CN102321667B - Virulence auxiliary vector for agrobacterium tumefaciens-mediated high molecular weight T-DNA (Transfer-Deoxyribonucleic Acid) transformation and preparation method as well as application thereof - Google Patents

Abstract

The invention relates to the field of gene engineering, in particular to a virulence auxiliary vector for agrobacterium tumefaciens-mediated high molecular weight T-DNA (Transfer-Deoxyribonucleic Acid) transformation. In the invention, VirE2 protein can be over-expressed in agrobacterium tumefaciens by constructing a vector containing virE-virG chimeric operon and agrobacterium tumefaciens replicon oriV. The invention also discloses a preparation method of the vector. The vector disclosed by the invention is used in agrobacterium tumefaciens-mediated genetic transformation, so that a sufficient amount of VirE2 protein wraps high molecular weight T-DNA when the T-DNA with length of over 25kb is transformed into plant genome, and the high molecular weight T-DNA is not degraded by nuclease and can be completely integrated into the plant genome to realize multi-gene genetic transformation.

Description

Toxicity assistant carrier that agriculture bacillus mediated macromolecule T-DNA transforms and its preparation method and application

Technical field

The present invention relates to the genetically engineered field, particularly the toxicity assistant carrier of agriculture bacillus mediated macromolecule T-DNA conversion the invention also discloses construction process and the application of this toxicity assistant carrier.

Background technology

Agriculture bacillus mediated Genetic Transformation in Higher Plants is a natural process that foreign gene is imported Plant Genome by Agrobacterium.In agrobacterium tumefaciens, exist a size to be about the large-scale plasmid of 150-240 kb, i.e. Ti-plasmids.There are two important zones at Ti-plasmids: T-DNA district and toxicity district (virulence region, Vir district).When being subjected to the aldehydes matters such as Syringylethanone of plants wound secretion, Agrobacterium induces down, be positioned on the agrobatcerium cell film VirA albumen by autophosphorylation and be activated, subsequently its phosphate group is passed to the VirG albumen with functional transcription factor, and by the promoter region of VirG albumen specific combination Vir district upstream region of gene, start the expression of all Vir district genes.Wherein, the VirD2 albumen of expressing is under the assistance of the albumen such as VirC1 and VirD1,5 ' end to the lower chain of T-DNA right margin (RB) cuts, and covalent attachment, consist of the ssT-DNA/VirD2 mixture, the ssT-DNA mixture is rapidly by the VirE2 protein encapsulation, enters behind the plant not by nuclease degradation to guarantee ssT-DNA.

Limited by the VirE2 protein quantity of the protein induced expression of VirG in the Wild Type Agrobacteria, 1000 molecules are arranged approximately, the T-DNA about 23kb is effectively protected.When T-DNA molecular weight during greater than 25 kb, owing to there is not the VirE2 albumen of capacity in the agrobatcerium cell, the ssT-DNA/VirD2 mixture can not get wrapping up fully, causes when transforming large fragment T-DNA to go out active, fracture, is integrated in the Plant Genome and macromolecule T-DNA can not be complete.In order to make being integrated in the Plant Genome that macromolecule T-DNA can be complete, need to be in Agrobacterium overexpression VirE2 albumen.Because VirE2 albumen is unstable, must have the VirE1 molecular chaperones to keep its structure.The present invention consists of the chimeric operon of virE-virG with Promoter-VirE1-VirE2 fragment and VirG-terminator splicing, and structure does not contain the carrier that the macromolecule T-DNA T-DNA district, auxiliary agriculture bacillus mediated transforms.

Summary of the invention

In view of this, in order to solve the complete problem that is integrated into Plant Genome of macromolecule T-DNA, the toxicity assistant carrier that provides agriculture bacillus mediated macromolecule T-DNA to transform, this carrier can the reporter molecule amount transform greater than the T-DNA of 25 kb; The preparation method of the toxicity assistant carrier of agriculture bacillus mediated macromolecule T-DNA conversion is provided, and it is simple to utilize present method to make up toxicity assistant carrier method.

One of the object of the invention is to provide the toxicity assistant carrier of above-mentioned preparation method's acquisition, and described toxicity assistant carrier comprises the chimeric operon of virE-virG, and the chimeric operon nucleotide sequence of described virE-virG is shown in SEQ ID NO:16.

Be preferably, described toxicity assistant carrier also comprises oriV Agrobacterium replicon and spec gene, and described oriV Agrobacterium replicon nucleotide sequence is shown in SEQ ID NO:17, and described spec gene nucleotide series is shown in SEQ ID NO:18.

More preferably, described toxicity assistant carrier nucleotide sequence is shown in SEQ ID NO:7.

Two of the object of the invention is to provide the preparation method of the toxicity assistant carrier that agriculture bacillus mediated macromolecule T-DNA transforms, and for achieving the above object, the invention provides following technical scheme:

The preparation method of toxicity assistant carrier specifically may further comprise the steps:

A. prepare the restructuring binary expression vector;

A1. prepare Pnos::nptII fragment and enzyme and cut, the Pnos::nptII endonuclease bamhi is inserted pCAMBIA1302 carrier polyclone enzyme cut site, get the pVCT2020 carrier;

A2. enzyme is cut the pEGFP-N1 carrier, and preparation eGFP gene inserts pET-32a (+) carrier polyclone restriction enzyme site with the eGFP gene, gets the pVCT2190 carrier;

A3. enzyme is cut gained pVCT2190 carrier, prepares the fragment of described eGFP gene, enzyme is cut gained eGFP gene insert gained pVCT2020 carrier restriction enzyme site place, gets the pVCT2210 carrier;

A4. enzyme is cut gained pVCT2210 carrier, identifies and recovery through agarose electrophoresis, and the fragment that reclaims is connected, and gets the pVCT2212 carrier;

A5. enzyme is cut gained pVCT2212 carrier, identifies with agarose gel electrophoresis, reclaims to contain the eGFP gene fragment, enzyme is cut gained eGFP gene insert pHells gate8 carrier restriction enzyme site place, gets the restructuring binary expression vector, called after pVCT2268 carrier;

B. preparation contains the underlying carrier of the chimeric operon of virE-virG;

B1. prepare the Promoter-VirE1-VirE2 gene, gained Promoter-VirE1-VirE2 gene is connected p Easy-Blant carrier gets the pVCT1213 carrier;

B2. prepare the VirG-terminator gene, gained VirG-terminator fragment is inserted the pVCT1213 carrier, must contain the underlying carrier of the chimeric operon of virE-virG, called after pVCT1244 carrier;

C. prepare the toxicity assistant carrier;

C1. enzyme is cut gained pVCT1244 carrier, and the chimeric operon of preparation virE-virG inserts gained pVCT2268 carrier with the chimeric operon of gained virE-virG, gets recombinant vectors, called after pVCT2270 carrier;

C2. enzyme is cut gained pVCT2270 carrier, contain the chimeric operon fragment of described virE-virG, Agrobacterium replicon oriV and described resistant maker gene fragment through agarose gel electrophoresis evaluation and recovery, the endonuclease bamhi that reclaims the pVCT2270 carrier is connected again, get described toxicity assistant carrier, called after pVCT2272 carrier.

Be preferably, the preparation of the described Pnos::nptII fragment of step a1 specifically comprises the steps:

Take sequence shown in the SEQ ID NO:1 as upstream primer, take sequence shown in the SEQ ID NO:2 as downstream primer, take the pBIN19 carrier as template, carry out pcr amplification, the pcr amplification condition is: 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, annealed 20 seconds for 50 ℃, 72 ℃ were extended 2 minutes 10 seconds, and 3 circulations were annealed 20 seconds for 60 ℃, 72 ℃ were extended 2 minutes 10 seconds, 27 circulations, 72 ℃ were extended 10 minutes, and got the Pnos::nptII fragment.

Be preferably, the preparation of the described Promoter-VirE1-VirE2 gene of step b1, concrete steps are as follows:

Take the pTiC58 plasmid DNA of described Agrobacterium C58 as template, the nucleotides sequence of upstream primer is classified as shown in the SEQ ID NO:3, the nucleotides sequence of downstream upstream primer is classified as shown in the SEQ ID NO:4, adopt PrimStar HS archaeal dna polymerase to carry out pcr amplification, get the VirE2 gene fragment, described pcr amplification condition is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, annealed 15 seconds for 56 ℃, 72 ℃ were extended 2 minutes 30 seconds, 28 circulations, 72 ℃ were extended 10 minutes, and got the Promoter-VirE1-VirE2 gene.

