CN105026563A

CN105026563A - Cotton casein kinase, and coding gene and application thereof

Info

Publication number: CN105026563A
Application number: CN201280076369.XA
Authority: CN
Inventors: 王建胜; 崔洪志; 何云蔚; 刘捷; 林余
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2012-10-23
Filing date: 2012-10-23
Publication date: 2015-11-04
Anticipated expiration: 2032-10-23
Also published as: CN105026563B; WO2014063269A1

Abstract

Provided are a casein kinase CIPK1-1 derived from cotton and its coding gene, as well as its application in culturing transgenic plants with improved drought tolerance.

Description

Cotton casein kinase, and coding gene and application thereof

The present invention relates to protein kinase and its encoding gene and application with applied technical field for individual cotton protein kinase and its encoding gene, the more particularly to one cotton protein kinase C IPK1-1 and its encoding gene from cotton, and its application in the genetically modified plants that drought tolerance is improved are cultivated.Background technology 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, dry early, the half-dried early area in the world accounts for the 34% of land area;The dry early, semiarid zone of China accounts for the 52% of area, and year, the national billion cubic meter of the annual water shortage in irrigation district about 30 received 350 40 hundred million kilograms of grain because of water shortage less by early area up to 20 270 ten thousand hectares；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 with further understanding (the Two transcrip tion facto rs of Liu Q. 1998., DREB1 and DREB2, w ith an EREBP/AP2 DNA binding domain, separate two cel lular signal transduction pathways in drought—and low temperature-responsive gene exp ression, respectively, in A rabidopsis. Plant Cel l, 10 : 1391-1406； KAN GJY. 2002. A rabid op sis basic leucine zipper p ro teins that mediate stress2responsive abscisic acid signal ing. Plant Cel l, 14 : 343 - 357； ABE H. 2003. A rabid op sis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcrip tional activato rs in abscisic acid signal ing. Plant Cel l, 15 : 63 - 78. ) .

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.Research in terms of the function and expression regulation of degeneration-resistant response gene occupies the majority, but the mechanism of contact between degeneration-resistant related signaling pathways and whole signal transmission network system need further research.

Content of the invention the present inventor has cloned a protein kinase for cotton using SSH with the RACE methods being combined（Be named as CIPK1-1 herein) encoding gene DNA sequence dna.And find to be conducted into after transfer-gen plant, the drought tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a protein kinase C IPK1-1 for cotton encoding gene, and its sequence is SEQ ID NO: 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 the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector；Preferably, the carrier is the rd29A-GhCIPKl-l-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 nucleotide sequence 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 nucleotide sequence 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：Cultivated under conditions of plant is effectively produced 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 described in invention first aspect, such as SEQ ID N0:Shown in 1.Brief description of the drawings Fig. 1 is GhCIPKl-1 plant expression vector（Rd29A-GhCIPKl-l-2300 structure flow).Fig. 2 is GhCIPKl-1 plant expression vector（Rd29A-GhCIPKl-l-2300 plasmid figure).Fig. 3 be GhCIPKl-1 transgenosis 1 for tobacco plant（In figure, T S4;Figure is right, T S7) and be used as the non-transgenic tobacco plants compareed（Figure is left）Drought-enduring simulated experiment result.

Fig. 4 be GhCIPKl-1 transgenosis 1 protein expression the result for tobacco plant and non-transgenic reference plant on transcriptional level.M is Marker, and 1-8 is transfer-gen plant, and 9_12 is adjoining tree, and 13 be positive control（GhCIPKl_l ).

