Tri-parent conjugation method for transforming agrobacterium rhizogenes
A technology of Agrobacterium rhizogenes and hybridization method, which is applied in the biological field, can solve the problems of large demand for bacterial liquid, complicated operation, restricting the application of Agrobacterium rhizogenes, etc., and achieves the effect of low solution efficiency.
Inactive Publication Date: 2010-12-15
ANKANG UNIV
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The conversion efficiency of this method is higher than that of the chemical method, but the operation is complicated and requires repeated washing with ultrapure water, which requires a large amount of bacterial liquid, and expensive instruments such as electric conversion apparatus and refrigerated centrifuge are required.
The three-parent hybridization method can complete the introduction only with the participation of auxiliary bacteria, which is the most efficient of the three methods, but requires that the recipient bacteria have special screening markers relative to the donor bacteria and auxiliary bacteria. Agrobacterium has no chromosomal resistance markers and cannot be applied, thus largely restricting the application of Agrobacterium rhizogenes in plant transgenesis
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[0023] figure 1 The PCR of the plasmid extracted from a single colony for transformation; where M represents the molecular weight standard, and the size (bp) of each band is marked on the left, and lanes 1, 3, and 5 are the rol B gene, rol C gene, and attacin gene in A4( pCAMBIA2300-attacin) strain extracted plasmid as template for PCR; swimming lanes 2 and 4 are PCRs carried out with templates extracted from Escherichia coli strain plasmids of rol B gene and rol C gene respectively; swimming lane 6 is respectively A4 of attacin gene The PCR detection of the plasmid template of (pCAMBIA2300-attacin) bacterial strain and Escherichia coli strain plasmid template;
[0024] figure 2 False positive rate for single colony rol B. rol C and attacin The PCR amplification of the gene; where A is the PCR amplification of the rol B gene; B is the PCR amplification of the rol C gene; C is the PCR amplification of the attacin gene; M represents the molecular weight standard, and the ...
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The invention discloses a tri-parent conjugation method for transforming agrobacterium rhizogenes. The key technical points include: among three types of strains, namely donor strains, acceptor strains and auxiliary strains, the donor strains and the auxiliary strains are auxotrophic and have resistance marks, the acceptor strains are wild agrobacterium rhizogenes, and tri-parent conjugation is performed by taking auxotrophic culture medium and corresponding antibiotic as a screening mark to complete the genetic transformation of genes into wild agrobacterium rhizogenes. The method of the invention has the advantages that: the operation is simple; the transformation efficiency is high; the limit that the wild agrobacterium rhizogenes without chromosome marks cannot be applied in tri-parent conjugation transformation is broken through; the situation that the wild agrobacterium rhizogenes can only be transformed electrically or transformed by competent cells generated by a chemical method, the situation that the operation is complicated and the situation that the transformation efficiency is low are changed; and the application of the agrobacterium rhizogenes to plant genetic engineering is greatly accelerated.
Description
technical field [0001] The invention relates to the genetic transformation of wild-type Agrobacterium rhizogenes without chromosomal resistance markers, and belongs to the field of biotechnology. Background technique [0002] With the development of genetic engineering technology, the Agrobacterium-mediated method has become more and more widely used in the genetic transformation of exogenous genes, the improvement of crop quality, and the cultivation of engineering plants with strong disease resistance, insect resistance, and stress resistance. There are two kinds of Agrobacterium tumefaciens and Agrobacterium rhizogenes currently used. The integrity of Agrobacterium tumefaciens mediating the insertion of foreign genes is good, but the T-DNA of the Ti plasmid has disease-causing genes, the expression of foreign genes is easily inactivated, gene expression is incompatible with plant regeneration, and the operation is complicated , limited material selection, low transformat...
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Login to View More IPC IPC(8): C12N15/09C12N1/21
Inventor 孔卫青杨金宏
Owner ANKANG UNIV