CN102443574B - Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector - Google Patents
Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector Download PDFInfo
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Abstract
The invention provides a recombinase which is an ntCre gene and has a sequence as represented by SEQ ID No: 1, and a binary expression vector containing the recombinase; T-DNA structure of the binary expression vector contains an RD29A:: ntCre:: Tnos gene and a LoxP sequence, wherein RD29A is a cold induction specific promoter for the recombinase ntCre, and LoxP is an identification, cutting and recombination sequence for the recombinase ntCre. According to the invention, the recombinase ntCre gene provided in the invention expresses under low temperature induction of the induction type promoter RD29A; after the binary expression vector containing the recombinase ntCre gene transforms a plant, a selective marker gene and the RD29A:: ntCre:: Tnos gene are highly efficiently deleted under low temperature induction, and a transgenic plant without the selective marker gene is obtained.
Description
Technical field
The present invention relates to a kind of recombinase gene, binary expression vector and construction process thereof and application, relate in particular to plant gene binary expression vector and relevant construction process and the application in transformation of tobacco thereof of a kind of recombinase ntCre gene and low temperature induction deleting selectable marker genes.
Background technology
In plant transgene process; for transgenic cell and non-transgenic cell are separated; usually can introduce a selectable marker gene; and the albumen of selectable marker gene coding can make vegetable cell have the antibiotic ability of opposing, thereby make under transgenic cell survives in having antibiotic environment.Because selectable marker gene has been incorporated in the genome of plant, resistant gene may shift (genetic drift) and vertical heredity by occurred level in new ecotope, and its potential hazardness is not found out completely.Meanwhile, once transfer-gen plant acquisition, necessity that selectable marker gene does not just exist.Therefore, can maybe should they be eliminated and be removed with suitable animal nutrition.The transgenic plant of non-resistant marker gene not only can promote the development of transgenic technology, and can get rid of the potential Biosafety problem of being brought by it.
According to recombinase gene, introduce mode, the transfer-gen plant that the marker gene deleting technique that utilizes site-specific restructuring to mediate obtains above-mentioned marker-free gene has following two kinds of methods:
First, indirectly introduce restructuring enzyme process, by the mode of sexual hybridization or twice transformation, recombinase gene is introduced in acquired transgenic plant genome, made that in this transfer-gen plant, the selectable marker gene between recombinase recognition site is deleted in the same way at two.Sexual hybridization introducing method is exactly with the Agrobacterium that contains recombinase gene and contain target gene and identical or different selectable marker gene, transform respectively same vegetable material, obtain transfer-gen plant male parent and maternal material, then by hybridization, recombinase gene in male parent is introduced maternal, delete in maternal material two selectable marker genes between recombinase recognition site in the same way, then separated by selfing, remove the recombinase gene and the selectable marker gene that are derived from male parent, finally obtain only containing the transfer-gen plant of target gene.Twice comversion is first with the Agrobacterium-mediated Transformation plant that contains target gene and selectable marker gene (between two recombinase recognition sites), obtain after transfer-gen plant, with the Agrobacterium that contains recombinase gene and another kind of marker gene, carry out twice transformation again, delete and transform for the first time the selectable marker gene in the transfer-gen plant obtaining, the transfer-gen plant that twice transformation obtains is separated by selfing again, remove the recombinase gene and the selectable marker gene that are derived from twice transformation, obtain only containing transfer-gen plant (the Dale E of target gene, Ow D W. Gene transfer with subsequent removal of the selection gene from the host genome[J]. Proceedings of the National Academy of Science of the USA, 1991, 88:10558-10562.).Indirectly introduce the resulting transfer-gen plant of restructuring enzyme process and all will just can obtain the transfer-gen plant of marker-free gene by further selfing separation, Breeding Process length consuming time, program is loaded down with trivial details, can only be confined in the plant of sexual hybridization simultaneously, and this reduces its practicality greatly.However, indirectly introduce restructuring enzyme process still occupies an important position in transgenic plant research.
The second, directly introduce restructuring enzyme process and be when introducing selectable marker gene between two recombinase recognition sites and introduce recombinase gene.When construction of expression vector (expression vector), selectable marker gene is placed between two recombinase recognition sites in the same way, and target gene is placed on outside them, while having like this recombinase gene to express in vegetable cell, the selectable marker gene goal gene deleted and outside recognition site between two recombinase recognition sites is in the same way retained.Recombinase gene generally by one can induction type promotor control.Transforming in early days, marker gene is given the ability of transgenic cell antiviral antibiotic or weedicide, make its proliferation and differentiation in having the environment of selective pressure, obtain after the transfer-gen plant of resistance, by certain condition, induce, make recombinase gene start to express, under the effect of recombinase, in plant materials, start to occur site-specific recombining reaction, thereby selectable marker gene is deleted.
Prior art is being introduced selectable marker gene and recombinase gene in the direct introducing restructuring enzyme process of a plant binary expression vector simultaneously, and the promotor of controlling recombinase gene expression adopts heat-inducible promoter conventionally.(the Wang Y such as Wang, Chen B, Hu Y, et a1. Inducible excision of select able marker gene from transgenic plants by the cre/loxP site-specifie recombination system [J] .Transgenic Res, 2005, 14 (5): 605-6l4.) built the carrier of controlling recombinase Cre genetic expression with heat shock protein, import to transgene tobacco after Agrobacterium, Arabidopis thaliana, the plants such as potato, after high temperature induction, recombinase Cre expresses, two marker gene between loxP site have in the same way successfully been deleted, obtain the transgenic plant of marker-free.The inventor carries out finding in heat shock delete flag gene studies at the above-mentioned heat-inducible promoter of application: (1) adopts the Cre/LxoP recombination system of the heat-inducible promoters such as HSP18.2 and HSP70m, in Arabidopis thaliana and tobacco, the effect of deleting selectable marker genes can, but need the treatment time of nearly 1 month, and active low in tomato, heat shock that need to be longer is deleted and is processed.(2) the indoor temperature environment control of tissue culture requires tighter, prevent from having a power failure in high temperature season, cause cultivating room temp and reach the start-up temperature of heat-inducible promoter and the Cre/LxoP recombination system that is integrated into Plant Genome is activated, thereby affect the acquisition of resistant transgenic plant.(3) when the heat-shock temperature with 30～37 ℃ is deleted the selectable marker gene of resistance or visual transfer-gen plant, substratum is easily evaporated and changes nutritive ingredient, unfavorable to the Proliferation and differentiation of the plants such as cucumber and tomato.
Summary of the invention
The first object of the present invention is to provide a kind of recombinase.
The second object of the present invention is to provide a kind of construction process of above-mentioned recombinase.
The 3rd object of the present invention is to provide a kind of plant binary expression vector for low temperature induction deleting selectable marker genes.
The 4th object of the present invention is to provide a kind of construction process of above-mentioned plant binary expression vector.
The 5th object of the present invention is to provide the application of plant binary expression vector in transgene tobacco.
