CN103243108B - Calcium ion binding protein derived from stem nodule as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses a calcium ion binding protein derived from stem nodule as well as an encoding gene and application thereof. The calcium ion binding protein gene BjCBP1 has a base sequence as shown in SEQ NO.1; the constructed recombinant prokaryotic expression vector pET32a-BjCBP1 consists of a sequence SEQ ID NO.1 and a prokaryotic expression vector pET32a(+); the recombinant expression vector pCAMBIA1302-BjCBP1 consists of the sequence SEQ ID NO.1 and a plant expression vector pCAMBIA 1302; the plant expression vector is used for plant genetic transformation; the BjCBP1 genes are subjected to over-expression under the start of a CaMV35S promoter, and lots of BjCBP1 proteins are synthesized, so that the adverse situation resistance of plants is improved.

Description

Calcium ion-binding protein and encoding gene and the application in a kind of tumorous stem mustard source

Technical field

The present invention relates to genetically engineered field, be specifically related to calcium ion-binding protein and encoding gene and the application in tumorous stem mustard source.

Background technology

Calcium Signal is at many cellular activities, as played an important role in the processes such as cellular metabolism, genetic expression, cytoskeleton dynamic change, cell cycle, necrocytosis, signal conduction.

In vegetable cell, one of effect of calcium ion is as second messenger, and a lot of external environmental signals, as light, biology are coerced, abiotic stress, plant hormone etc. all can stimulate Ca in cytosol ²⁺the increase of concentration, thus Calcium Signal conduction caused.What in Calcium Signal pathway, first respond is calcium ion-binding protein, and it is in conjunction with Ca ²⁺can there is conception later and change, hydrophobic surface is exposed, thereby activate or suppress the activity of other albumen, thus priming signal conduction.

The abiotic stress environment such as high salt, arid and low temperature are to the output of crop and grow and have a significant impact, and therefore, the research of relevant stress resistance of plant gene is one of focus of phytology research field always.

Tumorous stem mustard (Brassica juncea var.tumida Tsen et Lee), claims again green vegetables head, is Cruciferae brassica plant, is the main raw material of hot pickled mustard tube, is one of main cash crop in China Chongqing, Sichuan, zhejiang and other places district.For many years, the correlative study of tumorous stem mustard mainly concentrates on the fields such as genetic breeding, stock breeding, cultivation technique, Quality Safety, in recent years just there is successively the report of biology aspect research, clone, functional study and the tumorous stem mustard warty stem that comprises gene expands correlative study, and attempt improves the Yield and quality of tumorous stem mustard and disease resistance, resistance etc. by gene engineering method.

For tumorous stem mustard, yet there are no the sequence of its calcium ion-binding protein and the report of gene function, the present invention has cloned first tumorous stem mustard calcium ion-binding protein, and show through lot of experiments, this albumen has the effect that improves stress resistance of plant, for research and the application of stress resistance of plant gene provides fresh content and new basis.

Summary of the invention

Given this, one of object of the present invention is to provide tumorous stem mustard (English: the Brassica juncea var.tumida Tsen et Lee) calcium ion-binding protein (English: calcium-binding protein) gene in source, by its called after BjCBP1, its nucleotides sequence is classified as shown in SEQ ID NO.1, this gene length 627bp.

Two of object of the present invention is to provide the calcium ion-binding protein BjCBP1 in tumorous stem mustard source, its aminoacid sequence is shown in SEQ ID NO.3, have 208 amino acid, and sequence knows to have 4 EFh structural domains by conserved domain (conserved domains) detection of NCBI.

Three of object of the present invention is to provide the recombinant prokaryotic expression vector pET32a-BjCBP1 of tumorous stem mustard calcium ion-binding protein gene BjCBP1, and it consists of SEQ ID NO.1 sequence and prokaryotic expression carrier pET-32a (+).

Four of object of the present invention is to provide the recombinant plant expression vector pCAMBIA1302-BjCBP1 of tumorous stem mustard calcium ion-binding protein gene BjCBP1, and it consists of SEQ ID NO.1 sequence and plant expression vector pCAMBIA1302.

Five of object of the present invention is to provide tumorous stem mustard calcium ion-binding protein gene BjCBP1 in the application that improves the resistance to adverse circumstance characteristic of plant, especially improves plant at the tolerance of high salt condition.