Embodiment

The present invention is further described with reference to non-limiting example.Cotton SSH library constructions under embodiment 1, drought stress：

Specific method is：

Standing grain IJ builds subtracted library with the method shown in the PCR-selectTM cDNA Subtraction Kit of Clontech companies by Subtractive hybridization method.MRNA using the leaf of the cotton seedling of Osmotic treatment in experimentation is used as sample（), tester control (driver) is used as using the mRNA of the leaf of untreated cotton seedling.Specific steps are summarized as follows：

(1) material to be tested：

African cotton（National Cotton mid-term storehouse, obtains unit Cotton research institute, Unified number：ZM-06838) it is seeded on sterilized vermiculite, is cultivated under the conditions of 25 °C, photoperiod 16h/8h (Lx of light intensity 2000-3000), 1/2MS culture mediums are poured weekly（9. 39 mM KN0₃, 0. 625 mM KH₂P0₄, 10. 3 mM N N0₃, 0. 75 mM MgS0₄, 1. 5 mM CaCl₂, 50 μ Μ KI, 100 μ Μ H₃B0₃, 100 μ Μ MnS0₄, 30 μ Μ ZnS0₄, 1 μ Μ N Mo0₄, 0. 1 μ Μ CoCl₂, 100 μ Μ N EDTA, 100 μ Μ FeS0₄) once.It is used to test when seedling plant height is up to 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, normal to pour.Second group is Osmotic treatment group, and 25 °C, illumination cultivation stop pouring, handled 10 days, the blade of two groups of seedling apicals 1/3 of timely clip after being disposed, after liquid nitrogen quick freeze, preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and the 5g of the cotton leaf of Osmotic treatment group 0. are taken respectively, use plant RNA extracts kit

(invitrogen) total serum IgE of cotton is extracted.Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, 0D260/0D280 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 neat [the J boxes of Oligotex mRNA purifying examinations of Qiagen companies（Purification of polyA+ RNA from total RNA) separation mRNA.

(4) suppressed subtractive hybridization：

Method as shown in the PCR-selectTM cDNA Subtraction Kit kits of Clontech companies carries out Subtractive hybridization.Using Clontech PCR-Select cDNA Subtraction Kits subtractive hybridization kits, Driver and Tester mRNA is first distinguished into reverse transcription, obtain double-strand cDNA, then the progress subtractive hybridization using 2 μ g Tester and 2 μ g Driver cDNA as parent material.Then the Tester cDNA after digestion are divided into joints different in two equal portions, connection by Tester cDNA and Driver the cDNA h of Rsa I digestions 1. 5 respectively under 37 °C of water-baths, and Driver cDNA are not connected to head.Two kinds of Tester cDNA for being connected with different joints are mixed with excessive Driver respectively, carry out first time subtractive hybridization.The product of two kinds of first time subtractive hybridization is mixed, then second of subtractive hybridization is carried out with Fresh denatured Driver cDNA, the fragment of differential expression is expanded by inhibition PCR twice, it is enriched with.

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

Second of PCR primer of positive subtractive hybridization cDNA fragments（QIAquick PCR Purification Kit are purified, purchased from Qiagen) (it is purchased from Promega kits with pGEM_T Easy）Carrier is connected, and according to the program of pGEM_T Easy kits, is comprised the following steps that：Following ingredients are sequentially added with the PCR pipes of 200 μ 1：The positive subtractive of purifying hybridizes the μ 1 of 4 ligase buffer solution of second of PCR primer 3 μ 1, Τ 5 of cDNA fragments, PGEM-T Easy carriers 1 μ 1, the μ of T4 DNA ligases 1, are stayed overnight in 4 °C of connections.10 μ L coupled reaction products are taken, are added in 100 competence Escherichia coli JMI09 (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 nutrient solutions（1% Tryptone is purchased from 0X0ID, and 0 5% Yeast Extract are purchased from 0X0ID, and 1% NaCl is purchased from traditional Chinese medicines）Put in 37 °C of water-baths, with the min of 225 r/min shaken cultivations 30,200 μ L bacterium solutions are taken to plant on LB (ibid)/X-gal/IPTG (X- gal/IPTG are purchased from TAKARA) culture plate containing 50 μ g/mL ampicillins, 37 °C are cultivated 18 h.Count diameter in culture plate>1 mm clear white and blue colonies number, random 540 white colonies of picking (numbering：Gh-D2-001 to Gh-D2-540).All white colonies are chosen in 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, glycerol adding is saved backup to final concentration 20% in -80 °C after 37 °C of overnight incubations.With nest-type PRC primer Primer 1 and Primer 2R, (the PCR-se l ectTM cDNA Subtract ion Ki t kits of Clontech companies are carried）Bacterium solution PCR amplifications are carried out, 452 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, 405 effective EST (uni gene) are obtained.The cotton protein kinases encoding gene GhCIPKl-1 of embodiment 2 clone