Be preferably, the preparation of the described VirG-terminator gene of step b2, concrete steps are as follows:

Take the pTiC58 carrier of described Agrobacterium Agrobacterium C58 as template, the upstream primer nucleotide sequence is shown in SEQ ID NO:5, and the downstream primer nucleotide sequence adopts PrimStar HS archaeal dna polymerase to carry out pcr amplification shown in SEQ ID NO:6, get the VirG gene fragment, described pcr amplification condition is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, 56 ℃ of annealing 15 seconds, 72 ℃ were extended 1 minute, 28 circulations, 72 ℃ were extended 10 minutes, and got the VirG-terminator gene.

Be preferably, the preparation of the chimeric operon of the described virE-virG of step c1, concrete steps are as follows:

Cut gained pVCT1244 carrier with restriction enzyme Spe I, Xba I and Ema1105 I enzyme simultaneously, identify through agarose gel electrophoresis, reclaim the fragment of 3465bp size, get the chimeric operon of virE-virG.

The object of the invention three is to provide the application of toxicity assistant carrier in agriculture bacillus mediated genetic transformation.

Be preferably, with the agriculture bacillus mediated pBiBAC-CBF carrier transformation of tobacco that contains described toxicity assistant carrier.

Beneficial effect of the present invention is: the present invention is building up to Promoter-VirE1-VirE2-VirG-Terminator on the expression vector, and the VirG albumen that is activated in Agrobacterium acts on this VirDuring operon, VirG albumen will obtain overexpression, and overexpression VirG albumen will promote the overexpression of VirE2 albumen, satisfy macromolecule T-DNA packing demand, promote macromolecule T-DNA to the genetic transformation of plant.VirE1 albumen is the molecular chaperones of VirE2 albumen, can keep VirE2 albumen and be in solvable state, prevents that the VirE2 albumen of overexpression from forming inclusion body.The toxicity assistant carrier that consists of does not contain the T-DNA district, prevents that homologous recombination is occuring binary expression vector in the Agrobacterium.The toxicity assistant carrier Agrobacterium replicon that makes up simultaneously is the oriV replicon, can be compatible in bacillus with the pVS1 replicon, realize complete being integrated in the Plant Genome of macromolecule T-DNA.

Other advantage of the present invention, target and feature will be set forth to a certain extent in the following description, and to a certain extent, based on being apparent to those skilled in the art to investigating hereinafter, perhaps can obtain from the practice of the present invention instruction.The objectives and other advantages of the present invention can be passed through following specification sheets, claims, and the specifically noted structure realizes and obtains in the accompanying drawing.

The spectinomycin gene referred to as spec, has the activity of anti-spectinomycin (spe).

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 is pVCT2020 carrier figure of the present invention;

Fig. 2 is pVCT2190 carrier figure of the present invention;

Fig. 3 is pVCT2210 carrier figure of the present invention;

Fig. 4 is pVCT2212 carrier figure of the present invention;

Fig. 5 is pVCT2268 carrier figure of the present invention;

Fig. 6 is pVCT1213 carrier figure of the present invention;

Fig. 7 is pVCT1244 carrier figure of the present invention;

Fig. 8 is pVCT2270 carrier figure of the present invention;

Fig. 9 is pVCT2272 carrier figure of the present invention;

Figure 10 is transgene tobacco figure of the present invention;

Figure 11 is that the AtCBF1 gene PCR of kalamycin resistance tobacco is identified electrophorogram.

Embodiment

Below with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail; Should be appreciated that preferred embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

E.Z.N.A. Plasmid Miniprep Kit and E.Z.N.A. Gel Exteaction Kit test kit are available from Omega(USA) company; PfuDNA polysaccharase, calf small intestine Phosphoric acid esterase CIAP, Wide Range DNAmarker, Lambda DNA/Hind III+EcoR I Marker, DL2000 Marker give birth to the biological company limited (China) of worker available from Shanghai; Taq DNA polymerase, PrimeStar HS DNA polymerase, dNTP, restriction enzyme, p Easy-Blant carrier is given birth to biotechnology company limited (Japan) available from Beijing Quanshijin Biotechnology Co., Ltd, T4 DNA ligase available from the Dalian treasured; PET-32a (+) carrier is available from Novagen company; Kantlex (Kanamycin, Kan), Rifampin (Rifampicin, Rif), Streptomycin sulphate (Streptomycin, Str), spectinomycin (spectinomycin, Spe) etc. are available from Beijing ancient cooking vessel state biotech development center; PCAMBIA1302 carrier, pCAMBIA2300 carrier, BiBAC2 carrier and pEGFP-N1 carrier are preserved by this laboratory.Synthetic and the dna sequencing of primer is given birth to the biological company limited of worker by Shanghai and is finished.

Embodiment 1

The structure of restructuring binary expression vector

The a1.pVCT2020 Vector construction

According to pBIN19 carrier sequence (GenBank accession No:U09365) design primer, upstream primer is shown in SEQ ID NO:1, and sequence is: 5 '-cg GaattcAgggagtcacgttatgac-3 ' (line place is EcoR I site), downstream primer is shown in SEQ ID NO:2, and sequence is: 5 '-cg CtcgagTcccgctcagaagaac-3 ' (line place is Xho I site).Take the pBIN19 carrier as template, the PfuDNA polysaccharase, carry out pcr amplification, the pcr amplification condition is 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, 50 ℃ of annealing 20 seconds, 72 ℃ were extended 2 minutes 10 seconds, 3 circulations, annealed 20 seconds for 60 ℃, 72 ℃ were extended 27 circulations 2 minutes 10 seconds, 72 ℃ were extended 10 minutes, identify recovery 1116 bp fragments through agarose electrophoresis, the gained fragment contains rouge alkali synthetase gene promotor Pnos and kantlex coding region nptII, called after Pnos::nptII.Cut the Pnos::nptII fragment with EcoR I and Xho I enzyme, reclaim the Pnos::nptII fragment of 1107 bp; Use simultaneously identical enzymic digestion carrier pCAMBIA1302(GenBank accession AF234298), reclaim 8425 bp fragments, reclaim fragment with two and be used in the lower 16 ℃ of connections of spending the night of T4 ligase enzyme effect, get the pVCT2020 carrier of 9532 bp, as shown in Figure 1.

A2. pVCT2190 Vector construction.

Cut pET-32a (+) carrier with EcoR I and Not I enzyme, identify and reclaim pET-32a (+) carrier framework of 5874 bp fragments through agarose gel electrophoresis; Cut pEGFP-N1 carrier (GenBank accession No.U55762) with same enzyme simultaneously, through green fluorescent protein mutantion line (the called after eGFP gene) coding region that agarose gel electrophoresis is identified and reclaimed 772 bp; PET-32a (+) carrier framework that reclaims spent the night in 16 ℃ with the T4 dna ligase with the eGFP gene to be connected, and gets the pVCT2190 carrier, as shown in Figure 2.

The a3.pVCT2210 vector construction

Cut carrier gained pVCT2190 carrier with Xho I enzyme, after Klenow Fragment enzyme fills, use again Bgl II enzymic digestion, identify and reclaim the 833 bp fragments that contain the eGFP gene through agarose gel electrophoresis; Cut the pVCT2020 carrier with PmaC I and Xba I enzyme simultaneously, reclaim the large skeleton of pVCT2020 carrier of 8788 bp; The eGFP gene that reclaims spent the night in 16 ℃ with the T4 dna ligase with the large skeleton of pVCT2020 carrier to be connected, and gets the pVCT2210 carrier, as shown in Figure 3.