The present invention transcribes group order-checking by tumorous stem mustard and obtains sequence fragment SEQ ID NO.4, then according to fragment SEQ ID NO.4 design nested primer and by the amplification of RACE method, obtain first calcium ion-binding protein gene of tumorous stem mustard BjCBP1,208 amino acid of genes encoding, NCBI comparison prediction has 4 EF-hand structural domains, belongs to calcium ion-binding protein family member; By building the prokaryotic expression carrier of BjCBP1 gene, carry out prokaryotic expression and protein purification, find that BjCBP1 can, in conjunction with calcium ion, prove calcium ion-binding protein; By building BjCBP1 gene plant expression vector, can be directly used in agriculture bacillus mediated genetic transformation, formulate the new germ plasm of resistance to adverse circumstance, improve adverse circumstance tolerance characteristic, the especially salt-tolerant trait of plant, can carry out plant species improvement.

Accompanying drawing explanation

Fig. 1 is the agarose gel electrophoresis figure of BjCBP1 coding sequence pcr amplification of the present invention;

Fig. 2 is the SDS-PAGE gel electrophoresis figure of the BjCBP1 albumen after Prokaryotic expression, purification of the present invention;

Fig. 3 is the structure schema of recombinant plant expression vector pCAMBIA1302-BjCBP1 of the present invention;

Fig. 4 is the BjCBP1 gene expression dose detection figure of transgenosis type Arabidopis thaliana of the present invention and wild-type;

Fig. 5 is wild-type and transgenosis type Arabidopis thaliana rate of emergence result figure under NaCl of the present invention, Mannitol and ABA condition;

Fig. 6 is the growing state graph of wild-type and transgenosis type Arabidopis thaliana under NaCl of the present invention, Mannitol and ABA condition;

Fig. 7 is the expression level detected result figure of the adversity gene of wild-type and transgenosis type Arabidopis thaliana under NaCl of the present invention and ABA condition

Fig. 8 is the survival rate statistical graph of wild-type and transgenosis type Arabidopis thaliana under NaCl of the present invention.

Embodiment

Below in conjunction with embodiment and accompanying drawing, describe the present invention in detail, these embodiment and accompanying drawing only play illustrative effect, are not limited to range of application of the present invention.The invention is not restricted to following embodiment or embodiment, all modifications of making without prejudice to spirit of the present invention and distortion, within all should being included in the scope of the invention.

Experimental example 1: the clone of tumorous stem mustard calcium ion-binding protein gene BjCBP1

1 main agents: pillar a small amount of plant total RNA extraction agent box (W7021) is purchased from Shanghai Hua Shun Bioisystech Co., Ltd; DNA purifying reclaims test kit purchased from Tian Gen biochemical technology company limited; 5 '-Full RACE Kit test kit, M-MLV ThermoScript II, Premix Ex Taq, DL2000DNA Marker, pMD19-T carrier are all purchased from Dalian precious biotechnology company limited.

2 experimental techniques and step:

BjCBP1 gene order fragment SEQ ID NO.4 obtains: adopt pillar total RNA of the total RNA extraction agent of plant cassette method extraction acquisition tumorous stem mustard knurl stem in a small amount, it is 1.92 that total RNA gets the ratio of small portion by UV spectrophotometer measuring OD260/OD280, brightness by agarose gel electrophoresis 28S is approximately 2 times of 18S, illustrate that the purity of RNA is high and without degraded, reaches requirement of experiment.Send Huada Gene Research Center, Beijing to transcribe group order-checking (RNA-seq) in total RNA sample, method is for using with Oligo(dT) the total RNA of enrichment with magnetic bead tumorous stem mustard in mRNA, add fragmentation buffer that mRNA is broken into short-movie section, take mRNA as template, with the synthetic article one cDNA chain of hexabasic base random primer, then add damping fluid, dNTPs, RNase H and DNA polymerase I synthesize second cDNA chain, through QiaQuick PCR test kit (Qiagen company) purifying and after adding EB buffer solution elution, doing end reparation, add poly(A) and connect sequence measuring joints, then with agarose gel electrophoresis, carry out clip size selection, finally carry out pcr amplification, the sequencing library of building up (200bp) is used Illumina HiSeq ^tM2000 check order.By sequencing result is annotated, choose possible calcium ion-binding protein fragment sequence SEQ ID NO.4, then according to this fragment sequence, carry out clone and the functional analysis of full length gene.

The amplification of 2.1BjCBP1 gene 5 ' end unknown nucleotide sequence

2.1.1 total RNA extracts and reverse transcription: adopt the total RNA extraction agent of pillar a small amount of plant cassette method to extract the total RNA that obtains tumorous stem mustard knurl stem, then get the total RNA of approximately 1 μ g by 5 '-Full RACE Kit kit method dephosphorylation, remove cap, add joint, last reverse transcription obtains cDNA the first chain.