The object of the invention is to be achieved through the following technical solutions:
A recombinase, is characterized in that: it is ntCre gene, and its nucleotide sequence is as shown in SEQ ID.NO.1.
By 1274 Nucleotide, formed with appraise and decide position (nuclear a targeted, be abbreviated as nt) sequence of the recombinase ntCre gene through improvement of signal encoding sequence and intron, this gene can be started by the promotor of Arabidopis thaliana low temperature induction gene RD29A under 4 ℃ ~ 10 ℃ cold condition.Wherein, the 29th to the 52nd nucleotides sequence classified the sequence of coding nuclear localization signal as.The 4th to the 48th and the 605th to the 794th nucleotide sequence are respectively the sequence of intron.The 1st to the 3rd nucleotides sequence classified the translation initiation codon of ntCre as.The 1272nd to the 1274th nucleotides sequence classified translation stop codon of ntCre as.
The mRNA sequence being become by this genetic transcription is SEQ ID.NO.2;
The aminoacid sequence of being translated by this gene is: SEQ ID.NO.3;
The preparation method of above-mentioned recombinase, is characterized in that:
By primer and sequence that sequence is SEQ ID.NO.4, it is the primer of SEQ ID.NO.5, PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from coli strain BM25.8(Clontech, the U.S.) in genomic dna, pcr amplification goes out the Cre gene that is derived from P1 phage (GenBank accession X03453) of 1040 bp sizes, reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 3 circulations, 95 ℃ of sex change 10 sec, 60 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 27 circulations, last 72 ℃ are extended 10 min.With Taq archaeal dna polymerase, add after " A " tail, TA clones into T carrier pMD18-T(TaKaRa, Japan), transform intestinal bacteria XL1-Blue competent cell, Amp resistance recon screens through pcr amplification, primer sequence is that SEQ ID.NO.6 and sequence are SEQ ID.NO.7, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 898 bp sizes, sequencing result shows that the gene of cloning is with the sequence of announcing in GenBank, obtain positive colony carrier pVCT1251 and recombinase Cre gene.
With Xba I and Nco I enzyme, cut carrier pVCT1251, reclaim 3727 bp fragments, abandon 11 bp fragments.Synthetic sequence SEQ ID.NO.8 and synthetic sequence SEQ ID.NO.9, in 3 minutes after annealings of 94 ℃ of sex change, are formed to the Xba I-Nco I joint of the encoding sequence that contains Arabidopis thaliana At4g01735 gene intron and monkey SV40 virus capsid protein T antigen nuclear localization signal.To reclaim fragment is connected with T4 DNA ligase with joint, transform intestinal bacteria XL1-Blue competent cell, Amp resistance recon screens through pcr amplification, and primer is SEQ ID.NO.10 and SEQ ID.NO.11, and PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 30 sec, 35 circulations, last 72 ℃ are extended 10 min, and electrophoresis detection obtains 293 bp and expects big or small product.Through sequence verification, obtain positive colony carrier pVCT1252, make First Intron and the sequence with nuclear localization signal coding on recombinase Cre upstream region of gene band.
With Xba I and Sac I enzyme, cutting carrier pVCT2027(is provided by laboratory, contriver place), reclaim 3106 bp fragments, abandon 1889 bp fragments.With Xba I and Sac I enzyme, cut carrier pVCT1252, reclaim 1096 bp fragments, abandon 17 bp and 2669 bp fragments.Two fragments are connected with T4 DNA ligase, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, primer SEQ ID.NO.5 upper with the LacZ that lays respectively at heat-inducible promoter HSP70m upstream and that be positioned at Cre gene downstream carries out pcr amplification screening, 1315 bp expect big or small product, through the enzyme evaluation of cutting and check order, obtain positive colony carrier pVCT2136 again.
With EcoRV enzyme, cut carrier pVCT2136, electrophoresis reclaims 4196 bp fragments.Adopt PCR method, with primer 5 '-gtaaatttctagtttttctccttc-3 ' (being positioned at intron upstream) and primer 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from pCAMBIA1301 carrier DNA, amplify the intron sequences of 190 bp sizes, and electrophoresis reclaims.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, with the primer SEQ NO.4 and the SEQ ID.NO.5 (being positioned at ntCre gene downstream) that lay respectively at Cre gene two ends, carry out pcr amplification evaluation and sequence verification, obtain the ntCre gene of positive colony carrier pVCT2117 and expection.
A plant binary expression vector, is characterized in that: the T-DNA structure of described plant binary expression vector comprises RD29A::ntCre::Tnos gene and LoxP sequence.Wherein RD29A is the low temperature induction specificity promoter of recombinase ntCre, and LoxP is the sequence of identification, cutting and the restructuring of recombinase ntCre.
The main structure element of the T-DNA structure of plant binary expression vector pVCT2221 of the present invention comprises: LB(T-DNA left margin), the terminator of T35s(CaMV 35S gene), the identification of LoxP(recombinase ntCre, the sequence of cutting and restructuring), the neomycin phosphotransferase gene II with prokaryotic promoter and protokaryon terminator of nptIIm(transformation, it is kalamycin resistance gene, there is prokaryotic expression characteristic), the promotor of Pnos(rouge alkali synthetase gene), the terminator of Tnos(rouge alkali synthetase gene), ntCre(is with the modified form recombinase Cre gene of nuclear localization signal encoding sequence and intron), RD29A (promotor of Arabidopis thaliana low temperature induction gene RD29A), 35S (promotor of CaMV 35S gene), mGFP (green fluorescence protein gene of modified form), RB (T-DNA right margin).
The main structure element of the T-DNA structure of plant binary expression vector pVCT2224 of the present invention comprises: LB(T-DNA left margin), the terminator of T35s(CaMV 35S gene), the identification of LoxP(recombinase ntCre, the sequence of cutting and restructuring), the neomycin phosphotransferase gene II with prokaryotic promoter and protokaryon terminator of nptIIm(transformation, it is kalamycin resistance gene, there is prokaryotic expression characteristic), the promotor of Pnos(rouge alkali synthetase gene), the terminator of Tnos(rouge alkali synthetase gene), ntCre(is with the modified form recombinase Cre gene of nuclear localization signal encoding sequence and intron), RD29A (promotor of Arabidopis thaliana low temperature induction gene RD29A), 35S (promotor of CaMV 35S gene), ipt (isopentenyl transferase genes, the expression of ipt gene is subject to the control of the promotor 35S of CaMV 35S gene), Tipt (terminator of isopentenyl transferase genes), RB (T-DNA right margin).
The T-DNA common structure of above-mentioned two kinds of concrete plant binary expression vector pVCT2221 provided by the present invention and pVCT2224 is in the same way between LoxP recognition site, to contain Pnos::nptIIm and RD29A::ntCre gene at 2.Different structure is that both are respectively with 35S::mGFP and 35S::ipt gene.Plant binary expression vector pVCT2221 and pVCT2224 develop by commercialization carrier pCAMBIA1302 (the GenBank number of logging in is AF234298), there is sequence outside common T-DNA, contain protokaryon Kan resistant gene nptIII(neomycin phosphotransferase gene III), Agrobacterium replicon REP and the agrobacterium tumefaciens Ti-plasmids pVS1 STA district of intestinal bacteria replicon ColE1, agrobacterium tumefaciens Ti-plasmids pVS1.These sequences be guarantee binary vector can be in intestinal bacteria and Agrobacterium equal reproducibles, and give Host Strains with Kan resistance.