Clone Gh-D2-057 removes after redundant DNA, and sequence is SEQ ID NO:3, sequence analysis shows that the amino acid sequence of the coding of the sequence belongs to protein kinase, and the clone Gh-D2-057 full-length genes encoded are named as into GhCIPKl-l herein, and corresponding albumen is named as CIPK1-1.

SEQ ID NO: 3

1 ACCTTAGACT CGTTAATTAC CGACGATAGT TCTGGTTCTT GGGAGCCGAG AAGCCCGGTT

61 AGGCCGAGTT ATTTCAATGC TTTCGACATT ATATCTCTTT CACAAGGTTT AAACTTATCA

121 GGTTTGTTTG AGAAAGATTT GAACCAAAGG GATTGCTCAC GGTTCACCAC TAGAAAACCA

181 GCCAGCGATA TCGTTTCAAA ATTTCAACAA ATAGCCCAGA CCGAGAGTTT TAGCATCAAG

241 AACAAGGATG GGAAGGTGAA ATTGCAAGGC AGTAAGGAAG GGAGAAAGGG ACAGCTTGGT

301 ATAGATGCTG AGATCTTTGA AGTTACCGCT TCGTTTTATG TGGTGGAGTT GAAGAAAACT

361 GCTGGGGATA CTTTGGAATA CAAGAATTTC TGTAACAAAG AATTGAAGCC GTCGCTCAAG

421 GATATAGTGT GGGCTTGGCA AGGCAGCAAC AATTATACCC AGAGCCTGGT TTGAGTTTTC

481 GTATTCCAAC CAACAACTAT ACTGTGGTAG CAGCTAATTA GGGAGCTTCT TACCCTCTCC

541 GAATTTCAAT TTCTTTTCTT TTCTCGCCTT TTGTAATTAA GCTCCAAATT TTAGTTTATG

601 AATGTAAGAA GATGTGTAAA TAATTAACAA ATTAACCTTT CTACTAAAAA AAAAAAAAAA

661 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA The clone of GhCIPKl-1 full-length genes

According to the Gh-D2-057 genetic fragments obtained, two specific primers are designed, 5'RACE 3' end primers are used as.

GhCIPKl- 1 GSP1 : SEQ ID NO: 4：

CTTCAAAGATCTCAGCATCTAT

GhCIPKl— 1 GSP2: SEQ ID NO: 5：

ATTTCACCTTCCCATCCTTGTT

GhCIPKl— 1 GSP3: SEQ ID NO: 6：

AAGCATTGAAATAACTCGGCCT

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）, with cDNA (the reverse transcription primer SEQ ID NO of mRNA reverse transcriptions:4) amplification of first round PCR is carried out for template, comprised the following steps that：Ex Taq are purchased from TAKARA, the PCR reaction systems of 50 μ 1：5 μ 1 Ι Ο Χ Ε χ Buffer, 3 μ 125 mM dNTP, the cDNA of the mRNA reverse transcriptions of 2. 0 μ 1, the Ex Taq of 1. 0 μ 1,10 μM of primer SEQ ID NO:Each 2. 0 μ 1 of 7 and AAP, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of 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 1 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：The PCR reaction systems of 50 μ 1：The first round PCR product of 5 μ Ι Ο Χ Ε χ Buffer, 3 μ 125 mM dNTP, 2. 0 μ 1 dilution, the Ex Taq of 1. 0 μ 1,10 μM of primer SEQ ID NO:Each 2. 0 μ 1 of 8 and AUAP, and 35 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 2 min of extension, after 33 circulations, 72 °C of 10 min of extension.It is about 900bp bands that second of PCR primer, which reclaims fragment,（Gel Extraction Kit are purchased from OMEGA) pGEM_T Easy Vector are connected to, being transformed into JM109, (specific method is ibid）, random 10 white colonies of picking cultivate in the LB fluid nutrient mediums containing 50 g/mL ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID N0:8 carry out bacterium solution PCR amplifications with 3' ends primer AUAP（Reaction system and reaction condition are ibid）, 4 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd Sequencing sequencing, obtains the cDNA of gene 5' ends.