2.1.2 design of primers: according to two reverse nested primers of known array SEQ ID NO.4 design of transcribing group order-checking and obtaining:

BjCBP1-AOUT：5'-TCGTCTCCAATAGCCGAGAAAACT-3'

BjCBP1-AIN：5'-TACCGTCTCCGTCGCAGTCCAC-3'

2.1.3 nest-type PRC amplification:

First be OUT amplification: cDNA the first chain in step 2.1.1 of take is template, adopting primer BjCBP1-AOUT and 5 ' this primer of RACE Outer Primer(is the primer in 5 '-Full RACE Kit test kit) do pcr amplification, reaction system is: ddH2O7.4 μ L, Premix Ex Taq10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min.

Do again IN amplification: the PCR product of the OUT of take amplification is template, and adopting primer BjCBP1-AIN and 5 ' this primer of RACE Inner Primer(is also the primer in 5 '-Full RACE Kit test kit) do pcr amplification.20 μ L reaction systems are: ddH ₂o7.4 μ L, Premix Ex Taq10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 10min.

2.1.4 electrophoresis: the PCR product of IN amplification is had to a specific band through 1% agarose gel electrophoresis in 450bp left and right.

2.1.5TA clone: IN amplification is done to 3 20 μ L reaction systems, after electrophoresis, with DNA, purify and reclaim test kit recovery specific band and be connected to pMD19-T carrier, then transform bacillus coli DH 5 alpha competent cell, through PCR screening, by restructuring positive plasmid, send Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking.

2.1.6 sequencing result: 3 ' terminal sequence of sequence and the 5 ' terminal sequence of SEQ ID NO.4 that order-checking obtains fit like a glove, and both successfully splice, and the 5 ' terminal sequence that successfully obtains BjCBP1 gene is described.

3 ' the end unknown nucleotide sequence amplification of 2.2BjCBP1 gene

2.2.1 total RNA extracts and reverse transcription: adopt the total RNA extraction agent of pillar a small amount of plant cassette method to extract the total RNA that obtains tumorous stem mustard knurl stem; then getting the total RNA of approximately 1 μ g is template; adopt joint primer 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) 30MN-3'(N=A; G, C or T; M=A, G or C) (this joint primer is from the SMART RACE cDNA Synthesis Kit test kit of Clontech company) and M-MLV ThermoScript II reversion acquisition cDNA the first chain.

2.2.2 design of primers: according to two forward nested primers of known array SEQ ID NO.4 design of transcribing group order-checking and obtaining:

BjCBP1-SOUT：5'-ATGCTCAGGGAGGTGGACT-3'

BjCBP1-SIN：5'-GCTATTGGAGACGAGCGGTGC-3'

2.2.3 nest-type PRC amplification:

First be OUT amplification: cDNA the first chain in step 2.2.1 of take is template, adopting primer BjCBP1-SOUT and 3 ' this primer of RACE Primer(is the fore portion of step 2.2.1 center tap primer, be 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-3') do pcr amplification, 20 μ L reaction systems are: ddH ₂o7.4 μ L, Premix Ex Taq10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ of 60s, 30 circulations; 72 ℃ are extended 10min.

Do again IN amplification: the PCR product of the OUT of take amplification is template, adopt primer BjCBP1-SIN and 3 ' RACE Primer to do pcr amplification.20 μ L reaction systems are: ddH ₂o7.4 μ L, Premix Ex Taq 10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 50s, 32 circulations; 72 ℃ are extended 10min.

2.2.4 electrophoresis: the PCR product of IN amplification is had to a specific band through 1% agarose gel electrophoresis in 300bp left and right.

2.2.5TA clone: IN amplification is done to 3 20 μ L reaction systems, after electrophoresis, with DNA, purify and reclaim test kit recovery specific band and be connected to pMD19-T carrier, then transform bacillus coli DH 5 alpha competent cell, through PCR screening, by restructuring positive plasmid, send Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking.

2.2.6 sequencing result: 5 ' terminal sequence of sequence and the 3 ' terminal sequence of SEQ ID NO.4 that order-checking obtains fit like a glove, and both successfully splice, and the 3 ' terminal sequence that successfully obtains BjCBP1 gene is described.

The amplification of 2.3BjCBP1 coding sequence

2.3.1 sequence assembly: 3 ' terminal sequence and known array SEQ ID NO.4 that the 5 ' terminal sequence that 2.1.6 is obtained, 2.2.6 obtain splice, obtain BjCBP1 full length gene sequence SEQ ID NO.2, its total length 905bp, coding region is that SEQ ID NO.1 sequence has 627bp, there is 92bp 5 ' end non-coding region, and there is 186bp 3 ' end non-coding region.For further verifying whether the total length of splicing is real full length sequence, so carry out PCR checking from the non-coding region design primer of 5 ' end and 3 ' end.