The a4.pVCT2212 vector construction

Cut gained pVCT2210 carrier with Nco I enzyme, electrophoresis reclaims the fragment of 1679 bp and 7838 bp sizes; Use the T4 dna ligase in 16 ℃ of connections two fragments that reclaim, get the pVCT2212 carrier, as shown in Figure 4.Gained pVCT2212 carrier contains the expression cassette that is made of the promotor Pnos of rouge alkali synthetase gene, kantlex coding region nptII and CaMV 35S gene terminator pA sequence, called after: Pnos::nptII::pA; By the expression cassette that 35S promoter, eGFP gene and rouge alkali synthetase gene terminator Tnos sequence consist of, called after 35S::eGFP::Tnos gene.

The a5.pVCT2268 Vector construction

With restriction enzyme Sph I single endonuclease digestion pHells gate8 carrier, the agarose gel electrophoresis with 2% is identified, reclaims the large fragment of the 9370bp that contains Agrobacterium replicon oriV; Cut gained pVCT2212 carrier with the SphI enzyme simultaneously, agarose gel electrophoresis with 1% is identified, recovery contains the large fragment of the 1915bp of eGFP, connect the 1915bp fragment of recovery and the fragment of 9370bp, 16 ℃ of connections of spending the night under the effect of T4 ligase enzyme, the binary expression vector of must recombinating, called after pVCT2268 carrier, as shown in Figure 5.

Present embodiment is take pHells gate8 carrier as example, and the process enzyme is cut to connect and to be got the restructuring binary expression vector.This restructuring binary expression vector carrier Agrobacterium replicon is the oriV replicon, different from the pVS1 Agrobacterium replicon that the binary expression vector pCAMBIA series of commonly using adopts, contain these two kinds of replicons get carrier can be compatible in Agrobacterium, therefore can also be other binary expression vectors, select the principle of replicon can compatibility to be advisable with the replicon that carries the binary expression vector of target gene for the Agrobacterium replicon of restructuring binary expression vector.

Embodiment 2

The structure that contains the underlying carrier of the chimeric operon of virE-virG

The b1.pVCT1213 Vector construction

PTiC58 carrier sequence (GenBank accession:NC_003308) according to the agrobacterium strains C58 that reports, design contains promotor Promoter, the PCR special primer of VirE1 gene coding region and VirE2 gene coding region (referred to as Promoter-VirE1-VirE2), wherein upstream primer is shown in SEQ ID NO:3, sequence is 5 '-gaattacattcacacggcacc-3 ', downstream primer is shown in SEQ ID NO:4, and sequence is 5 '-tcacacgtggtggtggtggtggtgcagactgtttacggttgg-3 '.Take the pTiC58 carrier of agrobacterium strains C58 as template, utilize PrimeStar HS DNA polymerase to carry out pcr amplification, reaction conditions is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, 56 ℃ of annealing 15 seconds, 72 ℃ were extended 2 minutes 30 seconds, 28 circulations, 72 ℃ were extended 10 minutes.The PCR product is identified through 1% agarose electrophoresis and is reclaimed 2481bp purpose fragment, gets the Promoter-VirE1-VirE2 fragment.Gained Promoter-VirE1-VirE2 fragment, by promotor Promoter, VirE1 gene coding region and VirE2 gene coding region are followed in series to form.As shown in Figure 2, gained Promoter-VirE1-VirE2 fragment is connected p Easy-Blant carrier, Promoter-VirE1-VirE2 fragment and p EasyAfter-Blant carrier mixes in the 3:1 ratio,, connect product and transform intestinal bacteria in 16 ℃ of connections of spending the night with the T4 dna ligase E. coliThe XL1-BLUE competent cell filters out the positive colony bacterium colony of Amp and Kan resistance, extracts the p that plasmid must contain the Promoter-VirE1-VirE2 fragment Easy-Blant carrier, called after pVCT1213, as shown in Figure 6.

The b2.pVCT1244 Vector construction

PTiC58 carrier sequence (GenBank accession:NC_003308) according to the agrobacterium strains C58 that reports, the terminator terminator(that design contains VirG gene coding region and VirG gene is called for short: PCR special primer VirG-terminator), wherein upstream primer is shown in SEQ ID NO:5, sequence is 5 '-ggagatctgttgagctgcaaatggctg-3 ', downstream primer is shown in SEQ ID NO:6, and sequence is 5 '-gc Tctaga(the underscore place is taggacccatccaatcac-3 ' XbaThe I restriction enzyme site), take the pTiC58 carrier of agrobacterium strains C58 as template, utilize PrimeStar HS DNA polymerase to carry out pcr amplification, reaction conditions is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, annealed 15 seconds for 56 ℃, 72 ℃ were extended 1 minute, 28 circulations, and 72 ℃ were extended 10 minutes, identify recovery 919 bp fragments through 1% agarose electrophoresis, get the VirG-terminator fragment.Gained VirG-terminator fragment is followed in series to form by the terminator terminator of VirG gene coding region and VirG gene.Use restriction enzyme XbaThe I enzyme cuts back to close gained VirG-terminator fragment, cuts the pVCT1213 carrier with restriction enzyme NotI enzyme simultaneously, fills through Klenow Fragment enzyme, then cuts with restriction enzyme XbaI enzyme, gets the pVCT1213 endonuclease bamhi; The VirG-terminator fragment that enzyme is cut is connected with the pVCT1213 endonuclease bamhi, and ligation is 16 ℃ of connections of spending the night under the effect of T4 ligase enzyme, must contain VirGThe recombinant vectors of gene, called after pVCT1244, as shown in Figure 7.

In the pVCT1244 carrier, Not I/XbaI enzyme incised notch mouth is positioned at the end of VirE2 gene coding region, when therefore the VirG gene fragment is inserted the pVCT1213 carrier, the coding region of VirG gene and terminator splicing are at the end of VirE2 gene coding region, consisting of structure is the Promoter-VirE1-VirE2-VirG-Terminator sequence, the chimeric operon of called after virE-virG.

Embodiment 3

The structure of toxicity assistant carrier

The c1.pVCT2270 Vector construction

As shown in Figure 5, with restriction enzyme Nru I single endonuclease digestion gained pVCT2268 carrier, identify through agarose gel electrophoresis, get pVCT2268 carrier framework fragment; Cut carrier pVCT1244 with restriction enzyme Spe I, Xba I, Ema1105 I three enzymes simultaneously, identify through agarose gel electrophoresis, recovery contains the fragment of the 3465bp size of the chimeric operon of virE-virG, with the chimeric operon of virE-virG and pVCT2268 carrier framework fragment under the effect of T4 ligase enzyme 16 ℃ spend the night and be connected, get recombinant vectors, called after pVCT2270, as shown in Figure 8.

The c2.pVCT2272 Vector construction

With restriction enzyme Sal I single endonuclease digestion pVCT2270 carrier, T-DNA district on the excision pVCT2270 carrier, agarose gel electrophoresis with 1% reclaims the target stripe of the 11473bp of the chimeric operon of virE-virG, get pVCT2270 carrier endonuclease bamhi, with pVCT2270 carrier endonuclease bamhi 16 ℃ of connections of spending the night under the effect of T4 ligase enzyme of reclaiming, get the toxicity assistant carrier that agriculture bacillus mediated macromolecule T-DNA transforms, has nucleotide sequence shown in SEQ IDNO:7, called after pVCT2272 carrier, as shown in Figure 9.Nucleotide sequence shown in the SEQ IDNO:7, wherein 22-462 position nucleotides sequence is classified the agrobacterium tumefaciens plasmid replicon as, 1323-2111 position nucleotides sequence is classified the spectinomycin resistant maker gene as, 3215-3977 position nucleotides sequence is classified intestinal bacteria ColE1 replicon as, 4133-7528 position nucleotide sequence is followed successively by the terminator of the VirG gene of agrobacterium tumefaciens C58, the VirG gene coding region of agrobacterium tumefaciens C58, the VirE2 gene coding region of agrobacterium tumefaciens C58, the promotor of the VirE1 gene coding region of agrobacterium tumefaciens C58 and the VirE operon of agrobacterium tumefaciens C58,2277-918 position nucleotides sequence is classified the Tn7 transposon as, and 7582-11473 position nucleotide sequence derives from the sequence of pHellsgate 8 carriers.4133-7528 position nucleotide sequence has consisted of the chimeric operon of virE-virG of Promoter-VirE1-VirE2-VirG-Terminator structure.