After the 5'RACE product clonings sequencing of gained, with SEQ ID NO:3 results are spliced.Obtain GhCIPKl-1 cDNA sequence SEQ ID NO: 7.

SEQ ID NO: 7：

1 TGACTGATTT GTGGAAACTC AGGAAAAACA ACAAAAAGAG TGTGTTGGTT GTAGGATCCT

61 CAGCCTAAAA TTTTGGTAGC AGTGAGGAGA TCATCACAAA ATGGAGAAGA AAGGGACAGT

121 ATTGATGCAA AGGTTTGAGG TGGGACGATT ACTGGGTCAA GGGACATTCG CCAAGGTTTA

181 CCAAGCCAGG AATCTAAGAA CCGGCGAAAG CTGCGCCATT AAAACCATCG ATAAAGAGAA

241 GATAATGAAA GGAGGTTTGA TAGATCAAAT CAAGCGTGAA ATCTCAGTTA TGCGCCTCGT

301 TAAACATCCC AATGTTGTTC GACTCTATGA GGTAATGGCT AGCAAATCAA AGATATATTT

361 CGTGATGGAA TATGTTAAAG GCGGTGAGCT TTTCAACAAG ATCGCTAAAG GGAAGCTCAA

421 GGAAGATGAC GCCCGACGCT ATTTCCAGCA ATTGATAGCT GCCGTTGATT ACTGCCATAG

481 CAGAGGTGTT TATCACCGGG ATTTGAAGCC CGAGAATCTC CTCCTCGATG AAAATGGGAA

541 TCTCAAGGTT TCGGATTTTG GGCTGAGTGC GTTTATAGAA TCAAGCAGAC AAGATGGGCT

601 TCTTCACACC ACTTGCGGAA CTCCAGCTTA TGTTGCACCC GAAGTCATTC ACCACAAAGG

661 CTACGACGGA GCCAAGGCTG ATATTTGGTC TTGTGGAGTC ATTCTTTACG CTCTGTTGGC

721 TGGTTTTCTC CCTTTTCAAC ACTCCAATCT CATGGAACTG TATAGAAAGA TAAGTAGAGG

781 AGAATTCAAG TGCCCACAGT GGTTCTCTCC CCAAGTTCGG AAGCTTCTTT CCAGGATTCT

841 TGAACCCAAT CCAATCCAAA GAATCACTGT GGCTAAGCTA ATGGAAAATT GTTGGTTTAG

901 GAAAGGGTAT AAACATATTG ATATCCCACC ACCATCCCCT CAACCCCGTA CCTTAGACTC

961 GTTAATTACC GACGATAGTT CTGGTTCTTG GGAGCCGAGA AGCCCGGTTA GGCCGAGTTA

1021 TTTCAATGCT TTCGACATTA TATCTCTTTC ACAAGGTTTA AACTTATCAG GTTTGTTTGA

1081 GAAAGATTTG AACCAAAGGG ATTGCTCACG GTTCACCACT AGAAAACCAG CCAGCGATAT

1141 CGTTTCAAAA TTTCAACAAA TAGCCCAGAC CGAGAGTTTT AGCATCAAGA ACAAGGATGG

1201 GAAGGTGAAA TTGCAAGGCA GTAAGGAAGG GAGAAAGGGA CAGCTTGGTA TAGATGCTGA

1261 GATCTTTGAA GTTACCGCTT CGTTTTATGT GGTGGAGTTG AAGAAAACTG CTGGGGATAC

1321 TTTGGAATAC AAGAATTTCT GTAACAAAGA ATTGAAGCCG TCGCTCAAGG ATATAGTGTG

1381 GGCTTGGCAA GGCAGCAACA ATTATACCCA GAGCCTGGTT TGAGTTTTCG TATTCCAACC

1441 AACAACTATA CTGTGGTAGC AGCTAATTAG GGAGCTTCTT ACCCTCTCCG AATTTCAATT

1501 TCTTTTCTTT TCTCGCCTTT TGTAATTAAG CTCCAAATTT TAGTTTATGA ATGTAAGAAG

1561 ATGTGTAAAT AATTAACAAA TTAACCTTTC TACTAAAAAA AAAAAAAAAA AAAAAAAAAA

1621 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

According to SEQ ID NO: 7：Sequences Design pair of primers is as follows：

GhCIPKF: SEQ ID NO: 8：

ATGGAGAAGAAAGGGACAGTAT

GhCIPKR: SEQ ID NO: 9：

TCAAACCAGGCTCTGGGTATAA

Pass through SEQ ID NO:8 and SEQ ID NO:9 clone GhCIPKl-1 total lengths. Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 125 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:8 and SEQ ID NO:9 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 2min, after 33 circulations, 72 °C of 10 min of extension.

Pcr amplification product adds A tails：PCR primer adds 2. 5 times of absolute ethyl alcohol, and _ 20 °C are placed 10 minutes, and centrifugation is removed supernatant, dried, and is dissolved with 21 μ distilled waters.Add 2. 5 μ Ι Ο Χ Ε χ Buffer, 0. 5 μ 15 mM dATP, the Ι Ο Χ Ε χ Taq of 2. 5 μ 1.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 1300 bp is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers（Obtain GhCIPKl-1-pGEM plasmids）, (method is ibid by conversion JM109）, random 10 white colonies of picking cultivate in the LB fluid nutrient mediums containing 50 g/mL ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:8 and SEQ ID NO:9 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 3 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID N0:2, the amino acid sequence of its albumen encoded is SEQ