2.3.2 design of primers: according to the sequence SEQ ID NO.2 of 2.3.1, design a pair of upstream and downstream primer from the non-coding region at two ends:

Upstream primer BjCBP1-F:5'-TTCCCCATCAAAAATAAATCTT-3'

Downstream primer BjCBP1-R:5'-CAAACGATACTCATAACCCTCA-3'

2.3.3 the pcr amplification of full length sequence: cDNA the first chain in step 2.2.1 of take is template, adopts primer BjCBP1-F and BjCBP1-R to do pcr amplification.20 μ L reaction systems are: ddH ₂o7.4 μ L, Premix Ex Taq 10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 10min.

2.3.4 electrophoresis: the PCR product of 2.3.3 amplification is found to specific band as shown in Figure 1 through 1% agarose gel electrophoresis, and M is MarkerIII, and 1 be amplified band, has a specific band as seen about 750bp, conforms to expected results.

2.3.5TA clone: the amplification in 2.3.3 is done to 3 20 μ L reaction systems, after electrophoresis, with DNA, purify and reclaim test kit recovery specific band and be connected to pMD19-T carrier, then transform bacillus coli DH 5 alpha competent cell, through PCR screening, by restructuring positive plasmid, send Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking.

2.3.6 sequencing result: the sequence that order-checking obtains is in full accord with the respective segments of splicing sequence, illustrates and successfully obtains BjCBP1 gene.The albumen of BjCBP1 genes encoding contains 208 amino acid, as shown in SEQ ID NO.3 sequence, on NCBI, carry out conserved domain and predict that it contains 4 EF-hand motifs, EF-hand motif may have the ability in conjunction with calcium ion, therefore predict that BjCBP1 albumen can be in conjunction with calcium ion, further experimental example 2 is shown in experimental verification.

Experimental example 2: prokaryotic expression and the Characteristics Detection of tumorous stem mustard calcium ion-binding protein gene BjCBP1 in e. coli bl21 (DE3)

1, main agents: His-Tag protein purification test kit is purchased from Kang Wei reagent bio tech ltd, Beijing; T4DNA ligase enzyme, restriction enzyme BamH I and Hind Ш II are purchased from Dalian precious biotechnology company limited; DH5 α bacterial classification, coli strain containing prokaryotic expression plasmid pET-32a (+) are preserved by this laboratory.

2, the structure of recombinant prokaryotic expression vector pET32a-BjCBP1

2.1 design of primers: according to the coding region sequence SEQ ID NO.3 design upstream and downstream primer of BjCBP1 gene, and introduce respectively restriction enzyme site BamH I(GGATCC) and Hind Ш (AAGCTT) sequence, primer is:

Upstream primer pET-F:5 '-CGGATCCATGAAATTCGCAAAACTG-3 '

Downstream primer pET-R:5 '-CAAGCTTTCATCGCTGGAGATCCA-3 '

2.2PCR amplification: cDNA the first chain in step 2.2.1 of take in experimental example 1 is template, and primer pET-F and pET-R do pcr amplification: system is ddH ₂o7.4 μ L, Premix Ex Taq10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 10min.

2.3 electrophoresis: the PCR product of 2.2 amplifications is had to a specific band through 1% agarose gel electrophoresis in 630bp left and right, conform to expected results.

2.4TA clone obtains recombinant vectors pMD-T-BjCBP1: the amplification in 2.2 is done to 3 20 μ L reaction systems, after electrophoresis, with DNA, purify to reclaim test kit recovery specific band and be connected to pMD19-T carrier and obtain recombinant vectors pMD-T-BjCBP1, then transform bacillus coli DH 5 alpha competent cell, through PCR screening, by the positive DH5 αJun Yesong Beijing Liuhe Huada Genomics Technology Co., Ltd order-checking containing recombinant vectors pMD-T-BjCBP1.

2.5 sequencing results: in full accord with expected results.

The structure of 2.6 recombinant prokaryotic expression vector pET32a-BjCBP1: by the 2.4 positive DH5 α bacterium liquid enlarged culturing containing recombinant vectors pMD-T-BjCBP1, and adopt the method for common plasmid extraction kit to extract the recombinant plasmid pMD-T-BjCBP1 in bacterium liquid; The DH5 α strain expanded culture containing prokaryotic expression plasmid pET-32a (+) that laboratory is preserved, and the extraction plasmid pET-32a (+) that uses the same method.With BamH I and Hind Ш double digestion plasmid pMD-T-BjCBP1 and pET-32a (+), two kinds of plasmids of specification sheets of reference enzyme are respectively done 2 50 μ L enzymes and are cut system.Enzyme is cut rear electrophoresis, and cuts the large fragment of small segment and pET-32a (+) plasmid of pMD-T-BjCBP1 plasmid, utilizes glue to reclaim test kit and reclaims.(linked system is: small segment 7 μ L with T4DNA ligase enzyme, to connect small segment and large fragment, large fragment 1 μ L, T4DNA ligase enzyme 1 μ L, 10 * T4DNA Ligase Buffer1 μ L) at least 24 hours, then utilize thermal shock method to transform intestinal bacteria competence DH5 α; PCR screening positive clone, then carry out sequence verification, obtains the right-on recombinant prokaryotic expression vector pET32a-BjCBP1 of sequence and containing the intestinal bacteria of this expression vector.

