Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium

A technology of pollen tube channeling and Agrobacterium toxin, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., to achieve the effect of improving transformation efficiency and genetic stability

Inactive Publication Date: 2007-12-26
SHIHEZI UNIVERSITY
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The advantages and disadvantages of the two are e

Method used

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  • Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium
  • Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium
  • Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of a plant expression vector for a plant-enhanced green fluorescent protein gene (EGFP) (see Figure 1).

Embodiment 2

[0041] virD 1 , virD 2 and virE 2 gene cloning

[0042] 1. Cloning of the VirD1 gene in the toxic region of the Agrobacterium tumefaciens Ti plasmid (see Figure 2)

[0043] According to the complete sequence of Agrobacterium tumefaciens C58 VirD1 protein gene published by U.Washington, a pair of primers were designed by ourselves, as follows:

[0044] P1 is: 1,

[0045] 5'-CG GATATC ATGTCGCAAGGCAGTAGG-3'Eco V (underline) 26bp;

[0046] P2 is: 2,

[0047] 5'-CG AAGCTT TTACAAGGCGTCTTTCAGCA-3'HindIII (underlined) 28bp,

[0048] The span between the two primers is 459bp, and the size of the amplified fragment after adding restriction sites and protective bases is 474bp.

[0049] 2. Cloning of the VirD2 gene in the toxic region of the Agrobacterium tumefaciens Ti plasmid (see Figure 3)

[0050] According to Agrobacterium tumefaciens C58 VirD published by U.Washington 2 The full sequence of the gene, a pair of primers designed by ourselves, synthesized by Shanghai Sangon B...

Embodiment 3

[0062] virD 1 , virD 2 and virE 2 Gene plant transient expression vector pUC-pBin-VirD 1 , pUC-pBin-VirD 2 , pUC-pBin-VirE 2 And the construction of pUC-pBin-GFP. The technical roadmap is shown in Figure 5.

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Abstract

This invention relates to a method for increasing the conversion efficiency of pollen tube pathway method by using agrobacterium tumefaciens virulence gene. The method comprises: combining or mixing agrobacterium tumefaciens virulence gene and the target gene to be converted (green fluorescent protein gene) in vitro, packaging with liposome, and converting plants, especially cotton by using pollen tube pathway method. The method combines agrobacterium-liposome-pollen tube pathway technique, and has obviously increased protoplast conversion efficiency. Plasmids are not only protected by liposome when entering embryo sacs, but also encapsulated by the toxic protein to be expressed when entering egg cells, which can improve the stability of exogenous DAN in cell conversion process, realize precise cutting in T-DNA region, and increase the conversion efficiency.

Description

Technical field: [0001] The invention relates to a method for improving the transformation efficiency of the pollen tube passage method by using the Agrobacterium toxin gene. Background technique: [0002] The Agrobacterium-mediated method is currently the most clear research mechanism and the most widely used transgenic method {Wu Xia, Jiao Gaili, etc. The mechanism and research progress of Agrobacterium-mediated transformation of cotton[J]. Shanxi Agricultural Sciences, 2001, 29(1 ): 27~30; Fu Yongcai et al. New progress in genetic transformation of grass crops mediated by Agrobacterium [J]. Acta Biological Technology, 1999, 10(3): 1~5; Mediated Plant Ttansformation: the Biology behind the "Gene-Jockying" Tool [J]. Microbiology and Molecular Biology reviews, 2003, 16-37}. Because it can realize high-frequency autonomous transformation by means of the enzyme system of Agrobacterium and plants, it has always been one of the hot spots of research {Michielse CB, et al.Agrobac...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/31A01H1/04A01H1/02
Inventor 祝建波张煜星崔百明王爱英刘红玲李小红王彦芹
Owner SHIHEZI UNIVERSITY
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