Gained toxicity assistant carrier contains the chimeric operon of virE-virG, and the Agrobacterium replicon is the oriV replicon, resistant maker gene and intestinal bacteria replicon ColE1, Tn7 transposon; Wherein the chimeric operon structure of virE-virG is Promoter-VirE1-VirE2-VirG-Terminator, and resistant maker gene is spectinomycin resistance gene, and resistant maker gene also can be other resistant maker genes.The pVCT2272 carrier changes in the Agrobacterium, when the VirG albumen that is activated in the Agrobacterium acts on the chimeric operon of this virE-virG, VirG albumen will obtain overexpression, and overexpression VirG albumen will promote the overexpression of VirE2 albumen, satisfy macromolecule T-DNA packing demand, make that macromolecule T-DNA is complete to be integrated in the Plant Genome.

Embodiment 4

The application of carrier pVCT2272

Adopt freeze-thaw method that gained toxicity assistant carrier pVCT2272 carrier is transformed agrobacterium tumefaciens bacterial strain EHA105, containing spectinomycin 50mg/L, screen on the YEB solid medium of Streptomycin sulphate 100mg/L and Rifampin 100mg/L resistance, the agrobacterium tumefaciens EHA105 that must contain the pVCT2272 carrier, called after EHA105/ pVCT2272, adopt freeze-thaw method with pCAMBIA2300(GenBank accession no. AF234315) Plasmid Transformation EHA105/ pVCT2272, kantlex 50mg/L, spectinomycin 50mg/L, screen on the YEB solid medium of Streptomycin sulphate 100mg/L and Rifampin 100mg/L, get the EHA105 that contains simultaneously pCAMBIA2300 carrier and pVCT2272 carrier, called after EHA105/ pVCT2272/ pCAMBIA2300.Cultivate EHA105/ pVCT2272/ pCAMBIA2300 three generations, extract the plasmid of gained EHA105/ pVCT2272/ pCAMBIA2300, get pVCT2272 carrier and pCAMBIA2300 carrier mixture, transform intestinal bacteria XL1-Blue with the gained carrier mixture, screen at the LB solid medium that contains kantlex 50mg/L, the XL1-Blue that must contain the pCAMBIA2300 carrier, called after XL1-Blue/ pCAMBIA2300; Screen at the LB solid medium that contains spectinomycin 50mg/L, contain pVCT2272 carrier XL1-Blue, called after XL1-Blue/ pVCT2272.According to the pVCT2272 carrier design primer shown in SEQ ID NO:7, upstream primer is positioned at the upstream of VirG gene, sequence is: 5 '-ggagatctgttgagctgcaaatggctg-3 ' (SEQ ID NO:8), downstream primer is positioned at the upstream of intestinal bacteria replicon ColE1, sequence is 5 '-tccttctagtgtagccgtag-3 ' (SEQ ID NO:9), carry out pcr amplification take XL1-Blue/ pVCT2272 bacterium liquid as template, the PCR reaction conditions is: 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, annealed 20 seconds for 53 ℃, 72 ℃ were extended 1 minute 30 seconds, 35 circulations, last 72 ℃ were extended 10 minutes, and agarose gel electrophoresis detects the band that obtains 1675 bp sizes, shows the structure that has the VirG gene to link to each other with the ColE1 replicon in this bacterial strain.According to pCAMBIA2300 carrier primers, upstream primer is positioned at 35S promoter inside, sequence is: 5 '-ggatagtgggattgtgcgtca-3 ' (SEQ ID NO:10), it is inner that downstream primer is positioned at the 35S terminator, sequence is 5 '-catgagcgaaaccctataggaacc-3 ' (SEQ ID NO:11), carry out pcr amplification to contain XL1-Blue/ pCAMBIA2300 bacterium liquid as template, the PCR reaction conditions is: 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, annealed 20 seconds for 56 ℃, 72 ℃ were extended 1 minute, 35 circulations, last 72 ℃ were extended 10 minutes, and electrophoresis detection obtains expecting and the product of 1064 bp sizes shows the nptII gene that 35S promoter and terminator and control thereof are arranged in this bacterial strain.The PCR detected result shows, pVCT2272 carrier and pCAMBIA2300 carrier are present in simultaneously homologous recombination does not occur among the agrobacterium tumefaciens EHA105, have compatibility in agrobacterium tumefaciens bacterial strain EHA105.

In Arabidopis thaliana, contain cold inducible transcription incitant (C-repeat binding factor, AtCBF), there are three members of AtCBF1, AtCBF2 and AtCBF3 to be reported that in this family and find that after deliberation these three genes become interlocked arrangement, put in order to be CBF1 → CBF3 → CBF2.The ecotypic gDNA of Arabidopis thaliana Columbia is template, and is partially digested with BamHI, and electrophoresis reclaims the dna fragmentation of 50-100 Kb sizes; Cut the BiBAC2 carrier with the BamHI enzyme simultaneously, behind calf small intestine Phosphoric acid esterase CIAP dephosphorization, cross column purification; BiBAC2 carrier endonuclease bamhi mixes in the ratio of 1:5 with the gDNA endonuclease bamhi, in 16 ℃ of connections of spending the night, transforms intestinal bacteria XL1-Blue through the T4 dna ligase, screens at the LB solid medium that contains 50mg/L Kan, gets the recon of Kan resistance.Arabidopsis gene group sequence (GenBank accession No:CP002687.1) according to report, the cold inducible transcription incitant of design Arabidopis thaliana AtCBF1 gene primer, the upstream primer sequence is: AtCBF1 F:5 '-cttggatccttcgcttagtcctgtcctgg-3 ' (SEQ ID NO:12), downstream primer is: AtCBF1 R:5 '-tggaaaatagaaagtaaagagtgaagtgac-3 ' (SEQ ID NO:13), carry out pcr amplification take the recon of Kan resistance as template, the PCR reaction conditions is: 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, annealed 20 seconds for 55 ℃, 72 ℃ were extended 2 minutes, 35 circulations, last 72 ℃ were extended 10 minutes, the screening electrophoresis detection obtains the product of 2102 bp sizes, and namely the recon of Kan resistance contains the AtCBF1 gene; Arabidopsis gene group sequence (GenBank accession No:CP002687.1) according to report, the cold inducible transcription incitant of Arabidopis thaliana AtCBF2 gene and AtCBF3 gene detect primer AtCBF2/ AtCBF3 jointly, upstream primer is positioned at the AtCBF3 coding region, primer sequence is: AtCBF2/ AtCBF3 F:5 '-ggtgattatattccgacgcttgcga-3 ' (SEQ ID NO:14), downstream primer is positioned at AtCBF2 gene downstream, primer sequence is: AtCBF2/ AtCBF3 R:5 '-ttaatagctccataaggacacgtcatc-3 ' (SEQ ID NO:15), carry out pcr amplification take the recon of the Kan resistance that contains the AtCBF1 gene as template, reaction conditions is: 95 ℃ of denaturations 2 minutes, 95 ℃ of sex change 10 seconds, annealed 20 seconds for 58 ℃, 72 ℃ were extended 3 minutes 40 seconds, 35 circulations, last 72 ℃ were extended 10 minutes, the screening electrophoresis detection obtains the product of 3502 bp sizes, therefore filter out and contain simultaneously AtCBF3, the recon of AtCBF3 and three genes of AtCBF2, recon contains recombinant vectors, this recombinant vectors called after pBiBAC-CBF.

Adopt electric shocking method with pBiBAC-CBF carrier Transformed E HA105/ pVCT2272, screen at the YEB solid medium that contains kantlex 50mg/L, spectinomycin 50mg/L, Streptomycin sulphate 100mg/L and Rifampin 100mg/L, acquisition contains the engineering bacteria of the EHA105/pVCT2272 of pBiBAC-CBF carrier, called after EHA105/pVCT2272/pBiBAC-CBF.Gained EHA105/pVCT2272/pBiBAC-CBF leaf dish method transformation of tobacco is induced at the MS solid medium that contains 2.5 mg/L 6-BA and 150mg/L kantlex and to be sprouted, and obtains the transgene tobacco of anti-kantlex, as shown in Figure 10.Take kalamycin resistance tobacco gene group DNA as template, take sequence shown in the SEQ ID NO:12 as upstream primer, sequence is downstream primer shown in the SEQ ID NO:13, carries out pcr amplification and detects the AtCBF1 gene, as shown in Figure 11.The result shows, can detect the AtCBF1 amplification at the 2102bp place the kalamycin resistance tobacco, and the result shows that the T-DNA district of pBiBAC-CBF plasmid is integrated into the tobacco gene group.Further specify, the toxicity assistant carrier pVCT2272 carrier that the present invention obtains can realize making that macromolecule T-DNA is complete is integrated in the Plant Genome.