2.7pET32a-BjCBP1 recombinant prokaryotic expression vector transforms e. coli bl21 (DE3) bacterial strain and carries out abduction delivering, purifying protein and protein characteristic and detect: from 2.6, containing in the bacterial strain of pET32a-BjCBP1 carrier, extract recombinant plasmid, by heat shock method, transform BL21 (DE3) bacterial strain competence, PCR screening obtains positive colony.By this positive colony, being cultured to bacterial concentration OD600 containing 37 ℃, the jolting of 180rpm rotating speed in the LB liquid nutrient medium of 50mg/L penbritin, be 0.4-0.8, then add IPTG(Chinese name: sec.-propyl-β-D-sulfo-galactopyranoside; Final concentration is 1mM) induction makes to express BjCBP1 albumen for 5 hours.Then according to His-Tag protein purification test kit specification sheets purifying, obtain BjCBP1 albumen.The BjCBP1 albumen of acquisition is detected respectively at 12% polyacrylamide gel electrophoresis (SDS-PAGE) containing carrying out under 10mM calcium ion and 10mM EGTA metal ion chelation agent condition, and as shown in Figure 2, BjCBP1 albumen is containing Ca to electrophoresis result ²⁺condition under the rate of migration of electrophoresis faster, illustrate that BjCBP1 albumen can be in conjunction with Ca ²⁺, be calcium ion-binding protein.

Experimental example 3: the structure of the recombinant plant expression vector pCAMBIA1302-BjCBP1 of tumorous stem mustard calcium ion-binding protein gene BjCBP1

1 main agents: common plasmid extraction kit is purchased from Tian Gen biochemical technology company limited; T4DNA ligase enzyme, restriction enzyme BamH I, Bgl II and BstE II are purchased from Dalian precious biotechnology company limited; Bacterial classification containing plasmid pCAMBIA1302 is preserved by this laboratory.

The structure of 2 recombinant plant expression vector pCAMBIA1302-BjCBP1

2.1 design of primers: according to the coding region sequence SEQ ID NO.3 design upstream and downstream primer of BjCBP1 gene, and introduce respectively restriction enzyme site BamH I(GGATCC) and BstE II(GGTCACC) sequence, primer is:

Upstream primer BjCBP1-Bam:5 '-GGATCCATGAAATTCGCAAAACTG-3 '

Downstream primer BjCBP1-Bst:5 '-GGTCACCTCATCGCTGGAGATCCA-3 '

2.2PCR amplification: cDNA the first chain in step 2.2.1 of take in experimental example 1 is template, and primer BjCBP1-Bam and BjCBP1-Bst do pcr amplification: system is ddH ₂o7.4 μ L, Premix Ex Taq10 μ L, template 1 μ L, each 0.8 μ L of upstream and downstream primer.PCR program is: 94 ℃ of 3min, 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 10min.

2.3 electrophoresis: the PCR product of 2.2 amplifications is had to a specific band through 1% agarose gel electrophoresis in 630bp left and right, conform to expected results.

2.4TA clone obtains recombinant vectors pMD19-T-BjCBP1: the amplification in 2.2 is done to 3 20 μ L reaction systems, after electrophoresis, with DNA, purify to reclaim test kit recovery specific band and be connected to pMD19-T carrier and obtain recombinant vectors pMD19-T-BjCBP1, then transform bacillus coli DH 5 alpha competent cell, through PCR screening, by the positive DH5 αJun Yesong Beijing Liuhe Huada Genomics Technology Co., Ltd order-checking containing recombinant vectors pMD19-T-BjCBP1.

2.5 sequencing results: in full accord with expected results.