ID NO: l o

The amino acid sequence of CIPK1- -1 albumen： SEQ ID NO: 1

1 MEKKGTVLMQ RFEVGRLLGQ

21 GTFAKVYQAR NLRTGESCAI

41 KTIDKEKIMK GGLIDQIKRE

61 ISVMRLVKHP NVVRLYEVMA

81 SKSKIYFVME YVKGGELFNK

101 IAKGKLKEDD ARRYFQQLIA

121 AVDYCHSRGV YHRDLKPENL

141 LLDENGNLKV SDFGLSAFIE

161 SSRQDGLLHT TCGTPAYVAP

181 EVIHHKGYDG AKADIWSCGV

201 ILYALLAGFL PFQHSNLMEL

221 YRKISRGEFK CPQWFSPQVR

241 KLLSRILEPN PIQRITVAKL

261 MENCWFRKGY KHIDIPPPSP

281 QPRTLDSLIT DDSSGSWEPR

301 SPVRPSYFNA FDIISLSQGL

321 NLSGLFEKDL NQRDCSRFTT

341 RKPASDIVSK FQQIAQTESF

361 SIKNKDGKVK LQGSKEGRKG

381 QLGIDAEIFE VTASFYVVEL 401 KKTAGDTLEY KNFCNKELKP

421 SLKDIVWAWQ GSNNYTQSLV

441 *

The nucleotide sequence of GhCIPKl-1 encoding genes： SEQ ID NO: 2

1 ATGGAGAAGA AAGGGACAGT ATTGATGCAA AGGTTTGAGG TGGGACGATT ACTGGGTCAA

61 GGGACATTCG CCAAGGTTTA CCAAGCCAGG AATCTAAGAA CCGGCGAAAG CTGCGCCATT

121 AAAACCATCG ATAAAGAGAA GATAATGAAA GGAGGTTTGA TAGATCAAAT CAAGCGTGAA

181 ATCTCAGTTA TGCGCCTCGT TAAACATCCC AATGTTGTTC GACTCTATGA GGTAATGGCT

241 AGCAAATCAA AGATATATTT CGTGATGGAA TATGTTAAAG GCGGTGAGCT TTTCAACAAG

301 ATCGCTAAAG GGAAGCTCAA GGAAGATGAC GCCCGACGCT ATTTCCAGCA ATTGATAGCT

361 GCCGTTGATT ACTGCCATAG CAGAGGTGTT TATCACCGGG ATTTGAAGCC CGAGAATCTC

421 CTCCTCGATG AAAATGGGAA TCTCAAGGTT TCGGATTTTG GGCTGAGTGC GTTTATAGAA

481 TCAAGCAGAC AAGATGGGCT TCTTCACACC ACTTGCGGAA CTCCAGCTTA TGTTGCACCC

541 GAAGTCATTC ACCACAAAGG CTACGACGGA GCCAAGGCTG ATATTTGGTC TTGTGGAGTC

601 ATTCTTTACG CTCTGTTGGC TGGTTTTCTC CCTTTTCAAC ACTCCAATCT CATGGAACTG

661 TATAGAAAGA TAAGTAGAGG AGAATTCAAG TGCCCACAGT GGTTCTCTCC CCAAGTTCGG

721 AAGCTTCTTT CCAGGATTCT TGAACCCAAT CCAATCCAAA GAATCACTGT GGCTAAGCTA

781 ATGGAAAATT GTTGGTTTAG GAAAGGGTAT AAACATATTG ATATCCCACC ACCATCCCCT

841 CAACCCCGTA CCTTAGACTC GTTAATTACC GACGATAGTT CTGGTTCTTG GGAGCCGAGA

901 AGCCCGGTTA GGCCGAGTTA TTTCAATGCT TTCGACATTA TATCTCTTTC ACAAGGTTTA

961 AACTTATCAG GTTTGTTTGA GAAAGATTTG AACCAAAGGG ATTGCTCACG GTTCACCACT

1021 AGAAAACCAG CCAGCGATAT CGTTTCAAAA TTTCAACAAA TAGCCCAGAC CGAGAGTTTT

1081 AGCATCAAGA ACAAGGATGG GAAGGTGAAA TTGCAAGGCA GTAAGGAAGG GAGAAAGGGA

1141 CAGCTTGGTA TAGATGCTGA GATCTTTGAA GTTACCGCTT CGTTTTATGT GGTGGAGTTG

1201 AAGAAAACTG CTGGGGATAC TTTGGAATAC AAGAATTTCT GTAACAAAGA ATTGAAGCCG

1261 TCGCTCAAGG ATATAGTGTG GGCTTGGCAA GGCAGCAACA ATTATACCCA GAGCCTGGTT

The GhCIPKl-1 gene plant expression vector establishments of 1321 TGA 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 promoter that Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.Inducible promoter rd29A and Tnos is selected as the promoter and terminator of GhCIPKl-1 genes.

With primer SEQ ID NO:10 and SEQ ID NO:11 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector PBI 121）For template amplification Pnos, using TaKaRa PrimeSTAR HS DNA polymerases.The PCR reaction systems of 50 μ 1：10 μ 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, the PrimeSTAR of 1. 0 μ, 1 121,1. 0 μ of PBI 1,10 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 min of pre-degeneration 5, 94 °C of denaturation 30 s, 56 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.PCAMBIA2300 (promega, T4 ligase boxes are connected to by EcoRI, Bglll digestion）Obtain pCAMBIA2300-l.

SEQ ID NO: 10 ：

GCACGAATTCGGCGGGAAACGACAATCTGA

SEQ ID NO: 11：

ATCCAGATCTAGATCCGGTGCAGATTATTTG

SEQ ID NO:12 standing grain P SEQ ID NO:13 with PBI 121 be template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 μ 5 X PS Buffer, 3 μ 125 mM dNTP, the PrimeSTAR of 1. 0 μ, 1 121,1. 0 μ of PBI 1,10 μ Μ primer SEQ ID NO:12 and SEQ ID NO:13 each 2. 0 μ 1, and 31 μ 1 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.PCAMBIA2300_l (promega T4 ligase boxes are connected to by KpnI, EcoRI digestion）Obtain pCAMBIA2300-2