The above is the preferred embodiments of the present invention only, is not limited to the present invention, and obviously, those skilled in the art can carry out various changes and modification and not break away from the spirit and scope of the present invention the present invention.Like this, if of the present invention these are revised and modification belongs within the scope of claim of the present invention and equivalent technologies thereof, then the present invention also is intended to comprise these changes and modification interior.

＜110〉Southwestern University

＜120〉toxicity assistant carrier of agriculture bacillus mediated macromolecule T-DNA conversion and its preparation method and application

<160> 18

<210> 1

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉Pnos::nptII upstream primer

<400> 1

cggaattcag ggagtcacgt tatgac 26

<210> 2

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

＜223〉Pnos::nptII downstream primer

<400> 2

cgctcgagtc ccgctcagaa gaac 24

<210> 3

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉VirE2 upstream region of gene primer

<400> 3

gaattacatt cacacggcac c 21

<210> 4

<211> 42

<212> DNA

＜213〉artificial sequence

<220>

＜223〉VirE2 gene downstream primer

<400> 4

tcacacgtgg tggtggtggt ggtgcagact gtttacggtt gg 42

<210> 5

<211> 25

<212> DNA

＜213〉artificial sequence

<220>

＜223〉VirG upstream region of gene primer

<400> 5

ggagatctgt tgagctgcaa atggctg 27

<210> 6

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉VirG gene downstream primer

<400> 6

gctctagata ggacccatcc aatcac 26

<210> 7

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉toxicity assistant carrier sequence

