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

An Agrobacterium-mediated and auxiliary vector technology, applied in the field of genetic engineering, can solve the problems of fragmentation, loss, and incomplete integration of large molecular weight T-DNA, and achieve the effect of promoting genetic transformation.

Inactive Publication Date: 2012-01-18
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of this, in order to solve the problem of the complete integration of large molecular weight T-DNA into the plant genome, a toxic helper vector for Agrobacterium-mediated transformation of large molecular weight T-DNA is provided, The carrier can mediate the transformation of T-DNA with a molecular weight greater than 25 kb; it provides a method for preparing a toxic auxiliary carrier for the transformation of large molecular weight T-DNA mediated by Agrobacterium, and the method for constructing a toxic auxiliary carrier with this method is simple

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of recombinant binary expression vector

[0051] Construction of a1.pVCT2020 vector

[0052] Design primers according to the pBIN19 vector sequence (GenBank accession No: U09365), the upstream primer is shown in SEQ ID NO: 1, the sequence is: 5'-cg gaattc agggagtcacgttatgac-3' (the underline is the EcoR I site), the downstream primer is shown in SEQ ID NO: 2, and the sequence is: 5'-cg ctcgag tcccgctcagaagaac-3' (the Xho I site is underlined). Using pBIN19 vector as template, PfuDNA polymerase, PCR amplification, PCR amplification conditions are 94°C pre-denaturation for 3 minutes, 94°C denaturation for 20 seconds, 50°C annealing for 20 seconds, 72°C extension for 2 minutes and 10 seconds, 3 cycles , anneal at 60°C for 20 seconds, extend at 72°C for 2 minutes and 10 seconds, 27 cycles, extend at 72°C for 10 minutes, and recover a 1116 bp fragment by agarose electrophoresis, which contains the nopaline synthase gene promoter Pnos and kanamycin The prime...

Embodiment 2

[0063] Construction of Basic Vector Containing virE-virG Chimeric Operator

[0064] Construction of b1.pVCT1213 vector

[0065] According to the reported pTiC58 vector sequence of Agrobacterium strain C58 (GenBank accession: NC_003308), design specific PCR primers containing promoter Promoter, VirE1 gene coding region and VirE2 gene coding region (referred to as Promoter-VirE1-VirE2), where the upstream primer As shown in SEQ ID NO: 3, the sequence is 5'-gaattacattcacacggcacc-3', and the downstream primer is shown in SEQ ID NO: 4, and the sequence is 5'-tcacacgtggtggtggtggtggtgcagactgtttacggttgg-3'. Using the pTiC58 vector of Agrobacterium strain C58 as a template, PCR amplification was performed using PrimeStar HS DNA polymerase. The reaction conditions were: 98°C pre-denaturation for 1 minute, 98°C denaturation for 10 seconds, 56°C annealing for 15 seconds, and 72°C extension for 2 minutes for 30 seconds, 28 cycles, 72°C extension for 10 minutes. The PCR product was identi...

Embodiment 3

[0070] Construction of toxic helper vector

[0071] Construction of c1.pVCT2270 vector

[0072] as attached Figure 5 As shown, the obtained pVCT2268 vector was single-digested with the restriction endonuclease Nru I, and was identified by agarose gel electrophoresis to obtain the backbone fragment of the pVCT2268 vector; at the same time, it was three-digested with the restriction endonucleases Spe I, Xba I, and Ema1105 I The vector pVCT1244 was identified by agarose gel electrophoresis, and a 3465bp fragment containing the virE-virG chimeric operon was recovered, and the virE-virG chimeric operon was ligated with the backbone fragment of the pVCT2268 vector under the action of T4 ligase overnight at 16°C. A recombinant vector was obtained, named pVCT2270, as attached Figure 8 shown.

[0073] Construction of c2.pVCT2272 vector

[0074] Digest the pVCT2270 vector with restriction endonuclease Sal I, excise the T-DNA region on the pVCT2270 vector, and use 1% agarose gel el...

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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

technical field [0001] The invention relates to the field of genetic engineering, in particular to a toxic auxiliary carrier for transformation of large molecular weight T-DNA mediated by Agrobacterium. The invention also discloses a construction method and application of the toxic auxiliary carrier. Background technique [0002] Agrobacterium-mediated plant genetic transformation is a natural process of introducing foreign genes into the plant genome with the help of Agrobacterium. In Agrobacterium tumefaciens, there is a large plasmid, the Ti plasmid, about 150-240 kb in size. There are two important regions on the Ti plasmid: T-DNA region and toxicity region (virulence region, Vir region). When Agrobacterium is induced by phenolic substances such as acetosyringone secreted by plant wounds, the VirA protein located on the cell membrane of Agrobacterium is autophosphorylated and activated, and then transfers its phosphate group to the VirG protein with transcription factor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/66A01H5/00
Inventor 张兴国杜小兵苏承刚高启国钱春陈吉裕黄国栋
Owner SOUTHWEST UNIV
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