Specifically, plant binary expression vector of the present invention is as shown in Fig. 1 a or Fig. 1 b.
The construction process of plant binary expression vector provided by the present invention, is characterized in that:
Positive colony by a series of carriers, comprising: pVCT1251, pVCT1252, pVCT2006, pVCT2027, pVCT2136, pVCT2117, pCR2.1-AtCBF3, pCR2.1m, pVCT1191, pVCT1202, pVCT2072, pVCT2102, pVCT2231, pVCT1009, pVCT2236, pVCT2020, pVCT2118, pVCT2237, pVCT1243, pVCT2130, pVCT2238, finally builds plant binary expression vector pVCT2221 and pVCT2224.(Fig. 2)
The construction step of carrier pVCT2006 of the present invention is as follows:
With EcoR I and Hind III enzyme, cut carrier pUC18(Sangon, Shanghai; GenBank accession L08752), electrophoresis reclaims 2635 bp, abandons 51 bp fragments.With identical enzymic digestion carrier pBI121(GenBank accession AF485783), reclaim 3032 bp, abandon 11726 bp fragments.By two, reclaiming fragment connects with T4 DNA ligase, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, with the primer 5 '-cttgtcgacgtccccagattagccttttc-3 ' on CaMV 35S promoter and primer 5 '-gatagtctgccagttcagttcgt-3 ' of being arranged in gus gene, carry out pcr amplification, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains 1315 bp and expects big or small product, show that 35S::GUS::Tnos gene recombinated in pUC18 carrier, through EcoR I and Xba I enzyme, cut evaluation again, obtain expecting the product of 2167 bp and 3500 bp sizes, obtain positive colony carrier pVCT2006.
The construction step of carrier pVCT2027 of the present invention is as follows:
With Xba I and Pst I enzyme, cut carrier pVCT2006, electrophoresis reclaims 4810 bp, abandons 870 bp fragments.Synthetic HSP70m heat-inducible promoter, sequence is SEQ ID.NO.12, with Xba I and Pst I enzyme, cuts, and reclaims 180 bp, abandons 7 bp and 8 bp fragments.By two, reclaiming fragment connects with T4 DNA ligase, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, with primer 5 '-gatttatatcccggtcggtgaatc-3 ' (being arranged in the upstream of HSP70m promotor) and 5 '-gatagtctgccagttcagttcgt-3 ' (being positioned at gus gene), carry out pcr amplification, 631 bp expect big or small product, show to obtain HSP70m::GUS::Tnos gene structure, sequencing result shows the accurate sequence between HSP70m and gus gene, obtains positive colony carrier pVCT2027.
The construction step of carrier pVCT2136 of the present invention is as follows:
With Xba I and Sac I enzyme, cutting carrier pVCT2027(is provided by laboratory, contriver place), reclaim 3106 bp fragments, abandon 1889 bp fragments.With Xba I and Sac I enzyme, cut carrier pVCT1252, reclaim 1096 bp fragments, abandon 17 bp and 2669 bp fragments.Two fragments are connected with T4 DNA ligase, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, primer SEQ ID.NO.5 upper with the LacZ that lays respectively at heat-inducible promoter HSP70m upstream and that be positioned at Cre gene downstream carries out pcr amplification screening, 1315 bp expect big or small product, through the enzyme evaluation of cutting and check order, obtain positive colony carrier pVCT2136 again.
The construction step of carrier pVCT2117 of the present invention is as follows:
With EcoRV enzyme, cut carrier pVCT2136, electrophoresis reclaims 4196 bp fragments.Adopt PCR method, with primer 5 '-gtaaatttctagtttttctccttc-3 ' (being positioned at intron upstream) and primer 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from pCAMBIA1301 carrier DNA, amplify the intron sequences of 190 bp sizes, and electrophoresis reclaims.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, with the primer SEQ NO.4 and the SEQ ID.NO.5 (being positioned at ntCre gene downstream) that lay respectively at Cre gene two ends, carry out pcr amplification evaluation and sequence verification, obtain the ntCre gene of positive colony carrier pVCT2117 and expection.
The construction step of carrier pCR2.1-AtCBF3 of the present invention is as follows:
Adopt PCR method, with primer 5 '-ccataccaacaaaaaagacagag-3 ' (being positioned at AtCBF3 upstream) and 5 '-gtactaaaaatggaaataataatctgag-3 ' (being positioned at AtCBF3 downstream), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from Arabidopsisecotype Columbia(
arabidopsis thalianal. Ecotype Columbia) in genomic dna, amplify the Gene A tCBF3 (GenBank accession No. AB013815) of 755 bp sizes, PCR reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 51 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 30 circulations.72 ℃ are extended 10 min.Reclaim product through adding with Taq archaeal dna polymerase after " A " tail, TA clones into T carrier pCR2.1-TOPO(Invitrogen, US), transform intestinal bacteria XL1-Blue competent cell, Amp and Kan resistance recon screen through pcr amplification, primer is SEQ ID.NO.6 and SEQ ID.NO.7, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 898 bp sizes, sequencing result shows the sequence that has obtained expection, obtain positive colony carrier pCR2.1-AtCBF3.
The construction step of carrier pCR2.1m of the present invention is as follows:
With EcoR I enzyme, cut carrier pCR2.1-AtCBF3, electrophoresis reclaims 3890 bp fragments, abandons 773 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp and Kan resistance recon, through pcr amplification, screen, primer is 5 '-gtggatccttctagtgtagccgtag-3 ' (being positioned at ColE1 upstream) and 5 '-gtaacgccagggttttccca-3 ' (being positioned at LacZ downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1048 bp sizes, through EcoR I enzyme, cut again, be shown as unit point, show to obtain positive colony carrier pCR2.1m.
The construction step of carrier pVCT1191 of the present invention is as follows:
Adopt PCR method, with primer 5 '-tcatgaccaaaatccct-3 ' (being positioned at the prokaryotic gene terminator upstream of pCR2.1m carrier) and primer 5 '-ttcagaagaactcgtcaagaagg-3 ' (being positioned at the downstream of the Kan resistant gene CDS of pCR2.1m carrier), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from pCR2.1m carrier DNA, amplify the Kan protokaryon resistant gene (nptII of 2919 bp sizes, neomycin phosphotransferase gene), f1 replicon, LacZ gene, the sequences such as intestinal bacteria replicon ColE1 and protokaryon resistant gene terminator, and remove Amp coding region, PCR reaction conditions: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 3 min, 30 circulations.72 ℃ are extended 10 min.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, show that the nptII gene (called after nptIIm, i.e. nptII modified) of restructuring has expection Kan resistance function.Through pcr amplification, screen, primer is 5 '-atactcgagctactgggctatctgg-3 ' (being positioned at nptII prokaryotic promoter upstream) and 5 '-tttctcgagttggtagctcttgatcc-3 ' (being positioned at nptII protokaryon terminator downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 3 circulations, 94 ℃ of sex change 20 sec, 59 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 32 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1240 bp sizes, confirm that the Amp gene coding region between nptII coding region and protokaryon terminator is eliminated, through EcoRI enzyme, cut again, be shown as unit point, show to obtain positive colony carrier pVCT1191.This carrier is deleted the Amp resistant gene of pCR2.1m carrier, and makes prokaryotic gene terminator on nptII gene end band, forms nptIIm gene, and this gene can be given intestinal bacteria Kan resistance.