<400> 7

gtcgaccctt tccgacgctc accgggctgg ttgccctcgc cgctgggctg gcggccgtct 60

atggccctgc aaacgcgcca gaaacgccgt cgaagccgtg tgcgagacac cgcggccggc 120

cgccggcgtt gtggatacct cgcggaaaac ttggccctca ctgacagatg aggggcggac 180

gttgacactt gaggggccga ctcacccggc gcggcgttga cagatgaggg gcaggctcga 240

tttcggccgg cgacgtggag ctggccagcc tcgcaaatcg gcgaaaacgc ctgattttac 300

gcgagtttcc cacagatgat gtggacaagc ctggggataa gtgccctgcg gtattgacac 360

ttgaggggcg cgactactga cagatgaggg gcgcgatcct tgacacttga ggggcagagt 420

gctgacagat gaggggcgca cctattgaca tttgaggggc tgtccacagg cagaaaatcc 480

agcatttgca agggtttccg cccgtttttc ggccaccgct aacctgtctt ttaacctgct 540

tttaaaccaa tatttataaa ccttgttttt aaccagggct gcgccctggc gcgtgaccgc 600

gcacgccgaa ggggggtgcc cccccttctc gaaccctccc ggcccgctaa cgcgggcctc 660

ccatcccccc aggggctgcg cccctcggcc gcgaacggcc tcaccccaaa aatggcagcc 720

aagctcctaa cattttatta gagagcaggc tagttgctta gatacatgat cttcaggccg 780

ttatctgtca gggcaagcga aaattggcca tttatgacga ccaatgcccc gcagaagctc 840

ccatctttgc cgccatagac gccgcgcccc ccttttgggg tgtagaacat ccttttgcca 900

gatgtggaaa agaagttcgt tgtcccattg ttggcaatga cgtagtagcc ggcgaaagtg 960

cgagacccat ttgcgctata tataagccta cgatttccgt tgcgactatt gtcgtaattg 1020

gatgaactat tatcgtagtt gctctcagag ttgtcgtaat ttgatggact attgtcgtaa 1080

ttgcttatgg agttgtcgta gttgcttgga gaaatgtcgt agttggatgg ggagtagtca 1140

tagggaagac gagcttcatc cactaaaaca attggcaggt cagcaagtgc ctgccccgat 1200

gccatcgcaa gtacgaggct tagaaccacc ttcaacagat cgcgcatagt cttccccagc 1260

tctctaacgc ttgagttaag ccgcgccgcg aagcggcgtc ggcttgaacg aattgttaga 1320

cattatttgc cgactacctt ggtgatctcg cctttcacgt agtgaacaaa ttcttccaac 1380

tgatctgcgc gcgaggccaa gcgatcttct tgtccaagat aagcctgcct agcttcaagt 1440

atgacgggct gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc 1500

ggcgcgattt tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt 1560

cgctcatcgc cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc 1620

tcaaatagat cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag 1680

gcaacgctat gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct 1740

ggctcgaaga tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc 1800

ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg 1860

cggagaatct cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct 1920

cgccgcgttg tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt 1980

ggcttcaggc cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg 2040

agatggcgct cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc 2100

gcttccctca tgatgtttaa ctcctgaatt aagccgcgcc gcgaagcggt gtcggcttga 2160

atgaattgtt aggcgtcatc ctgtgctccc gagaaccagt accagtacat cgctgtttcg 2220

ttcgagactt gaggtctagt tttatacgtg aacaggtcaa tgccgccgag agtaaagcca 2280

cattttgcgt acaaattgca ggcaggtaca ttgttcgttt gtgtctctaa tcgtatgcca 2340

aggagctgtc tgcttagtgc ccactttttc gcaaattcga tgagactgtg cgcgactcct 2400

ttgcctcggt gcgtgtgcga cacaacaatg tgttcgatag aggctagatc gttccatgtt 2460

gagttgagtt caatcttccc gacaagctct tggtcgatga atgcgccata gcaagcagag 2520

tcttcatcag agtcatcatc cgagatgtaa tccttccggt aggggctcac acttctggta 2580

gatagttcaa agccttggtc ggataggtgc acatcgaaca cttcacgaac aatgaaatgg 2640

ttctcagcat ccaatgtttc cgccacctgc tcagggatca ccgaaatctt catatgacgc 2700

ctaacgcctg gcacagcgga tcgcaaacct ggcgcggctt ttggcacaaa aggcgtgaca 2760

ggtttgcgaa tccgttgctg ccacttgttt aatagactgg atggaggcgg ataaagttgc 2820

aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2880

cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2940

tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 3000

cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 3060

tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 3120

ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 3180

ccccgtagaa aagatcaaag gatcttcttg atatcctttt tttctgcgcg taatctgctg 3240

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 3300

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 3360

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 3420

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 3480

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 3540

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca 3600

ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 3660

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 3720

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3780

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3840

gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3900

cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3960

gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 4020

ttcacaccgc atatgaagat cggcggcaat agcttcttag cgccatcccg gctgagaaag 4080

cccagtaagg aaacaactgt aggttcgagt cgctagatag gacccatcca atcactccgc 4140

agtgctgagt ttttcggata gtaccgagga aaggcagctt tgccaagccg catagcaatc 4200

tgctcacgtt gggaacagat tgctaaaggc gaaatgcacc tctacctcag gccgccatca 4260

cacccccgta cgaaacatcc acgtcagcgt caaagaaata gccagcacct cttgcagtct 4320

tgatcaactg aggggtcgtc ggatccccct caagcttccg gcgcagccgc aaaatgagga 4380

catcaatact tctgtcatac acctcctcct cgcgtacccg actggcgatc agaagctgct 4440

cccgggatag gacgtcgcgc ggcttctcca ggaaagcaac caggagatta aactcacctg 4500

ccgtgagttt cacctcactg ccctcttccg aaatcaagcg gcgtcgcctg agattaagtg 4560

tccagtcagc gaaactaaat gagcgtcgat ctttggttcg cgcgacactg ggccgcacgc 4620

gtaacgcaac acggatgcgc gccagaaatt cccgcgtccc aaaaggcttg gcaataaaat 4680

cggttgctcc caactcgagc gcaataactt tgtccgcctc ttcgaggcga gcgccgctaa 4740

taattatgat tggaacatcg gacttcgtgg ccagactacg aacaatttca agcccatctt 4800

cgcgacccaa attaagatcg acgaccacga catcgaccgt ctcggagcag agtacacgat 4860

tgaactgctt gctgtcggct accgcagtca ccttaaaggc atggatcgta agatactcga 4920

ctataagatg ccgcatagcg acatcgtcat cgatgacaag aacgtgtttc aacggttcac 4980

ctctcaatct aggatcctgg ccagccattt gcagctcaac agatctccgg ccgccagtgt 5040

gatggattca cacgtggtgg tggtggtggt gcagactgtt tacggttggg ccgcgcggtc 5100

ggctttcata agtccgccgg ctgtcactga tggggggcaa tgctggctcc ggccggacaa 5160

aagcacgtga atatctatcg gacggttcag cgagaagctg cgctgcgtcg ctcggtaatt 5220

tcatcatcag gcgctcatat tccacaacat tagtgtaagt gccggttctt tgcccctttt 5280

cattgcgatc cgctatcagg acatcttttt gagacagttg ttgcaacttt tccggggaca 5340

aatctggaac ctcacgtaag ttcgggccat ccgcaacaat aaccgctgcc tgtctgtcaa 5400

actgctcaaa cttgacacta ttcgcatgac tttgccgacc catcgacacg ggcagaccaa 5460

cttgatccat cagttgggta aagtgttcgg ggtcggaata ttccagtctt gaaggtgaca 5520

tgtaacggtt tccgtcgcga gcaaggacct gaactcgatc ccacaacttt ccctctttca 5580

aggcttgcgt ccaaggggag ttgggtaggt tttgcaatag gtagtcaacg cttacatggc 5640

gatcgcgagt catccctccc tggccatctg gctgccttcc catattcaca gtcgcagccg 5700

gaaactgctt gttgtgagct cgattctcgc cggcgaactc tgcgaaacgg atatccgcac 5760

tgccccgctc ccatgattcc aaatatttgg agtcatgcat aatcccagat ttggatttga 5820

gcttgatctc ggtatcgcta ccgtatttag ttttgattgc gcgttcgaat tcctcaaatc 5880

gcttgttagc gtaagcgtcg cccgcaaacc tgtacgagaa atgtatatca ttttgcgggg 5940

tcttgattaa gatatccggc agcagtgaac cagcttgaaa ctggtgctca tagcgttttt 6000

gaatttctct gcgcccgtat ttctctgttt gtatatatct atcctcaggc ctcaatttat 6060

aatttcggtc gagcttaata ttcttgtctg tttgatagaa gatgtcggtg ccagtgatgc 6120

ccatctctgc gtgagcgcga ttccagactt ccaacttgta ctgaaaacac tgtttactct 6180

gttctcttgg ctccaggtta tgcaattgtg cagccaattg ctccgggata tcgatcctct 6240

cgaatgcgag gggcggggga tagtagttgc cgactccatc cctatagagc cggcagctcg 6300

cattgaattc cgccttggtg ggcagatgat cgaggttaca tttagtagta cctgcgaact 6360