The structure of 2.6 recombinant plant expression vector pCAMBIA1302-BjCBP1: construction process as shown in Figure 3, by the 2.4 positive DH5 α bacterium liquid enlarged culturing containing recombinant vectors pMD19-T-BjCBP1, and adopt the method for common plasmid extraction kit to extract the recombinant plasmid pMD19-T-BjCBP1 in bacterium liquid; The DH5 α strain expanded culture that contains plant expression plasmid pCAMBIA1302 that laboratory is preserved, and the extraction plasmid pCAMBIA1302 that uses the same method.With BamH I and BstE II double digestion plasmid pMD19-T-BjCBP1, with Bgl II(Bgl II and BamH I, be isocaudarner) and BstE II double digestion plasmid pCAMBIA1302, two kinds of plasmids of specification sheets of reference enzyme are respectively done 2 50 μ L enzymes and are cut system.Enzyme is cut rear electrophoresis, and cuts the large fragment of small segment and the pCAMBIA1302 plasmid of pMD19-T-BjCBP1 plasmid, utilizes glue to reclaim test kit and reclaims.(linked system is: small segment 7 μ L with T4DNA ligase enzyme, to connect small segment and large fragment, large fragment 1 μ L, T4DNA ligase enzyme 1 μ L, 10 * T4DNA Ligase Buffer1 μ L) at least 24 hours, then utilize thermal shock method to transform intestinal bacteria competence DH5 α; PCR screening positive clone, then carry out sequence verification, obtains the right-on recombinant expression vector pCAMBIA1302-BjCBP1 of sequence and containing the intestinal bacteria of this recombinant expression vector.

2.7pCAMBIA1302-BjCBP1 recombinant plasmid transformed Agrobacterium: extract recombinant plasmid containing in the bacterial strain of pCAMBIA1302-BjCBP1 carrier from 2.6, by liquid nitrogen cold shock method, transform agrobacterium tumefaciens GV3101, method is: 1. in 200 μ L agrobacterium tumefaciens GV3101 competent cells, add 2 μ g(5-10 μ L) recombinant plasmid dna, ice bath 5min, then goes to freezing 8min in liquid nitrogen; 2. rapidly with 37 ℃ of water-baths in temperature bathe after 5min, add 3. 28 ℃ ﹑ 250rpm preculture 4-5h of 800 μ LLB liquid nutrient mediums, then coat and contain Kan (LB of 50mg/l) ﹑ Gent (50mg/l) is dull and stereotyped, cultivates after 24-28 hour for 28 ℃ and can occur bacterium colony; 4. picking thalline is thalline PCR and is identified, determines after correct and is stored in-70 ℃, is the engineering strain of Genetic Transformation in Higher Plants.

Embodiment 3: the genetic transformation of Arabidopis thaliana

1 main agents: PCR kit for fluorescence quantitative SYBR Premix Ex Taq ^tMkit is purchased from Dalian precious biotechnology company limited; Pillar is the total RNA extraction agent of plant box W7021 in a small amount, purchased from Shanghai Hua Shun Bioisystech Co., Ltd; DNA enzyme is purchased from Promega company.

Cultivation and the Agrobacterium of 2 Arabidopis thalianas are infected experiment

1. get wild-type Arabidopis thaliana seed, by sterile water wash after 75% ethanol disinfection 1min; Use 5%NaClO sterilizing 10min, sterile water wash 5 times, goes most NaClO debris again.Add a certain amount of sterilized water, vernalization 3-4 days under 4 ℃ of conditions;

2. by the Arabidopis thaliana seed of vernalization 3-4 days illumination cultivation 7 days on the blank substratum of MS, seedling to be grown, transplant to vermiculite substratum (vermiculite: Nutrition Soil: perlite=3:1:1), the pouring of 1/2MS liquid nutrient medium;

3. treat that plant culturing is to nascent inflorescence 10-15cm, when secondary inflorescence has just formed bud shape, remove nascent inflorescence; Be cultured to and can infect experiment;

4. by the Agrobacterium bacterium liquid enlarged culturing that transforms pCAMBIA1302-BjCBP1 plasmid preparing in experimental example 2 steps 2.7, in conversion, proceed to large bottle incubated overnight first 1 day evening; To bacterial concentration be OD600=2.0; Centrifugal, abandon supernatant, Agrobacterium precipitation is suspended in the infiltration nutrient solution of approximately 3 times of volumes, make OD600 in 0.8 left and right;

5. the over-ground part of plant is immersed in agrobacterium suspension and infected 5 minutes; With preservative film, the plant of infecting (T0 is for plant) cover is got up to keep humidity, put dark culturing normal condition cultivation after 12 hours in incubator; Within 2-3 days, throw off preservative film, after 7 days, can water; And regularly infect several times;

6. continue to be cultured to plant maturation, sowing (T1 is for seed).

MS substratum consists of by a certain percentage macroelement, trace element, molysite, organic substance, hormone etc.MS substratum is as the widest in vitro cell culture base of current use, because wherein inorganic salt concentration is higher, for the growth of explant provides enough mineral nutritions and can make callus accelerating growth.For ease of solution preparation and reduce error, first press the mother liquor of macroelement, trace element, molysite, organic substance preparation certain volume.Treat used time dilution mixing by a certain percentage again.