The construction step of carrier pVCT1202 of the present invention is as follows:
Adopt PCR method, with primer 5 '-ata
ctcgagctactgggctatctgg-3 ' (be positioned at the promotor upstream of nptIIm gene, line place is Xho I site) and primer 5 '-cattatacgaagttatcctgcaggcggccgcccagggccc
tggtagctcttgatccggc-3 ' (line part is complementary with the positive chain-ordering of protokaryon terminator end of nptIIm gene) amplifies the neomycin phosphotransferase gene nptIIm of 1265 bp sizes from pVCT1191 carrier DNA.PCR reaction conditions is: 95 ℃ of denaturation 2 min, and 95 ℃ of sex change 10 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 3 circulations, then through 95 ℃ of sex change 10 sec, 60 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 27 circulations, and last 72 ℃ are extended 10 min.With the PCR product of purifying, again carry out pcr amplification, primer pair is that 5 '-atactcgagctactgggctatctgg-3 ' (is positioned at the promotor upstream of nptIIm gene, line place is Xho I site) and 5 '-ataacttcgtatagcatacattatacgaagttatcc-3 ' (line part is LoxP site), reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 46 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 3 circulations, again through 95 ℃ of sex change 10 sec, 60 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 27 circulations, last 72 ℃ are extended 10 min.Electrophoresis reclaims 1283 bp fragments, with Taq archaeal dna polymerase, add after " A " tail, TA clones in T carrier pMD18-T, transforms intestinal bacteria XL1-Blue competent cell, obtain Amp and Kan resistance recon, show that cloned nptIIm has Kan resistance function in intestinal bacteria.Through pcr amplification, screen, primer is 5 '-atactcgagctactgggctatctgg-3 ' (to be positioned at nptII prokaryotic promoter upstream, line part is Xho I site) and 5 '-gtaacgccagggttttccca-3 ' (being positioned at LacZ downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting and the product of 1363 bp sizes confirms that nptIIm is cloned, and expresses the consistent of direction and lacZ.With Hind III and Xho I enzyme, cut, obtain expecting 1302 bp and 2675 bp products, with Sal I and Sac I, obtain expecting 1314 bp and 2664 bp products, and confirmed the direction of inserting.Sequencing result shows the sequence that obtains SEQ ID.NO.13 primer sequence and nptIIm gene, and the multiple clone site on T carrier, all in full accord with the sequence of expection, obtains positive colony carrier pVCT1202.
The construction step of carrier pVCT2072 of the present invention is as follows:
With Xho I enzyme, cut carrier pCAMBIA1302(GenBank accession No. AF234298), electrophoresis reclaims 9455 bp fragments, abandons 1094 bp fragments.With Xho I and Sal I enzyme, cut carrier pVCT1202, reclaim 1284 bp fragments (with nptII-LoxP), abandon 2693 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, Kan resistance recon screens through pcr amplification, and primer is 5 '-ata
ctcgagctactgggctatctgg-3 ' (is positioned at the prokaryotic promoter upstream of nptIIm gene, line place is Xho I site) and 5 '-catgagcgaaaccctataggaacc-3 ' (being arranged in T35s eucaryon terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1362 bp sizes, show by anticipated orientation, to inject the Xho I site of pCAMBIA1302 with the nptIIm gene of prokaryotic promoter and terminator.Xho I enzyme is cut evaluation, is shown as the unit point of expection, obtains positive colony carrier pVCT2072.PVCT2072, through agrobacterium mediation converted tobacco, has obtained having the plant of kalamycin resistance, and result shows that 2x35S::nptIIm::T35s genetic expression is normal, can give transgenic tobacco cells kalamycin resistance.
The construction step of carrier pVCT2102 of the present invention is as follows:
With PmaC I and XbaI enzyme cutting carrier pVCT2072, electrophoresis reclaims 8475 bp fragments, abandons 413 bp and 1124 bp fragments.With Not I enzyme, cut back to close fragment, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with EcoRI enzyme, reclaims 2732 bp fragments, abandons 1249 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp and Kan resistance recon, primer 5 '-tcgacgatccagatccggtgca-3 ' (being arranged in Pnos promotor downstream) and the 5 '-acggggagtcaggcaactat-3 ' (being positioned at Amp gene) of pcr amplification screening, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1610 bp sizes, show to contain Pnos and Amp Gene Partial sequence, and the structure for expection.Enzyme is cut qualification result and is shown, Sal I and EcoR I can cut out 2653 bp and 8504 bp to expect and obtain positive colony carrier pVCT2102 by big or small product.
The construction step of carrier pVCT2231 of the present invention is as follows:
With EcoR I and Sal I enzyme, cut carrier pVCT2072, electrophoresis reclaims 8504 bp fragments, abandons 54 bp and 2563 bp fragments.With identical enzyme, cut carrier pVCT2117, reclaim 1732 bp fragments, abandon 2653 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon.Pcr amplification screening, primer is 5 '-atcgcaagaccggcaacagg-3 ' (being arranged in Tnos terminator) and 5 '-gagctctaatcgccatcttccagca-3 ' (being positioned at ntCre gene downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting and the product of 1610 bp sizes shows that ntCre gene is inserted in pVCT2102.Enzyme is cut qualification result and is shown, SalI and EcoRI can cut out 1731 bp and 8504 bp, and NcoI can cut out the object band of 2613 bp and 7570 bp, shows that structure is correct.Sequencing result has shown between Pnos promotor upstream and Tnos terminator downstream and accurate connection the in Tnos upstream and ntCre gene downstream, obtains positive colony carrier pVCT2231.
The construction step of carrier pVCT1009 of the present invention is as follows:
With primer 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and primer 5 '-tccaatagaagtaatcaaaccct-3 ' (being positioned at RD29A low temperature induction promotor downstream), PfuDNA polysaccharase (Sangon, Shanghai) from Arabidopsisecotype Columbia (
arabidopsis thalianal. Ecotype Columbia) in genomic dna, pcr amplification goes out the AtRD29A low temperature induction promotor (GenBank accession No. D13044) of 891 bp sizes, with Taq archaeal dna polymerase, add after " A " tail, TA clones into T carrier pUCm-T(Sangon, Shanghai), transform intestinal bacteria XL1-Blue competent cell, Amp resistance recon, through pcr amplification screening and sequence verification, obtains positive colony carrier pVCT1009.