tgtcttgtcc ggtttgccgc acaagaatat actcgtcggt acgccgatca tggcctggta 6420

aagccatctt cgcgaataag ttggatagct cattttcaac ctcagaccga ttgccttgag 6480

cgctgctaga aacatgactt gactgagaat cgagcatgtc cgcatccaac cgcgggccga 6540

gatcatccgt gtgagtcggg cttccgtgca tgttgtgttc gggagtttct tggctacccg 6600

tcatatcaat ttcggacatc ccggtggaat ttacgccctc cctcttctgg cgcttcaaat 6660

tcacgaaatc agaagtttcg acattgcctg ctgcggtctc agtgatattt tcaccattgc 6720

cttcggcctt cggatccatc gtctaactcc ttttagccgg ctaggttttc ttcggggagc 6780

gggcagtgaa ggacaaaatt ctccagttca atcatttcga gatccaaact cgtaaagcca 6840

tttgactggt gatggtcgct caactcctct ttgtgaattt cttgcggctt ctcaaccgcc 6900

aagacaggca tgtttttatt cgcatttagc ttgatgatca ccatatcatt gtttctccta 6960

cagaaattac gattttccta gtgccttccg ctccgatcct gcccagccca aatccatcga 7020

acggaaaatt atcggtgcaa attaaggttt gctccatctt catggccccg aatggcccgg 7080

tgcgagtgta gggcctcctg cacaactctc ttcagacgtg gcgcatgctt tgccggaagc 7140

ccacaatcgt cgacggtttt catctttctc ttgaaccaaa agtcgctcaa aacaagtcac 7200

acagtgaggt tgtcggtttg ccgccaggtc aacttgacta tcgacgtaag cggcttcagc 7260

cggcagcctg cgcgggacga aagccgtccg atcaacatca tcaccgtgca ttgctttcta 7320

ggctgctgca gcggcagtca ctcttcggac acaatcaaga ctcctaatag aattgagcct 7380

tcggtgaatc taccatccga gcctccgtca ttaaacgaat atcgcgtttc atatgtaatt 7440

cttgccaagt cacggagtta gcacgacttt atcgaataag gccgcaggag atagtgatcg 7500

agtaaccggt gccgtgtgaa tgtaattcat ctgcagaatt gcccttaagg gcaattccag 7560

cacactggcg gccgttacta gcgagatccc ccggaaccaa aggaagtagg ttaaacccgc 7620

tccgatcagg ccgagccacg ccaggccgag aacattggtt cctgtaggca tcgggattgg 7680

cggatcaaac actaaagcta ctggaacgag cagaagtcct ccggccgcca gttgccaggc 7740

ggtaaaggtg agcagaggca cgggaggttg ccacttgcgg gtcagcacgg ttccgaacgc 7800

catggaaacc gcccccgcca ggcccgctgc gacgccgaca ggatctagcg ctgcgtttgg 7860

tgtcaacacc aacagcgcca cgcccgcagt tccgcaaata gcccccagga ccgccatcaa 7920

tcgtatcggg ctacctagca gagcggcaga gatgaacacg accatcagcg gctgcacagc 7980

gcctaccgtc gccgcgaccc cgcccggcag gcggtagacc gaaataaaca acaagctcca 8040

gaatagcgaa atattaagtg cgccgaggat gaagatgcgc atccaccaga ttcccgttgg 8100

aatctgtcgg acgatcatca cgagcaataa acccgccggc aacgcccgca gcagcatacc 8160

ggcgacccct cggcctcgct gttcgggctc cacgaaaacg ccggacagat gcgccttgtg 8220

agcgtccttg gggccgtcct cctgtttgaa gaccgacagc ccaatgatct cgccgtcgat 8280

gtaggcgccg aatgccacgg catctcgcaa ccgttcagcg aacgcctcca tgggcttttt 8340

ctcctcgtgc tcgtaaacgg acccgaacat ctctggagct ttcttcaggg ccgacaatcg 8400

gatctcgcgg aaatcctgca cgtcggccgc tccaagccgt cgaatctgag ccttaatcac 8460

aattgtcaat tttaatcctc tgtttatcgg cagttcgtag agcgcgccgt gcgtcccgag 8520

cgatactgag cgaagcaagt gcgtcgagca gtgcccgctt gttcctgaaa tgccagtaaa 8580

gcgctggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg tttgcaatgc 8640

accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag 8700

gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc gcacatgagg 8760

cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata gtcgaacatc 8820

cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt gtagtggtcg 8880

ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga cgttttcttg 8940

ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg cccaacgcgg 9000

tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg 9060

taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg 9120

atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca caccagttcg 9180

tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt gttgacgtgg 9240

aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg 9300

gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg 9360

aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc 9420

ttggcctcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc 9480

atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc ctgttcgaga 9540

cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc cagttgcacg 9600

ctgtcgcgct cgatcttggc cgtagcttgc tggaccatcg agccgacgga ctggaaggtt 9660

tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac 9720

cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt cattcaccct 9780

ccttgcggga ttgccccgac tcacgccggg gcaatgtgcc cttattcctg atttgacccg 9840

cctggtgcct tggtgtccag ataatccacc ttatcggcaa tgaagtcggt cccgtagacc 9900

gtctggccgt ccttctcgta cttggtattc cgaatcttgc cctgcacgaa taccagctcc 9960

gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc 10020

cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat 10080

gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc 10140

atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag 10200

gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc 10260

cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc 10320

cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg 10380

gctgcccttc ctggttgggt aatgactcca acttattgat agtgttttat gttcagataa 10440

tgcccgatga ctttgtcatg cagctccacc gattttgaga acgacagcga cttccgtccc 10500

agccgtgcca ggtgctgcct cagattcagg ttatgccgct caattcgctg cgtatatcgc 10560

ttgctgatta cgtgcagctt tcccttcagg cgggattcat acagcggcca gccatccgtc 10620

atccatatca ccacgtcaaa gggtgacagc aggctcataa gacgccccag cgtcgccata 10680

gtgcgttcac cgaatacgtg cgcaacaacc gtcttccgga gactgtcata cgcgtaaaac 10740

agccagcgct ggcgcgattt agccccgaca tagccccact gttcgtccat ttccgcgcag 10800

acgatgacgt cactgcccgg ctgtatgcgc gaggttaccg actgcggcct gagtttttta 10860

agtgacgtaa aatcgtgttg aggccaacgc ccataatgcg ggctgttgcc cggcatccaa 10920

cgccattcat ggccatatca atgattttct ggtgcgtacc gggttgagaa gcggtgtaag 10980

tgaactgcag ttgccatgtt ttacggcagt gagagcagag atagcgctga tgtccggcgg 11040

tgcttttgcc gttacgcacc accccgtcag tagctgaaca ggagggacag ctgatagaaa 11100

cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa gttggcagca 11160

tcacccctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg 11220

gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg 11280

acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccggctga 11340

cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt 11400

gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca 11460

gcggaaaaga tcc 11473

<210> 8

<211> 27

<212> DNA

＜213〉artificial sequence

<220>

＜223〉pVCT2272 carrier sense upstream primer

<400> 8

ggagatctgt tgagctgcaa atggctg 27

<210> 9

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉pVCT2272 carrier sense downstream primer

<400> 9

tccttctagt gtagccgtag 20

<210> 10

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉pCAMBIA2300 carrier sense upstream primer

<400> 10

ggatagtggg attgtgcgtc a 21

<210> 11

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

＜223〉pCAMBIA2300 carrier sense downstream primer

<400> 11

catgagcgaa accctatagg aacc 24

<210> 12

<211> 29

<212> DNA

＜213〉artificial sequence

<220>

＜223〉AtCBF1 upstream region of gene primer

<400> 12

cttggatcct tcgcttagtc ctgtcctgg 29

<210> 13

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

＜223〉AtCBF1 gene downstream primer

<400> 13

tggaaaatag aaagtaaaga gtgaagtgac 30

<210> 14

<211> 25

<212> DNA

＜213〉artificial sequence

<220>

＜223〉AtCBF3/AtCBF2 upstream region of gene primer

<400> 14

ggtgattata ttccgacgct tgcga 25

<210> 15

<211> 27

<212> DNA

＜213〉artificial sequence

<220>

＜223〉AtCBF3/AtCBF2 gene downstream primer

<400> 15

Ttaatagctc cataaggaca cgtcatc 27

<210> 16

<211> 3218

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the chimeric operon sequence of virE-virG