The homozygotic screening of 3 Arabidopis thaliana transgenosis

T1 is coated on equably after sterilization, sterilizing and vernalization treatment for seed and contains on the antibiotic MS substratum of Totomycin (25mg/L), to artificial climate incubator, cultivates, and observes the upgrowth situation of seedling (T1 is for plant).Within 10-12 days, obviously can observe afterwards non-transgenic Arabidopis thaliana can only grow 2 cotyledons, not have true leaf, root very short, and transgenic arabidopsis grows 2-4 sheet true leaf, root is long, so transgenic seedling is transplanted to vermiculite substratum, collects respectively each transgenic seedling seed (T2 is for seed) after maturation.T2 is coated on equably after sterilization, sterilizing and vernalization treatment for seed and contains on the antibiotic MS substratum of Totomycin (25mg/L), resistance seedling with the quantity of non-resistance seedling than being about 3:1's, again resistance seedling (T2 is for plant) is transplanted to vermiculite substratum, after maturation, collect respectively each transgenic seedling seed (T3 is for seed).Get each T2 for the small portion T3 of plant for seed, after sterilization, sterilizing and vernalization treatment, be coated on equably and contain on the antibiotic MS substratum of Totomycin (25mg/L), if be full the homozygote that is of resistance seedling, corresponding T3 can be for subsequent experimental for seed, and corresponding T2 is homozygote for plant.

Choose at random homozygote T2 for plant 6 strains, difference called after TL1, TL2, TL3, TL4, TL5 and TL6, adopt pillar total RNA that the total RNA extraction agent of plant cassette method extracts each strain plant leaf in a small amount, through DNA enzyme, remove DNA, according to quantitative PCR kit SYBR Premix Ex Taq ^tMthe expression amount of the BjCBP1 gene in each transgenic arabidopsis of Kit specification sheets operation detection, the primer of BjCBP1 gene is:

Upstream primer: 5 '-CGTTAGAGGAGTGCGAGCGTATG-3 '

Downstream primer: 5 '-CGAGAACTCAGTGAAGCACACGAAT-3 ',

Reference gene is Arabidopis thaliana 18S gene, and its primer is:

Upstream primer: 5 '-CGTCCCTGCCCTTTGTACAC-3 '

Downstream primer: 5 '-CGAACACTTCACCGGATCATT-3 ',

Quantitative PCR result as shown in Figure 4, X-coordinate WT represents wild-type Arabidopis thaliana, TL1 to TL6 represents the 6 strain transgenic arabidopsis plant of choosing at random, ordinate zou represents the relative expression quantity of BjCBP1 gene, know that TL3 and TL4 expression amount are higher, so the T3 that chooses TL3 and TL4 for seed for subsequent experimental, the T3 of TL3 and TL4 for seed and the plant of growth after germinateing all referred to as transgenosis TL3 and transgenosis TL4.

Experimental example 4: the adverse circumstance reaction experiment that turns BjCBP1 gene Arabidopis thaliana

Transgenosis TL3 and TL4 seed and wild type seeds are coated on equably on MS substratum after sterilization, sterilizing and vernalization treatment, to artificial climate incubator, cultivate, observe percentage of germination situation and plant-growth situation, transgenosis type and wild-type do not have notable difference.Transgenosis TL3 and TL4 seed and wild type seeds are coated on equably respectively containing on 100mM NaCl, 250mM N.F,USP MANNITOL (English mannitol) or the 0.5 μ M dormin MS substratum of (being called for short ABA) after sterilization, sterilizing and vernalization treatment, observe percentage of germination situation and plant-growth situation.Percentage of germination situation as shown in Figure 5, A is adding up from 0-9 days percentage of germination under 0.5 μ M ABA condition, B is the percentage of germination picture of the 5th day under 0.5 μ M ABA condition, C is adding up from 0-9 days percentage of germination under 100mM NaCl condition, C is adding up from 0-9 days percentage of germination under 250mM mannitol condition, and in A, C, D, black box, black circle, white circle represent respectively wild-type, transgenosis TL3, transgenosis TL4.Percentage of germination lab diagram 5 shows that transgenosis type seed is obviously lower than wild-type percentage of germination at 2-6 days, be that transgenosis type rate of emergence has super allergic phenomena to adverse environmental factor (NaCl, mannitol and ABA), illustrate that BjCBP1 gene plays an important role when plant adverse environmental factor is grown.