The construction step of carrier pVCT2236 of the present invention is as follows:
With Pst I and Xba I enzyme, cut carrier pVCT2117, electrophoresis reclaims 4196 bp fragments, abandons 188 bp fragments.By same enzyme, cut carrier pVCT1009, reclaim 927 bp fragments, abandon 16 bp and 2721 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, the primer of pcr amplification screening is 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and 5 '-aaccttcaaaatcaatccaagtatggacc-3 ' (being positioned on the First Intron of Cre gene), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 54 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 957 bp sizes, show to contain in recon RA29A::Cre sequence simultaneously, and by the structural arrangement of expection.Enzyme is cut qualification result and is shown, Hind III and Xba I enzyme are cut to obtain the expection product of 943 bp and 4188 bp sizes, obtain positive colony carrier pVCT2236.
The construction step of carrier pVCT2020 of the present invention is as follows:
With Pnos upstream primer 5 '-cg
gaattcagggagtcacgttatgac-3 ' (line place is EcoR I site) and nptII downstream primer 5 '-cg
ctcgagtcccgctcagaagaac-3 ' (line place is Xho I site), PfuDNA polysaccharase (Sangon, Shanghai) from carrier pBIN19(GenBank accession U09365) pcr amplification go out the Pnos::nptII gene fragment of 1116 bp sizes, after EcoR I and Xho I enzyme are cut, reclaim 1107 bp fragments, abandon 3 bp and 7 bp fragments.With identical enzymic digestion carrier pCAMBIA1302(GenBank accession AF234298), reclaim 8425 bp fragments, abandon 1030 bp and 1094 bp fragments.By two, reclaim fragment and connect with T4 DNA ligase, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, with Pnos upstream primer 5 '-cg
gaattcagggagtcacgttatgac-3 ' and the primer 5 '-catgagcgaaaccctataggaacc-3 ' being positioned on CaMV 35S terminator pA carry out pcr amplification, obtain 1184 bp and expect and show that Pnos::nptII end has connected terminator pA by big or small product.EcoR I and Xho I enzyme are cut and identify to be shown, obtain expecting the product of 1107 bp and 8425 bp sizes, and Sac I and BstE II be unit point, obtain the positive colony carrier pVCT2020 of 9532 bp sizes.
The construction step of carrier pVCT2118 of the present invention is as follows:
With EcoR I and+Hind III enzyme cuts carrier pVCT2020, electrophoresis reclaims 9481 bp fragments, abandons 51 bp fragments.With identical enzyme, cut carrier pVCT2136, reclaim 1559 bp fragments, abandon 2635 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is that 5 '-tcgacgatccagatccggtgca-3 ' (being positioned at Pnos promotor downstream) and 5 '-accatggccaatttactgaccgtacacc-3 ' .(are positioned at Cre upstream region of gene), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 50 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1609 bp sizes, show to contain in recon Pnos promotor and Cre gene order simultaneously, and by the structural arrangement of expection.Enzyme is cut qualification result and is shown, BstE II and BamH I enzyme are cut the expection product that obtains 2135 bp and 8905 bp sizes, obtain positive colony carrier pVCT2118.This carrier can be used for hybridizing rear heat shock and deletes the selectable marker gene in acceptor transgenic plant.
The construction step of carrier pVCT2237 of the present invention is as follows:
With Hind III and Sac I enzyme, cut carrier pVCT2118, electrophoresis reclaims 9756 bp fragments, abandons 1284 bp fragments.With identical enzyme, cut carrier pVCT2236, reclaim 2229 bp fragments, abandon 2902 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is that 5 '-tcgacgatccagatccggtgca-3 ' (being positioned at Pnos promotor downstream) and 5 '-accatggccaatttactgaccgtacacc-3 ' .(are positioned at Cre upstream region of gene), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 50 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1799 bp sizes, show to contain in recon Pnos promotor and Cre gene order simultaneously, and by the structural arrangement of expection.Enzyme is cut qualification result and is shown, EcoR I and Hind III enzyme are cut the expection product that obtains 2496 bp and 9481 bp sizes, obtain positive colony carrier pVCT2237.This carrier can be used for hybridizing rear low temperature induction and deletes the selectable marker gene in acceptor transgenic plant.
The construction step of carrier pVCT1243 of the present invention is as follows:
Adopt PCR method, with primer 5 '-a
ccatggatctgcgtctaattttcgg-3 ' (is positioned at upstream, ipt gene coding region, line place is Nco I site) and primer 5 '-cgttcgatgacgaaaatggaag-3 ' (being positioned at the downstream of ipt gene terminator), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from agrobacterium tumefaciens (
agrobacterium tumefaciens) in strain C58 thalline amplification be positioned at the isopentenyl transferase genes ipt (GenBank accession No. NC_003308) on pTiC58 carrier, reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 30 circulations, last 72 ℃ are extended 10 min.Electrophoresis reclaims 1001 bp fragments, with Taq archaeal dna polymerase, adds after " A " tail, and TA clones into T carrier pMD18-T(TaKaRa, Japan), transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon.The primer of pcr amplification screening is 5 '-a
ccatggatctgcgtctaattttcgg-3 ' (is positioned at upstream, ipt gene coding region, line place is Nco I site) and 5 '-gtaacgccagggttttccca-3 ' (being positioned at the downstream of LacZ), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1078 bp sizes, show to contain in recon ipt gene order simultaneously, and by the structural arrangement of expection.Enzyme is cut qualification result and is shown,
ecor I,
ncoi,
xhoi is the single endonuclease digestion site of expection.Sequence verification has obtained ipt gene clone, obtains positive colony carrier pVCT1243.
The construction step of carrier pVCT2130 of the present invention is as follows:
With EcoR I and Nco I enzyme, cut carrier pVCT1243, electrophoresis reclaims 3657 bp fragments, abandons 38 bp fragments.With identical enzyme, cut carrier pCAMBIA1302(GenBank accession No. AF234298), the CaMV 35S promoter fragment of recovery 813 bp, abandons 9736 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Amp resistance recon, the primer of pcr amplification screening is that 5 '-cccaagcttcatggagtcaaagattc-3 ' (is positioned at CaMV 35S promoter upstream, line place is HindIII site) and 5 '-ctaatacattccgaatggatgacc-3 ' (be positioned at the downstream of ipt coding region, line part is translation stop codon of ipt gene), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 20 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1277 bp sizes, show to contain in recon CaMV 35S promoter and ipt gene order simultaneously, and by the structural arrangement of expection.Enzyme is cut qualification result and is shown, HindIII enzyme is cut the expection product that obtains 1783 bp and 2686 bp sizes, obtains positive colony carrier pVCT2130.