SEQ ID NO: 12：

AAGGGTACCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 13：

TCAGAATTCCCAGTGAATTCCCGATCTAGTA

SEQ ID NO:14 and SEQ ID NO:15 with arabidopsis（Colombia's type, purchased from TARI, www. arabidopsis. org) DNA be 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.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 arabidopsis DNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:14 and SEQ ID NO:15 each 2. 0 μ 1, and 31 μ 1 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.It is connected to by HindIII, Sail digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300_3

SEQ ID NO: 14： ACTAAGCTTCCTTCTTGACATCATTCAATTTTA

SEQ ID NO : 15：

TGAGTCGACTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO :16 and SEQ ID NO:(template is the GhCIPKl-1-pGEM plasmids that embodiment 2 is obtained to 17 amplification GhCIPKl-1）, using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 125 mM dNTP, the GhCIPKl- 1- pGEM plasmids of 1. 0 μ 1,1. 0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:16 standing grain P SEQ ID NO:17 each 2. 0 μ 1, and 31 μ 1 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 2min, after 33 circulations, 72 °C of 10 min of extension.It is connected to by Kpnl, Sai l digestions（Connection method is ibid）PCAMBIA2300_3, obtains plant expression vector rd29A- GhCIPKl-l-2300.

SEQ ID NO : 16：

TGAGGTACCATGGAGAAGAAAGGGACAGTAT SEQ ID NO : 17：

The rd29A- GhCIPKl-1-2300 expression vectors of AAGGTCGACTCAAACCAGGCTCTGGGTATAA embodiments 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 μ g/ml rifampins and 50 μ g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in LB fluid nutrient mediums of 5 ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, and overnight incubation is shaken under 28 °C（About 12-16 h) to 0D600 values be 0. 4, formed seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）In the LB fluid nutrient mediums for being inoculated in the same concentration antibiotic of 100 ml, 28 °C are shaken the culture h of 2-2. 5 to 0D₆..=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 4000 g under certain glycerine resuspension thalline of head for precooling 10%, 4 °C and centrifuge 10 min, collect precipitation；Repeated to wash 3_4 times with 10% glycerine；Adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt competent cell on ice, 1 μ 1 plasmid, the min of ice bath about 10 after mixing are added into 40 μ 1 competent cell.Competence and DNA mixture are transferred to the electricity of precooling with rifle Hit in cup, 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. lcm 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 cell with rifle.Suspension is transferred to 1.5 ml centrifuge tube, 28 °C, 225 rpm cultivate 1 h.Take 100 200 μ 1 bacterium solution be coated with corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 g/ml rifampins, 50 y g/ml streptomysins, 50 μ g/ml kanamycins）, 28 °C of cultures.Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed 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 NH₄N0₃, 1.5 mM MgS0₄, 3.0 mM CaCl₂, 50 μM of KI, 100 μ Μ Η₃Β0₃, 100 μM of MnS0₄ , 30 μ M ZnS0₄, 1 μM of N Mo0₄, 0.1 μ Μ CoCl₂, 100 μM of N EDTA, 100 μM of FeS0₄, 7.4 g/L agar, the g/L of sucrose 30) under aseptic condition germinate, prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of 5 mmX5 mm sizes, with the min of During Agrobacterium leaf dish 10 of the rd29A-GhCIPKl-l-2300 containing expression vector in exponential phase, blot bacterium solution, co-cultured 2 days under dark condition（MS culture mediums）.Blade is gone into differential medium（The MS+l 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, cultivated 45 days or so under illumination condition, 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）, use SEQ IDN0: 9:Standing grain P SEQ IDN0:10 (the PCR reaction systems of 50 μ 1：5 μ 1 10 X Ex Buffer, 3 μ 125 mM dNTP, the Ex Taq of 2.0 μ 1 DNA, 1.0 μ 1,10 μM of primer SEQ ID NO:9 and SEQ ID NO:10 each 2.0 μ 1, and 35 μ 1 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 2 min of extension, after 33 circulations, 72V extends 10 min), PCR identifications preserve positive plant and T are numbered.S1_T.S20. Embodiment 6 is overexpressed GhCIPKl-1 transgene tobaccos T1 drought-enduring simulated experiment and the sterilized vermiculite of Function Identification is impregnated with 1/2MS culture mediums. T.S1-T.S20 and control tobacco seed are sowed on vermiculite respectively, 15 seeds are sowed per basin, a 1/2MS is poured in 25 °C, optical culture/10 hour light culture circulation in 14 hours for every 5 days, after cultivating 25 days, SEQ ID NO:8 and SEQ ID NO:9 do PCR detections, remove negative plant.Choose transgene tobacco of the same size and control tobacco is cooked drought-enduring experiment, per 4-5 more consistent seedling of basin reservation size.Transgene tobacco, control tobacco arid (are not watered for 14 days）, 25 °C, optical culture/10 hour light culture circulation in 14 hours.T1 is for transfer-gen plant（The plant that TO grows up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and T S4, T S7, T S9, T S10, T five strain tobaccos of S16 show obvious drought tolerance（Referring to Fig. 2, by T S4, T exemplified by S7, T S9, T S10, T S16 result and T S4, T S7 it is similar, be not shown here）.Embodiment 7 verifies that normal growth transfer-gen plant randomly selects 8 in CIPK1-1 protein expressions implementation 6 on transcriptional level, and adjoining tree randomly selects 4 in implementing 6, and 14 days 05g of blade 0. of arid use plant RNA extraction kit（Invitrogen) the total serum IgE extracted.Ultraviolet spectrophotometry total serum IgE calculates each RNA concentration in 260 nm and 280 nm absorbance.According to neat [the J box Superscript I I I Reverse Transcriptase institutes not method progress reverse transcription of invitrogen reverse transcriptions examination（1 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID N0: 9).Pass through SEQ ID NO:18 standing grain P SEQ ID NO:19 detection GhCIPKl_l, detect CIPKl-1 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.The PCR reaction systems of 50 μ 1：10 μ 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:18 and SEQ ID NO:19 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 28 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）, 1-8 is transfer-gen plant, and 9-12 is adjoining tree, and 13 be positive control（GhCIPKl_l, SEQ ID NO: 2).Stripe size shown in figure and positive control it is in the same size.As a result show that normal growth plant plant pair GhCIPKl-1 transcriptions are stronger, it is impossible to which normal growth plant is not transcribed or transcribed very weak.

