Application of cell-penetrating peptide TAT2 in transgenic cotton cultivation

A technology of TAT2 and membrane-penetrating peptide, which is applied in the field of genetic engineering to achieve the effects of low production cost, improved genetic transformation efficiency, and easy operation

Inactive Publication Date: 2016-05-11
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the use of CPPs in the cultivation of transgenic cotton

Method used

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  • Application of cell-penetrating peptide TAT2 in transgenic cotton cultivation
  • Application of cell-penetrating peptide TAT2 in transgenic cotton cultivation
  • Application of cell-penetrating peptide TAT2 in transgenic cotton cultivation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example mainly introduces the preparation process of the DNA fragments to be transformed as follows, which mainly involves the extraction of a large number of plasmids (plasmid DNA) and the amplification of linear DNA. The pCAMBIA1300-221 plasmid adopted in the present invention has GUS gene and hygromycin resistance gene, and its structure is as follows figure 1 As shown, the 6510bp part shown in the figure is the linear DNA part.

[0041]According to the conventional genetic engineering operation method, the functional exogenous gene is amplified by PCR, connected with the pCAMBIA1300-221 plasmid or linear DNA, then transformed into Escherichia coli, screened positive strains, amplified and cultured, and finally extracted the plasmid DNA in the positive strains Or linear DNA will do.

[0042] Plasmid extraction (i.e. plasmid DNA, linear DNA, plasmid DNA carrying exogenous DNA fragments or linear DNA carrying exogenous DNA fragments) can be performed using the en...

Embodiment 2

[0060] Using the DNA to be transformed prepared in Example 1 (in order to ensure the versatility of this method, only plasmid DNA and linear DNA were used for verification, and plasmid DNA or linear DNA carrying foreign DNA fragments were not used) to penetrate the membrane When the peptide TAT2 mediates the cultivation of new transgenic cotton, the relevant experimental steps are as follows.

[0061] (1) Preparation of transformation solution

[0062] It is composed of the membrane-penetrating peptide TAT2 and the DNA to be transformed. In terms of mass ratio, the membrane-penetrating peptide TAT2:the DNA to be transformed=1:1;

[0063] In this example, the concentration of DNA is 30 μg / mL, and the concentration of penetrating peptide TAT2 is 30 μg / mL;

[0064] The DNA to be transformed is the plasmid DNA and linear DNA prepared in Example 1.

[0065] (2) After the cotton is fertilized, the transformation solution is injected

[0066] After the cotton has bloomed and ferti...

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PUM

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to the application of cell-penetrating peptide TAT2 in transgenic cotton cultivation. The cell-penetrating peptide TAT2 comprises 18 amino acids, and the sequence is shown in SEQ ID NO.1. After cotton pistil fertilization is finished, DNA to be transformed and cell-penetrating peptide TAT2 solution which are evenly mixed are injected into an ovary through a pollen tube to be used for carrying a DNA fragment to be transformed to a cotton genome. Due to the fact that the cell-penetrating peptide can bring a foreign DNA into a fertilization cell directly and smoothly and the foreign DNA can enter a cell nucleus, genetic transformation efficiency is improved remarkably compared with the traditional pollen tube path way; besides, the method does not depend on a cotton tissue culture regeneration system, operation is convenient, expensive instruments are not needed, production cost is low, and therefore a more effective method worth application and popularization can be provided for cotton variety improvement and fundamental research.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of membrane-penetrating peptide TAT2 in the cultivation of transgenic cotton. Background technique [0002] Cotton is the most important natural textile fiber raw material in the world today, and it is also an important economic crop and strategic reserve in my country. China is not only a big country in textile export and production, but also a big country in textile consumption. However, due to factors such as the reduction of arable land, food security, and the price ratio of grain and cotton in China's cotton production, it is impossible to increase the planting area by a large margin. The only way to increase cotton production and efficiency is to strengthen scientific and technological innovation and improve the technological level and per unit yield level of cotton production. . In addition to cultivated land factors, biological an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8202
Inventor 郝福顺孙立荣李金侗张高峰苗琛
Owner HENAN UNIVERSITY
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