The construction step of carrier pVCT2238 of the present invention is as follows:
With primer 5 '-cccaagcttcatggagtcaaagattc-3 ', (be positioned at CaMV 35S promoter upstream, line place is HindIII site) and primer 5 '-cgttcgatgacgaaaatggaag-3 ' (being positioned at the downstream of ipt gene terminator), PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from pVCT2130 carrier DNA, amplify 35S::ipt gene fragment, reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 30 circulations, last 72 ℃ are extended 10 min.Electrophoresis reclaims 1552 bp fragments, after cutting, reclaims 1547 bp fragments with HindIII enzyme, abandons 4 bp fragments.With HindIII and PmaC I enzyme, cut carrier pVCT2237, reclaim 10464 bp fragments, abandon 413 bp and 1100 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect (downstream of ipt gene and Tnos upstream are connected together), transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon.The primer of pcr amplification screening is that 5 '-accatggatctgcgtctaattttcgg-3 ' (is positioned at upstream, ipt gene coding region, line place is Nco I site) and primer 5 '-atcgcaagaccggcaacagg-3 ' (being arranged in Tnos terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 57 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting and the product of 1094 bp sizes shows that in recon, containing ipt gene downstream is connected with Tnos simultaneously.Enzyme is cut qualification result and is shown, Sac I is the unit point of expection, and EcoR I and SalI enzyme are cut the expection product that obtains 3091 bp and 8921 bp sizes.Sequencing result has shown the ipt gene of restructuring and the sequence of Tnos terminator and both the junction sequence of accurately recombinating, and obtains positive colony carrier pVCT2238.
The construction step following (Figure 10) of plant binary expression vector pVCT2221 of the present invention:
With Sal I enzyme, cut carrier pVCT2231, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, and electrophoresis reclaims 8779 bp fragments, abandons 1456 bp fragments.With BstEII enzyme, cut carrier pVCT2237, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, reclaims 3744 bp fragments, abandons 8233 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1719 bp sizes, show to contain in recon RD29A::ntCre sequence simultaneously.With primer 5 '-cggaattcagggagtcacgttatgac-3 ' (being arranged in Pnos promotor upstream) and 5 '-catgagcgaaaccctataggaacc-3 ' (being positioned at CaMV 35S terminator pA), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 59 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1664 bp sizes, shows to contain in recon Pnos::ntpIIm::pA sequence simultaneously.Use again primer 5 '-atgacgcacaatcccactatcc-3 ' (being arranged in CaMV 35S promoter) and 5 '-atcgcaagaccggcaacagg-3 ' (being arranged in Tnos terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 56 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, and last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1004 bp sizes, shows to contain in recon 35S::mGFP::Tnos sequence simultaneously.Enzyme is cut qualification result and is shown, EcoR I and Hind III enzyme are cut the expection product that obtains 2496 bp and 10032 bp sizes.Through sequence verification, obtain positive colony plant binary expression vector pVCT2221.
The construction step following (Figure 11) of plant binary expression vector pVCT2224 of the present invention:
With Sal I enzyme, cut carrier pVCT2231, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, and electrophoresis reclaims 8779 bp fragments, abandons 1456 bp fragments; With BstEII enzyme, cut carrier pVCT2238, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, reclaims 3779 bp fragments, abandons 8233 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1719 bp sizes, show to contain in recon RD29A::ntCre sequence simultaneously.Use again primer 5 '-atgacgcacaatcccactatcc-3 ' (being arranged in CaMV 35S promoter) and 5 '-cgttcgatgacgaaaatggaag-3 ' (being positioned at the downstream of ipt gene terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, and last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1088 bp sizes, shows to contain in recon 35S::ipt sequence simultaneously.Enzyme is cut qualification result and is shown, BstE II and EcoR I are the unit point of expection, and Sal I and Sac I enzyme are cut the expection product that obtains 2816 bp and 9747 bp sizes.Sequencing result has shown the sequence that CaMV 35S terminator pA, LoxP are connected with nptIIm.Through sequence verification, obtain positive colony plant binary expression vector pVCT2224.
The application of above-mentioned plant binary expression vector aspect transgene tobacco.
The low temperature induction deleting selectable marker genes system that the RD29A::ntCre that the present invention is constructed and LoxP formed by different way and goal gene, selectable marker gene and promoter related etc. other thereof build the mode that element is fused into new gene and can adopt the conventional mode in this area, and the new gene being fused into can be transferred to and in the conventional carrier in this area, be built into expression vector and be transformed in plant.
" plant binary expression vector " of indication of the present invention refers to the miniature plasmid (or miniature Ti carrier) that does not contain Vir district and contain T-DNA, comprise the T-DNA structure and the T-DNA outboard structure that by T-DNA left margin (LB) and right margin (RB), are defined, in its T-DNA structure, with the selectable marker gene that can express in plant and goal gene, and T-DNA outside is with protokaryon resistant gene, intestinal bacteria replicon and Agrobacterium replicon.This carrier is a kind of shuttle vectors that can copy in Agrobacterium again intestinal bacteria, and can be through agriculture bacillus mediated, by the gene transfered plant genome in T-DNA.
" transgene tobacco " of indication of the present invention refers to by means such as molecular biology, biotechnologys, other biological gene is transferred in tobacco gene group by plant binary expression vector, thereby make some inherited character of the tobacco of being transformed be transformed and express, for the goal gene of transforming, can derive from the gene of plant, animal and microorganism or synthetic.
The present invention has following beneficial effect:
Recombinase ntCre gene provided by the invention, under the low temperature induction of inducible promoter RD29A, express, binary expression vector of the present invention containing described recombinase ntCre gene, by having solved in this expression vector conversion host, existing marker gene deletion is existing all will just can obtain the transfer-gen plant of marker-free gene by selfing separation, Breeding Process length consuming time, program is loaded down with trivial details, can only be confined to the problem in the plant of sexual hybridization simultaneously, particularly, utilizing low temperature induction promotor RD29A to control ntCre/LxoP recombination system expresses, under the effect of recombinase, thereby site-specific recombining reaction occurs in plant materials deletes selectable marker gene, thereby it is longer to have changed the available technology adopting HSP70m heat-inducible promoter delete flag gene time, to unfavorable some problems that exist that wait of transgenic plant growth, facilitate in summer utilizes ntCre/LxoP recombination system under cold condition, to delete the selectable marker gene in transfer-gen plant simultaneously, avoided obtaining resistance or visual transfer-gen plant because high temperature season during accidental power failure affects.
The construction process of plant binary expression vector provided by the present invention has excised selectable marker gene and recombinase gene self efficiently, has avoided recombinase overexpression to bring the potential hazard to plant-growth, thereby obtains the transfer-gen plant of marker-free.
Accompanying drawing explanation
Fig. 1 is the binary expression vector T-DNA structure of low temperature induction deleting selectable marker genes;
Fig. 2 is the structure schematic diagram of plant binary expression vector pVCT2221 and pVCT2224;
Fig. 3 is the principle of work of the binary expression vector pVCT2221 of low temperature induction deleting selectable marker genes;
Fig. 4 is the principle of work of the binary expression vector pVCT2224 of low temperature induction deleting selectable marker genes;
Fig. 5 is the GUS active mass dyeing of transgene tobacco;
Fig. 6 is the different tissues GUS active mass dyeing of transgene tobacco;
Fig. 7 is the functional verification of the low temperature induction deleting selectable marker genes after pVCT2221 transformation of tobacco;
Fig. 8 is the functional verification of the low temperature induction deleting selectable marker genes after pVCT2224 transformation of tobacco;
Fig. 9 is the binary expression vector T-DNA structure of carrying the low temperature induction deleting selectable marker genes of goal gene;
Figure 10 is structure and the evaluation of binary expression vector pVCT2221;
Figure 11 is structure and the evaluation of binary expression vector pVCT2224.
