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Method for improving transgenosis efficiency of cotton

A transgenic, high-efficiency technology, applied in the field of transgenics, can solve problems such as plant regeneration problems, and achieve the effects of fast stability, improved conversion rate, and improved conversion rate

Inactive Publication Date: 2015-10-28
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene gun method still suffers from the problem of plant regeneration in the introduction of cotton genes.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] S1. Operate on ice, adjust the concentrations of the expressed and purified Agrobacterium toxic proteins VirD1, VirD2, and VirE2 to 100 μg / μl;

[0020] S2. Digest DNA with restriction endonuclease Hind III.+EcoR I and connect it to pBin438 plasmid to construct exogenous plasmid DNA;

[0021] S3. Resuspend the obtained exogenous plasmid DNA in a plasmid DNA protectant to obtain a mixed solution with a concentration of exogenous plasmid DNA of 1 μg / μL; the plasmid DNA protectant consists of aluminum hydroxide sol protectant, calcium phosphate protectant and Tween20 The protective agent is obtained by mixing the volume ratio of 1:2:1;

[0022] S4. Take 2 μl of one or more of the solutions obtained in step S1, add in turn the mixed solution obtained in step S3 containing 8 μl of exogenous plasmid DNA, mix 6 μl of liposomes, add 30 μl of buffer solution, and place at 4°C After 2 hours, take it out, and introduce the gene of interest into cotton by the pollen tube passage me...

Embodiment 2

[0024] S1. Operate on ice, adjust the concentrations of the expressed and purified Agrobacterium toxic proteins VirD1, VirD2, and VirE2 to 100 μg / μl;

[0025] S2. Digest DNA with restriction endonuclease Hind III.+EcoR I and connect it to pBin438 plasmid to construct exogenous plasmid DNA;

[0026] S3. Resuspend the obtained exogenous plasmid DNA in a plasmid DNA protectant to obtain a mixed solution with a concentration of exogenous plasmid DNA of 1 μg / μL; the plasmid DNA protectant consists of aluminum hydroxide sol protectant, calcium phosphate protectant and Tween20 The protective agent is obtained by mixing the volume ratio of 1:2:1;

[0027] S4. Take 2 μl of one or more of the solutions obtained in step S1, add in turn the mixed solution obtained in step S3 containing 8 μl of exogenous plasmid DNA, mix 6 μl of liposomes, add 30 μl of buffer solution, and place at 4°C After 2 hours, take it out, and introduce the target gene into cotton by the pollen tube passage method;...

Embodiment 3

[0029] S1. Operate on ice, adjust the concentrations of the expressed and purified Agrobacterium toxic proteins VirD1, VirD2, and VirE2 to 100 μg / μl;

[0030] S2. Digest DNA with restriction endonuclease Hind III.+EcoR I and connect it to pBin438 plasmid to construct exogenous plasmid DNA;

[0031] S3. Resuspend the obtained exogenous plasmid DNA in a plasmid DNA protectant to obtain a mixed solution with a concentration of exogenous plasmid DNA of 1 μg / μL; the plasmid DNA protectant consists of aluminum hydroxide sol protectant, calcium phosphate protectant and Tween20 The protective agent is obtained by mixing the volume ratio of 1:2:1;

[0032] S4. Take 2 μl of one or more of the solutions obtained in step S1, add in turn the mixed solution obtained in step S3 containing 8 μl of exogenous plasmid DNA, mix 6 μl of liposomes, add 30 μl of buffer solution, and place at 4°C After 2 hours, take it out, and introduce the target gene into cotton by the pollen tube passage method;...

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Abstract

The invention discloses a method for improving the transgenosis efficiency of cotton. The method comprises the following steps: after mixing restriction enzyme digestion DNA with a plasmid DNA protective agent, introducing a target gene into cotton through the pollen tube pathway method by using lipidosome packaging to carry toxic protein genes on Ti plasmids. The method can effectively improve the transgenosis efficiency.

Description

technical field [0001] The invention relates to the field of transgenics, in particular to a method for improving the transgenic efficiency of cotton. Background technique [0002] It is an important way to improve cotton quality and obtain new germplasm by using transgenic technology to introduce exogenous genes into cotton cells and stably inherit them. There are mainly Agrobacterium tumefaciens mediated method and pollen tube (Pollen-Tube Pathway Method) introduction method in the aspect of cotton transgenic. Since Umbeck used the Agrobacterium-mediated method to transfer the neomycin phosphotransferase gene nap ll and the chloramphenicol acetyltransferase gene CAT into the cotton genome for the first time in 1987, the cotton genetic transformation method mediated by Agrobacterium has been obtained Wide range of applications. The genetic transformation mechanism of Ti plasmid carried by Agrobacterium has been studied clearly, and has obvious advantages, such as stable i...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 刘红玲祝建波朱华国孙燕飞邱红林王彦芹
Owner SHIHEZI UNIVERSITY
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