cttgcagtct tgatcaactg aggggtcgtc ggatccccct caagcttccg gcgcagccgc 60

aaaatgagga catcaatact tctgtcatac acctcctcct cgcgtacccg actggcgatc 120

agaagctgct cccgggatag gacgtcgcgc ggcttctcca ggaaagcaac caggagatta 180

aactcacctg ccgtgagttt cacctcactg ccctcttccg aaatcaagcg gcgtcgcctg 240

agattaagtg tccagtcagc gaaactaaat gagcgtcgat ctttggttcg cgcgacactg 300

ggccgcacgc gtaacgcaac acggatgcgc gccagaaatt cccgcgtccc aaaaggcttg 360

gcaataaaat cggttgctcc caactcgagc gcaataactt tgtccgcctc ttcgaggcga 420

gcgccgctaa taattatgat tggaacatcg gacttcgtgg ccagactacg aacaatttca 480

agcccatctt cgcgacccaa attaagatcg acgaccacga catcgaccgt ctcggagcag 540

agtacacgat tgaactgctt gctgtcggct accgcagtca ccttaaaggc atggatcgta 600

agatactcga ctataagatg ccgcatagcg acatcgtcat cgatgacaag aacgtgtttc 660

aacggttcac ctctcaatct aggatcctgg ccagccattt gcagctcaac agatctccgg 720

ccgccagtgt gatggattca cacgtggtgg tggtggtggt gcagactgtt tacggttggg 780

ccgcgcggtc ggctttcata agtccgccgg ctgtcactga tggggggcaa tgctggctcc 840

ggccggacaa aagcacgtga atatctatcg gacggttcag cgagaagctg cgctgcgtcg 900

ctcggtaatt tcatcatcag gcgctcatat tccacaacat tagtgtaagt gccggttctt 960

tgcccctttt cattgcgatc cgctatcagg acatcttttt gagacagttg ttgcaacttt 1020

tccggggaca aatctggaac ctcacgtaag ttcgggccat ccgcaacaat aaccgctgcc 1080

tgtctgtcaa actgctcaaa cttgacacta ttcgcatgac tttgccgacc catcgacacg 1140

ggcagaccaa cttgatccat cagttgggta aagtgttcgg ggtcggaata ttccagtctt 1200

gaaggtgaca tgtaacggtt tccgtcgcga gcaaggacct gaactcgatc ccacaacttt 1260

ccctctttca aggcttgcgt ccaaggggag ttgggtaggt tttgcaatag gtagtcaacg 1320

cttacatggc gatcgcgagt catccctccc tggccatctg gctgccttcc catattcaca 1380

gtcgcagccg gaaactgctt gttgtgagct cgattctcgc cggcgaactc tgcgaaacgg 1440

atatccgcac tgccccgctc ccatgattcc aaatatttgg agtcatgcat aatcccagat 1500

ttggatttga gcttgatctc ggtatcgcta ccgtatttag ttttgattgc gcgttcgaat 1560

tcctcaaatc gcttgttagc gtaagcgtcg cccgcaaacc tgtacgagaa atgtatatca 1620

ttttgcgggg tcttgattaa gatatccggc agcagtgaac cagcttgaaa ctggtgctca 1680

tagcgttttt gaatttctct gcgcccgtat ttctctgttt gtatatatct atcctcaggc 1740

ctcaatttat aatttcggtc gagcttaata ttcttgtctg tttgatagaa gatgtcggtg 1800

ccagtgatgc ccatctctgc gtgagcgcga ttccagactt ccaacttgta ctgaaaacac 1860

tgtttactct gttctcttgg ctccaggtta tgcaattgtg cagccaattg ctccgggata 1920

tcgatcctct cgaatgcgag gggcggggga tagtagttgc cgactccatc cctatagagc 1980

cggcagctcg cattgaattc cgccttggtg ggcagatgat cgaggttaca tttagtagta 2040

cctgcgaact tgtcttgtcc ggtttgccgc acaagaatat actcgtcggt acgccgatca 2100

tggcctggta aagccatctt cgcgaataag ttggatagct cattttcaac ctcagaccga 2160

ttgccttgag cgctgctaga aacatgactt gactgagaat cgagcatgtc cgcatccaac 2220

cgcgggccga gatcatccgt gtgagtcggg cttccgtgca tgttgtgttc gggagtttct 2280

tggctacccg tcatatcaat ttcggacatc ccggtggaat ttacgccctc cctcttctgg 2340

cgcttcaaat tcacgaaatc agaagtttcg acattgcctg ctgcggtctc agtgatattt 2400

tcaccattgc cttcggcctt cggatccatc gtctaactcc ttttagccgg ctaggttttc 2460

ttcggggagc gggcagtgaa ggacaaaatt ctccagttca atcatttcga gatccaaact 2520

cgtaaagcca tttgactggt gatggtcgct caactcctct ttgtgaattt cttgcggctt 2580

ctcaaccgcc aagacaggca tgtttttatt cgcatttagc ttgatgatca ccatatcatt 2640

gtttctccta cagaaattac gattttccta gtgccttccg ctccgatcct gcccagccca 2700

aatccatcga acggaaaatt atcggtgcaa attaaggttt gctccatctt catggccccg 2760

aatggcccgg tgcgagtgta gggcctcctg cacaactctc ttcagacgtg gcgcatgctt 2821

tgccggaagc ccacaatcgt cgacggtttt catctttctc ttgaaccaaa agtcgctcaa 2880

aacaagtcac acagtgaggt tgtcggtttg ccgccaggtc aacttgacta tcgacgtaag 2940

cggcttcagc cggcagcctg cgcgggacga aagccgtccg atcaacatca tcaccgtgca 3000

ttgctttcta ggctgctgca gcggcagtca ctcttcggac acaatcaaga ctcctaatag 3060

aattgagcct tcggtgaatc taccatccga gcctccgtca ttaaacgaat atcgcgtttc 3120

atatgtaatt cttgccaagt cacggagtta gcacgacttt atcgaataag gccgcaggag 3180

atagtgatcg agtaaccggt gccgtgtgaa tgtaattc 3218

<210> 17

<211> 441

<212> DNA

＜213〉artificial sequence

<220>

＜223〉oriV Agrobacterium replicon sequence

ccgggctggt tgccctcgcc gctgggctgg cggccgtcta tggccctgca aacgcgccag 60

aaacgccgtc gaagccgtgt gcgagacacc gcggccggcc gccggcgttg tggatacctc 120

gcggaaaact tggccctcac tgacagatga ggggcggacg ttgacacttg aggggccgac 180

tcacccggcg cggcgttgac agatgagggg caggctcgat ttcggccggc gacgtggagc 241

tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg cgagtttccc acagatgatg 300

tggacaagcc tggggataag tgccctgcgg tattgacact tgaggggcgc gactactgac 360

agatgagggg cgcgatcctt gacacttgag gggcagagtg ctgacagatg aggggcgcac 420

ctattgacat ttgaggggct g 441

<210> 18

<211> 789

<212> DNA

＜213〉artificial sequence

<220>

＜223〉spec gene order

ttatttgccg actaccttgg tgatctcgcc tttcacgtag tgaacaaatt cttccaactg 61

atctgcgcgc gaggccaagc gatcttcttg tccaagataa gcctgcctag cttcaagtat 121

gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg 181

cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg 241

ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc 301

aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc 361

aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg 421

ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt 481

agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg 541

gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg 601

ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg 661

cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag 721

atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc 781

ttccctcat 789

Claims (2)

1. the toxicity assistant carrier that transforms of agriculture bacillus mediated macromolecule T-DNA, it is characterized in that: described toxicity assistant carrier has nucleotide sequence shown in SEQ ID NO:7.

2. the preparation method of the described toxicity assistant carrier of claim 1 is characterized in that, specifically may further comprise the steps:

A. prepare the restructuring binary expression vector;

A1. prepare Pnos::nptII fragment and enzyme and cut, the Pnos::nptII endonuclease bamhi is inserted pCAMBIA1302 carrier polyclone enzyme cut site, get the pVCT2020 carrier;

A2. enzyme is cut the pEGFP-N1 carrier, and preparation eGFP gene inserts pET-32a (+) carrier polyclone restriction enzyme site with described eGFP gene, gets the pVCT2190 carrier;

A3. enzyme is cut gained pVCT2190 carrier, prepares the fragment of described eGFP gene, enzyme is cut gained eGFP gene insert gained pVCT2020 carrier restriction enzyme site place, gets the pVCT2210 carrier;

A4. enzyme is cut gained pVCT2210 carrier, identifies and recovery through agarose electrophoresis, and the fragment that reclaims is connected, and gets the pVCT2212 carrier;

A5. enzyme is cut gained pVCT2212 carrier, identifies with agarose gel electrophoresis, reclaims to contain the eGFP gene fragment, enzyme is cut gained eGFP gene insert pHells gate8 carrier restriction enzyme site place, gets the restructuring binary expression vector, called after pVCT2268 carrier;

B. preparation contains the underlying carrier of the chimeric operon of virE-virG;

B1. prepare the Promoter-VirE1-VirE2 gene, gained Promoter-VirE1-VirE2 gene is connected p Easy-Blant carrier gets the pVCT1213 carrier;

B2. prepare the VirG-terminator gene, gained VirG-terminator fragment is inserted the pVCT1213 carrier, must contain the underlying carrier of the chimeric operon of virE-virG, called after pVCT1244 carrier;

C. prepare the toxicity assistant carrier;

C1. enzyme is cut gained pVCT1244 carrier, and the chimeric operon of preparation virE-virG inserts gained pVCT2268 carrier with the chimeric operon of gained virE-virG, gets recombinant vectors, called after pVCT2270 carrier;

C2. enzyme is cut gained pVCT2270 carrier, contain the chimeric operon fragment of described virE-virG, Agrobacterium replicon oriV and resistant maker gene fragment through agarose gel electrophoresis evaluation and recovery, the endonuclease bamhi that reclaims the pVCT2270 carrier is connected again, get described toxicity assistant carrier, called after pVCT2272 carrier.

3. the preparation method of described toxicity assistant carrier according to claim 2, its spy is: the preparation of the described Pnos::nptII fragment of step a1 specifically comprises the steps:

Take sequence shown in the SEQ ID NO:1 as upstream primer, take sequence shown in the SEQ ID NO:2 as downstream primer, take the pBIN19 carrier as template, carry out pcr amplification, the pcr amplification condition is: 94 ℃ of denaturations 3 minutes, 94 ℃ of sex change 20 seconds, annealed 20 seconds for 50 ℃, 72 ℃ were extended 2 minutes 10 seconds, and 3 circulations were annealed 20 seconds for 60 ℃, 72 ℃ were extended 2 minutes 10 seconds, 27 circulations, 72 ℃ were extended 10 minutes, and got the Pnos::nptII fragment.

4. the preparation method of described toxicity assistant carrier according to claim 2 is characterized in that, the preparation of the described Promoter-VirE1-VirE2 gene of step b1, and concrete steps are as follows:

Take the pTiC58 plasmid DNA of Agrobacterium C58 as template, the nucleotides sequence of upstream primer is classified as shown in the SEQ ID NO:3, the nucleotides sequence of downstream upstream primer is classified as shown in the SEQ ID NO:4, adopt PrimStar HS archaeal dna polymerase to carry out pcr amplification, get the VirE2 gene fragment, described pcr amplification condition is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, annealed 15 seconds for 56 ℃, 72 ℃ were extended 2 minutes 30 seconds, 28 circulations, 72 ℃ were extended 10 minutes, and got the Promoter-VirE1-VirE2 gene.

5. the preparation method of described toxicity assistant carrier according to claim 2 is characterized in that, the preparation of the described VirG-terminator gene of step b2, and concrete steps are as follows:

Take the pTiC58 carrier of Agrobacterium C58 as template, the upstream primer nucleotide sequence is shown in SEQ ID NO:5, and the downstream primer nucleotide sequence adopts PrimStar HS archaeal dna polymerase to carry out pcr amplification shown in SEQ ID NO:6, get the VirG gene fragment, described pcr amplification condition is: 98 ℃ of denaturations 1 minute, 98 ℃ of sex change 10 seconds, 56 ℃ of annealing 15 seconds, 72 ℃ were extended 1 minute, 28 circulations, 72 ℃ were extended 10 minutes, and got the VirG-terminator gene.

6. the preparation method of described toxicity assistant carrier according to claim 2 is characterized in that, the preparation of the chimeric operon of the described virE-virG of step c1, and concrete steps are as follows:

Cut gained pVCT1244 carrier with restriction enzyme Spe I, Xba I and Ema1105 I enzyme simultaneously, identify through agarose gel electrophoresis, reclaim the fragment of 3465bp size, get the chimeric operon of virE-virG.

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