Embodiment
Following examples are further illustrating of by reference to the accompanying drawings the present invention being carried out, but below explanation does not limit the present invention, any trial is to change of the present invention and distortion, only otherwise exceed object of the present invention and spirit, all should be considered as belonging to the defined scope of claims of the present invention.
Embodiment mono-: the structure of the T-DNA of the preparation of recombinase ntCre gene, plant gene binary expression vector pVCT2221, be transformed in agrobacterium tumefaciens EHA105, conversion of plant also obtains transgenic plant (tobacco) and effect
The preparation method 1, with the recombinase ntCre gene of nuclear localization signal encoding sequence
With primer SEQ ID.4. and primer SEQ ID.NO.5, PrimStar HS archaeal dna polymerase (TaKaRa, Japan) from coli strain BM25.8(Clontech, the U.S.) in genomic dna, pcr amplification goes out the Cre gene that is derived from P1 phage (GenBank accession X03453) of 1040 bp sizes, reaction conditions is: 95 ℃ of denaturation 2 min, 95 ℃ of sex change 10 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 3 circulations, 95 ℃ of sex change 10 sec, 60 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 10 sec, 27 circulations, last 72 ℃ are extended 10 min.With Taq archaeal dna polymerase, add after " A " tail, TA clones into T carrier pMD18-T(TaKaRa, Japan), transform intestinal bacteria XL1-Blue competent cell, Amp resistance recon screens through pcr amplification, primer is SEQ ID.NO.6 and SEQ ID.NO.7, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 52 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 898 bp sizes, sequencing result shows that the gene of cloning is with the sequence of announcing in GenBank, obtain positive colony carrier pVCT1251 and recombinase Cre gene.
With Xba I and Nco I enzyme, cut carrier pVCT1251, reclaim 3727 bp fragments, abandon 11 bp fragments.Synthetic sequence SEQ ID.NO.8 and synthetic sequence SEQ ID.NO.9, in 3 minutes after annealings of 94 ℃ of sex change, are formed to the Xba I-Nco I joint of the encoding sequence that contains Arabidopis thaliana At4g01735 gene intron and monkey SV40 virus capsid protein T antigen nuclear localization signal.Through sequence verification, to reclaim fragment and be connected with T4 DNA ligase with joint, and transform intestinal bacteria XL1-Blue competent cell, Amp resistance recon screens through pcr amplification, primer is SEQ ID.NO.10 and SEQ ID.NO.11, PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 30 sec, 35 circulations, last 72 ℃ are extended 10 min, and electrophoresis detection obtains 293 bp and expects big or small product.Through sequence verification, obtain positive colony carrier pVCT1252, make First Intron and the sequence with nuclear localization signal coding on recombinase Cre upstream region of gene band.
2, the construction process (Fig. 2, Figure 10) of the T-DNA of plant gene binary expression vector pVCT2221
With Sal I enzyme, cut carrier pVCT2231, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, and electrophoresis reclaims 8779 bp fragments, abandons 1456 bp fragments.With BstEII enzyme, cut carrier pVCT2237, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, reclaims 3744 bp fragments, abandons 8233 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1719 bp sizes, show to contain in recon RD29A::ntCre sequence simultaneously.With primer 5 '-cggaattcagggagtcacgttatgac-3 ' (being arranged in Pnos promotor upstream) and 5 '-catgagcgaaaccctataggaacc-3 ' (being positioned at CaMV 35S terminator pA), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 59 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1664 bp sizes, shows to contain in recon Pnos::ntpIIm::pA sequence simultaneously.Use again primer 5 '-atgacgcacaatcccactatcc-3 ' (being arranged in CaMV 35S promoter) and 5 '-atcgcaagaccggcaacagg-3 ' (being arranged in Tnos terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 56 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1004 bp sizes, shows to contain in recon 35S::mGFP::Tnos sequence simultaneously.Enzyme is cut qualification result and is shown, EcoR I and Hind III enzyme are cut the expection product that obtains 2496 bp and 10032 bp sizes.Through sequence verification, obtain positive colony plant binary expression vector pVCT2221.
3, be transformed in agrobacterium tumefaciens EHA105, conversion of plant also obtains transgenic plant (tobacco) and effect
Through Agrobacterium EHA105, mediate, the T-DNA of plant binary expression vector pVCT2221 is integrated into after tobacco gene group, to give transformant and there is kalamycin resistance and demonstrate green fluorescence, obtain the transgene tobacco of low temperature induction deleting selectable marker genes simultaneously.Through low temperature induction, express and can navigate to endonuclear Cre recombinase, and delete two sequences between LoxP in the same way by Cre recombinase, net result is residual 1 LoxP, and comes from T35s and the Tnos terminator (Fig. 3) on T-DNA.
Concrete application method and compliance test result are as follows:
T-DNA in pVCT2221 expression vector is imported to tobacco nuclear gene group, on the solid medium of the additional 2.5 mg/L BA of MS, obtain the test-tube plantlet of anti-150 mg/L kantlex (Kan), on the substratum of 50 mg/L kantlex, can normally take root (Fig. 7 A).Under 400 nm light of fluorescent microscope, the tobacco plant root of Kan resistance presents bright green fluorescence (Fig. 7 B).Through the subzero treatment of 4 ℃ of left and right after 8 weeks, a part is transferred on the substratum that contains 150 mg/L kantlex by plant, originally anti-kantlex presents albefaction (Fig. 7 C), i.e. no longer anti-kantlex, show that the kalamycin resistance gene in transfer-gen plant is deleted, and another part transfer-gen plant is transferred on the substratum that does not add kantlex, can normal growth and root induction, the root generating no longer has green fluorescence (Fig. 7 D) under fluorescent microscope, shows that mGFP gene is also deleted.