SEQ ID NO : 18： CGCTATTTCCAGCAATTGATAGC

SEQ ID NO : 19： GTATTCCAAAGTATCCCCAGCA

Claims

Claims

1. an encoding protein kinase gene for cotton, its sequence is SEQ ID NO: 2.

2.-kind of recombinant expression carrier, its nucleotide sequence for containing gene described in claim 1 and the nucleotide sequence are operably connected with the expression control sequence of the expression vector.

3. the carrier described in claim 2, it is the rd29A-GhCIPKl-l-2300 carriers shown in accompanying drawing 2.

4. a kind of recombinant cell, it contains the recombinant expression carrier described in the nucleotide sequence or Claims 2 or 3 of gene described in claim 1；Preferably, the recombinant cell is restructuring agrobatcerium cell.

5. a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in the nucleotide sequence or Claims 2 or 3 of gene described in claim 1 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

6. a kind of method of prepare transgenosis plant, including：Plant or the plant tissue of recombinant expression carrier described in nucleotide sequence or Claims 2 or 3 containing gene described in claim 1 are cultivated under conditions of plant is effectively produced.

7. the method described in claim 6, wherein the plant is tobacco.

8. the recombinant expression carrier described in gene, Claims 2 or 3 described in claim 1 or the recombinant cell described in claim 4 are used to improve drought resistance in plants and the purposes for plant breeding.

9. the purposes described in claim 8, wherein the plant is tobacco.

10. the albumen of the gene code described in claim 1, such as SEQ ID NO:Shown in 1.