In addition, after transgenosis TL3 and TL4 seed and wild type seeds are germinateed 3 days on MS substratum, get part and be transferred to the NaCl containing 100mM, continued growth on the MS substratum of 250mM N.F,USP MANNITOL (English mannitol) or 0.5 μ M dormin (being called for short ABA), making plant growing state after 10 days, as shown in Figure 6, transgenosis type TL3, the growth on normal MS substratum of TL4 and wild-type is distinguished without obvious, but transgenosis type growth fraction wild-type under adverse environmental factor is obviously suppressed, be to grow to adverse environmental factor (NaCl after transgenosis type seed germination, mannitol and ABA) also there is super allergic phenomena, again illustrate that BjCBP1 gene plays an important role when plant adverse environmental factor is grown.

Embodiment 4: turn the degeneration-resistant border experiment of BjCBP1 gene Arabidopis thaliana

1 turns adversity gene RD29B and RD22 detection of expression in BjCBP1 gene Arabidopis thaliana

Arabidopis thaliana RD29B and RD22 gene are stress inducible gene, have the effect that improves plant adverse circumstance tolerance.Wild-type and transgenosis type seed are transplanted to vermiculite and grown when MS substratum germinates and grow 4 true leaves, when plant has 3 weeks when large, to plant complete stool spray water, NaCl (200mM) aqueous solution or ABA (the 10 μ M) aqueous solution, after 3 hours, extract total RNA separately, the expression of fluorescence quantitative PCR detection adversity gene RD29B and RD22.

The primer of RD29B gene is:

Upstream primer: 5 '-AAGATTTTCCGACAAGAGGTGAT-3 '

Downstream primer: 5 '-TTGGGACGAGATAGTTCTGGTGA-3 ',

The primer of RD22 gene is:

Upstream primer: 5 '-GTGGCTAAGAAGAACGCACCGAT-3 '

Downstream primer: 5 '-TGGCATACCGCAACTGCTTTAG-3 ',

Reference gene is still Arabidopis thaliana 18S gene, and its primer is:

Upstream primer: 5 '-CGTCCCTGCCCTTTGTACAC-3 '

Downstream primer: 5 '-CGAACACTTCACCGGATCATT-3 ',

Quantitative PCR result as shown in Figure 7, X-coordinate CK is contrast, it is the condition of spray water, ordinate zou is relative expression's level of adversity gene, result shows that adversity gene RD29B and RD22 are under collating condition, the expression level of wild-type and transgenosis type is basic identical, but when adverse environmental factor, in transgenosis type, the expression of RD29B and RD22 gene is all than the remarkable rising of wild-type.Illustrate thus under adverse environmental factor, BjCBP1 gene has the ability that improves the resistance to adverse circumstance growth of plant.

The impact of the survival rate of BjCBP1 gene pairs Arabidopis thaliana under 2 salt stresses

Wild-type WT seed and transgenosis type seed TL3, TL4 are coated on equably after growing 2 weeks on the MS substratum containing the NaCl of 300mM and are transplanted to making to recover growth without continuing in the vermiculite of salt stress to cultivate after sterilization, sterilizing and vernalization treatment, within 12 days, observe afterwards the plant quantity (it is dead that the whole albefactions of plant are considered to) of still survival.Result as shown in Figure 8, cultivate after 12 days after growing 2 weeks under standard state by high-salt stress, and wild-type plant mortality ratio is high, and survival rate is less than 10%, and the survival rate of transgenosis type reaches more than 30%, has significant difference.Illustrate that BjCBP1 gene contributes to the growth of plant under high salt, has improved the resistance of plant.

In sum, BjCBP1 gene has the effect that improves stress resistance of plant, promotes adaptive faculty and the energy for growth of plant under adverse circumstance.

Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.

Claims (5)

1. tumorous stem mustard calcium ion-binding protein gene BjCBP1, is characterized in that, its gene order is SEQ ID NO.1.

2. tumorous stem mustard calcium ion-binding protein, is characterized in that: its aminoacid sequence is shown in SEQ ID NO.3.

3. the recombinant prokaryotic expression vector pET32a-BjCBP1 of tumorous stem mustard calcium ion-binding protein gene BjCBP1, is characterized in that: by SEQ ID NO.1 sequence, be connected into prokaryotic expression carrier pET-32a (+) and form.

4. the recombinant plant expression vector pCAMBIA1302-BjCBP1 of tumorous stem mustard calcium ion-binding protein gene BjCBP1, is characterized in that: by SEQ ID NO.1 sequence, be connected into plant expression vector pCAMBIA1302 and form.

5. tumorous stem mustard calcium ion-binding protein gene BjCBP1 is improving the application of the resistance to adverse circumstance characteristic of plant, and wherein said plant is Arabidopis thaliana, and described resistance to adverse circumstance characteristic is salt-tolerant trait.