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Improved method for converting plant pollen tube

A plant pollen and plant technology, applied in the field of improved plant pollen tube transformation, can solve the problems of low transformation efficiency, easy degradation of exogenous plasmid DNA and the like

Inactive Publication Date: 2014-04-30
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide an improved plant pollen tube transformation method for solving the problem that the exogenous plasmid DNA is easy to degrade and the transformation efficiency is not high during the transformation process of the pollen tube passage method

Method used

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  • Improved method for converting plant pollen tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Extraction of Plant Expression Vector pCAMBIA1301 Plasmid DNA

[0032] The plasmid DNA was extracted from Escherichia coli containing the plant expression vector pCAMBIA1301 according to the conventional plasmid DNA extraction method-alkaline lysis method.

Embodiment 2

[0033] Embodiment 2 Preparation of Aluminum Hydroxide Sol Protective Agent

[0034] (1) Add 2-4mL 1% aluminum chloride solution to a glass test tube, then add 0.05-2 mL 10% ammonia water to form a precipitate;

[0035] (2) Discard the supernatant, wash the precipitate with distilled water for 3-5 times, remove the water by decantation, and separate the lotion from the precipitate by filtration for the last time;

[0036] (3) Transfer the precipitate into a 150 mL beaker, add 20-80 mL of distilled water, stir and heat to boil. During this period, add 0.1-0.2 mL 0.1 mol / L HCl solution and keep stirring to obtain aluminum hydroxide sol;

[0037] (4) Take a little aluminum hydroxide sol and resuspend the pCAMBIA1301 plasmid DNA precipitate extracted in Example 1, and the obtained mixture is aluminum hydroxide sol-pCAMBIA1301, and the concentration of the plasmid DNA of pCAMBIA1301 is 0.8-1.2 μg / μL.

Embodiment 3

[0038] Embodiment 3 Preparation of calcium phosphate protective agent

[0039] (1) Resuspend the plasmid DNA precipitate of pCAMBI1301 extracted in Example 1 with 5-15 μL of sterilized distilled water to make the concentration 1 μg / μL, then add 50-60 μL of 2 mol / L CaCl2 solution and mix well. Occasionally, a small amount of turbid substance will appear.

[0040] (2) Centrifuge at 12000 r / min for 10 min, take out the supernatant, and add it to 400-450 μL sterilized distilled water, which is called pCAMBIA1301-CaCl2 solution.

[0041] (3) Take a 2 mL sterilized centrifuge tube, add 500 μL 2×HEPES buffer solution (pH6.95-7.05) into it, and add pCAMBIA1301-CaCl2 solution drop by drop into the HEPES buffer solution by blowing air.

[0042] (4) Place the test tube in a 37°C water bath and incubate for 3-5 minutes, milky white turbidity can be seen.

[0043] (5) Centrifuge at 10000 r / min for 3 min, discard the supernatant. The resulting mixture was calcium phosphate-pCAMBIA1301, a...

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Abstract

The invention discloses an improved method for converting a plant pollen tube. Aluminum hydroxide sol, calcium phosphate and Tween20 are adopted respectively to serve as exogenous plasmid DNA protective agents, and a plant pollen tube channel method is utilized to convert an exogenous gene. Receptor tobacco and soybean GUS active tissue histochemical staining results show that the gus positive rates with the aluminum hydroxide sol serving as the DNA protective agent are respectively 11.1% and 3.78%; the gus positive rates with calcium phosphate serving as the DNA protective agent are respectively 8.89% and 2.91%; the gus positive rates with Tween20 serving as the DNA protective agent are respectively 10.0% and 1.41 %; and the gus positive rates with 1*SSC solution serving as the DNA protective agent are respectively 7.78% and 1.97 %, the problem that the conversion rate in the plant pollen tube channel method is low is solved, and the conversion rate is improved.

Description

technical field [0001] The invention relates to the field of biotechnology genetic breeding, in particular to an improved plant pollen tube transformation method. Background technique [0002] In 1981, Chinese scientist Zhou Guangyu introduced exogenous DNA into upland cotton for the first time by using the pollen tube passage method, and successfully bred a transgenic variety resistant to Fusarium wilt. In recent years, due to its unique advantages, the pollen tube passage method has been widely used by scientists for transgenic operations of plants. This method has the following advantages: First, the pollen tube passage method has a wide range of applications and can be applied to any flowering plants, and different gene transfers can be performed between different species, so it relaxes the target gene and receptor range of plants. Second, it does not rely on the cumbersome plant tissue culture and the long process of inducing plant regeneration. The pollen tube passag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
Inventor 王丕武付永平张君曲静姚丹马建王鑫雨单睿关淑艳
Owner JILIN AGRICULTURAL UNIV
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