Embodiment bis-: the construction process of the T-DNA of plant binary expression vector pVCT2224, be transformed in agrobacterium tumefaciens EHA105, transformation of tobacco also obtains transgene tobacco and effect
1, the construction process (Fig. 2, Figure 11) of the T-DNA of plant binary expression vector pVCT2224
With Sal I enzyme, cut carrier pVCT2231, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, and electrophoresis reclaims 8779 bp fragments, abandons 1456 bp fragments.With BstEII enzyme, cut carrier pVCT2238, Klenow Fragment enzyme fills, and crosses DNA recovery post and removes enzyme, then cut with Sac I enzyme, reclaims 3779 bp fragments, abandons 8233 bp fragments.After reclaiming fragment purification, with T4 DNA ligase, connect, transform intestinal bacteria XL1-Blue competent cell, obtain Kan resistance recon, the primer of pcr amplification screening is 5 '-ctgcaagaatctcaaacacgg-3 ' (being positioned at RD29A low temperature induction promotor upstream) and 5 '-ctgtaactatcatcatcatcatag-3 ' (being positioned at intron downstream), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 50 ℃ of annealing 20 sec, 72 ℃ are extended 1 min 40 sec, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1719 bp sizes, show to contain in recon RD29A::ntCre sequence simultaneously.Use again primer 5 '-atgacgcacaatcccactatcc-3 ' (being arranged in CaMV 35S promoter) and 5 '-cgttcgatgacgaaaatggaag-3 ' (being positioned at the downstream of ipt gene terminator), PCR reaction conditions is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 20 sec, 55 ℃ of annealing 20 sec, 72 ℃ are extended 1 min, 35 circulations, last 72 ℃ are extended 10 min, electrophoresis detection obtains expecting the product of 1088 bp sizes, shows to contain in recon 35S::ipt sequence simultaneously.Enzyme is cut qualification result and is shown, BstE II and EcoR I are the unit point of expection, and Sal I and Sac I enzyme are cut the expection product that obtains 2816 bp and 9747 bp sizes.Sequencing result has shown the sequence that CaMV 35S terminator pA, LoxP are connected with nptIIm, obtains positive colony plant binary expression vector pVCT2224.
2, be transformed in agrobacterium tumefaciens EHA105, transformation of tobacco also obtains transgene tobacco and effect
Through Agrobacterium EHA105, mediate, the T-DNA of plant binary expression vector pVCT2224 is integrated into after tobacco gene group, to give transformant and there is kalamycin resistance, and make transgenosis test-tube plantlet present deformity, lose apical dominance and can not take root because having promoted that phytokinin is synthetic.The visual transfer-gen plant of this kalamycin resistance is transferred to not containing on the plant culture of kantlex, and be placed in the interior week of the subzero treatment 2-4 in 4 ℃ of left and right of refrigerator, and repeat 1-2 generation.Abduction delivering can navigate to endonuclear Cre recombinase, and delete two sequences between LoxP in the same way by Cre recombinase, net result is by residual 1 LoxP, and come from T35s and the Tnos terminator (Fig. 4) on T-DNA, make the transfer-gen plant of original deformity in form, recover normal.Finally by PCR, detect and confirm no longer to contain nptIIm, ntCre and ipt gene, thereby the selectable marker gene of render transgenic plant is deleted.
Concrete application method and compliance test result are as follows:
Use agrobacterium strains EHA105(pVCT2224) transformation of tobacco.Do not adding the test-tube plantlet of growing thickly (Fig. 8 A) that obtains the anti-150 mg/L kantlex of a large amount of deformities on the MS substratum of any hormone.Deformity test-tube plantlet can suitably grow tall, but top high-quality is not obvious, easily raw side shoot, internode shorten, blade is long and narrow, can not take root (Fig. 8 B, Fig. 8 C).Through 4 ℃ of left and right subzero treatment after 8 weeks, a part is transferred on the substratum that contains 150 mg/L kantlex by plant, originally anti-kantlex presents albefaction (Fig. 8 D), i.e. no longer anti-kantlex, show that the kalamycin resistance gene in transfer-gen plant is deleted, and another part transfer-gen plant is transferred on the substratum that does not add kantlex, can normal growth and root induction (Fig. 8 E, Fig. 8 F), show that ipt gene is also deleted.With the primer being positioned on T35s and Tnos, after High fidelity PCR amplification, result shows nptIIm, ntCre and the ipt gene that does not have pVCT2224 between T35s and Tnos, TA clones into order-checking after pMD18-T and confirms, be integrated between 2 LoxP in the T-DNA of tobacco gene group accurate restructuring has occurred, deleted all sequences therebetween, and residual 1 LoxP, this result is completely with expection.
Embodiment tri-: after binary expression vector transformation of tobacco of the present invention, and the functional verification of RD29A promotor.
The carrier that contains RD29A::GUS reporter gene, by Agrobacterium EHA105 gene transfer tobacco, is obtained integrating the kalamycin resistance plant of RD29A::GUS gene.By normal temperature and subzero treatment transgenosis and non-transgenic plant, carry out the chemical staining analysis of GUS active mass, result shows: without the transgenosis single-strain blade of subzero treatment after X-Gluc dyeing, with 70% ethanol, fully decolour, only observe only a few locus coeruleus or occur without locus coeruleus, showing that RD29A promotor does not have the function that starts downstream gene expression at normal temperatures.The transgenosis individual plant that spends the night and process through 4 ℃ of left and right low temperature, after X-Gluc dyeing, fully decolours with 70% ethanol, can obviously observe the appearance of locus coeruleus, shows that pRD29A promotor can start the expression of downstream gene at low temperatures, is a low temperature induction type promotor.And no matter non-transgenic individual plant is normal temperature or subzero treatment, after X-Gluc dyeing, with 70% ethanol, fully decolour, all can not observe locus coeruleus and occur, show that non-transgenic plant does not have background effect.See Fig. 5.
Microscopy is observed root, stem, leaf, the petiole of Transgenic Tobacco plant and all by X-Glue solution, is dyed blueness, but petal is not dyed blueness, illustrates that gene all has expression in the root of plant, stem, leaf, petiole, does not express in petal.In root, tip of a root part gus gene is not expressed, and from the square section of root, the marrow GUS activity of root is stronger, and a little less than cortex gus gene is expressed.In stem, vascular bundle partly has higher GUS active.And cortex and marrow gus gene are not almost expressed.In leaf, gus gene activity is higher with the vascular bundle of vein, and leaf hollow billet gus gene is expressed also stronger.In petiole, gus gene is expressed also stronger.Show RD29A promotor Bu Shi tissue-specific promoter.(see figure 6)
RD29A promotor all transfer-gen plants under 4 ℃ of subzero treatment all have expression, and the experimental result that different transgenosis individual plants is expressed start-up temperature shows: individual plant T
0-5, T
0-9, T
0-11, T
0-15 just start to express at 10 ℃.
PVCT2221 of the present invention and pVCT2224 plant expressing vector are just for the effect of checking low temperature induction deleting selectable marker genes builds.For for goal gene is imported to plant, between LoxP site on the right of goal gene can being inserted into and rightmost Tnos (Fig. 1 a and Fig. 1 b), thereby in deleting selectable marker genes, retain goal gene, obtain transfer-gen plant marker-free gene, that only contain goal gene.For example, the pVCT2291 in Fig. 9 and pVCT2297 are respectively the expression vector being derived by pVCT2221 and pVCT2224.
Claims (3)
1. a recombinase gene, is characterized in that: it is ntCre gene, and described recombinase gene sequence is the sequence as shown in SEQ ID No.1.
2. comprise the plant binary expression vector of recombinase gene as claimed in claim 1, it is characterized in that: described plant binary expression vector has the structure as shown in Fig. 1 a or Fig. 1 b.
3. the application of plant binary expression vector as claimed in claim 2 in obtaining transgene tobacco.
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