respectively_QFor plant, drought-enduring notable transgene tobacco T_QFor plant（Upgrowth situation is good）The arid leaf 0.05g of 14 days, the total serum IgE extracted with plant RNA extraction kit (Invitrogen).Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, calculates each RNA concentration.According to Invitrogen reverse transcriptions examination, [method shown in J box Superscript III Reverse Transcriptase carries out reverse transcription, and (2^g total serum IgEs are used as template, reverse transcription primer SEQ ID NO together: 8).Pass through SEQ ID NO:7 and SEQ ID NO:8 amplification Ghcyspl-2, detect cyspl-2 albumen relative expression's situations.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin, after 29 circulations, 72 °C of 10 min of extension.Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-4 is control tobacco, and 5-18 is drought-enduring notable transgene tobacco T_QFor plant, 19-23 is drought-enduring not notable transgene tobacco T_QFor plant.Stripe size shown in figure is in the same size with G/zqy^'s -2（About 1.4 Kbp).As a result show to compare foreign gene Ghcyspl-2 in the mRNA for not having to transcribe Ghcyspl-2 in tobacco, drought-enduring notable transfer-gen plant and transcribe stronger, drought-enduring not notable transgene tobacco T_QTranscription for Ghcyspl-l in plant is very weak or does not transcribe.

Claims (10)

1. Claims

   1. a cysteine proteinase for cotton, its amino acid sequence such as SEQ ID NO:Shown in 1.

   2. encoding the gene of cysteine proteinase described in claim 1, its nucleotides sequence is classified as SEQ ID NO:2.

   3. a kind of recombinant expression carrier, it is obtained by the way that the gene described in claim 2 is inserted into a kind of expression vector, and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector, preferably, the expression vector is pCAMBIA2300.

   4. the recombinant expression carrier described in claim 3, it is the rd29A-Ghcysp 1-2-2300 carriers shown in accompanying drawing 2.

   5. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or claim 3 or 4 described in claim 2；Preferably, the recombinant cell is restructuring agrobatcerium cell.

   6. a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or claim 3 or 4 described in claim 2 is imported into plant or plant tissue and makes the gene expression;Preferably, the plant is tobacco.

   7. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or claim 3 or 4 described in claim 2 or plant tissue are cultivated under conditions of plant is effectively produced.

   8. the method described in claim 7, wherein the plant is tobacco.

   9. the recombinant expression carrier described in gene, claim 3 or 4 described in claim 2 or the recombinant cell described in claim 5 are used to improve drought resistance in plants and the purposes for plant breeding.

   10. the purposes described in claim 9, wherein the plant is tobacco.