A gene gun-mediated genetic transformation method of Tripterygium wilfordii

A technology of gene gun method and genetic transformation, applied to other methods of inserting foreign genetic materials, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems that have not been reported

Active Publication Date: 2020-08-25
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, transgenic plants or cells of tobacco, Arabidopsis, soybean, etc. have been obtained by applying the gene gun method, but there is no report in Tripterygium wilfordii

Method used

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  • A gene gun-mediated genetic transformation method of Tripterygium wilfordii
  • A gene gun-mediated genetic transformation method of Tripterygium wilfordii
  • A gene gun-mediated genetic transformation method of Tripterygium wilfordii

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Experimental program
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Embodiment 1

[0063] Embodiment 1, the optimization of the genetic transformation method of Tripterygium wilfordii mediated by gene gun

[0064] 1. Tripterygium wilfordii suspension cell culture

[0065] Cut the new leaves and stems of Tripterygium wilfordii, clean them, disinfect them, wash them several times with sterile water, cut the young leaves into small pieces of about 1.0cm×1.0cm, cut the young stems into small pieces of about 1.0cm long, and inoculate in the 1.0mg·L -1 On 2,4-D MS solid medium (25°C, cultured in the dark), after about half a month of culture, the callus began to grow at the incision of the explant, and the callus with white luster, loose texture and good growth was selected. Injured tissue, transferred to a solution containing 0.5mg·L -1 2,4-D+0.1mg·L -1 KT+0.5mg·L -1 IBA was subcultured on MS solid medium (25°C, dark culture).

[0066] The callus of Tripterygium wilfordii has been continuously subcultured for more than 3 generations, and the calli with good...

Embodiment 2

[0116]Example 2, application of gene gun-mediated genetic transformation of tripterygium wilfordii

[0117] 1. Construction of TwFPS1 overexpression vector

[0118] 1. Extract the total RNA of Tripterygium wilfordii and reverse transcribe it into cDNA.

[0119] 2. Use the cDNA obtained in step 1 as a template, and perform PCR amplification with primers FPS1-att-F and primers FPS1-att-R to obtain the amplified product.

[0120] FPS1-att-F: 5′-CACCATGAGCGACACCAAGTCCAAGT-3′;

[0121] FPS1-att-R: 5'-CTACTTCTCTCGCTTGTATATT-3'.

[0122] 3. Through the BP reaction, import the amplified product obtained in step 2 into the vector pENTR / SD / D-TOPO to obtain a positive entry clone plasmid containing the double-stranded DNA molecule shown in Sequence 1 of the Sequence Listing (sequencing verification).

[0123] BP reaction system: 0.5 μl of the amplification product obtained in step 2, 2 μl of Salt Solution, and 0.5 μl of vector pENTR / SD / D-TOPO.

[0124] BP reaction conditions: react a...

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Abstract

The invention discloses a gene gun mediated tripterygium wilfordii genetic transformation method. The gene gun mediated tripterygium wilfordii genetic transformation method disclosed by the inventioncomprises the following step: utilizing a gene gun method to guide plasmid containing target genes into tripterygium wilfordii suspension cells, wherein in the gene gun method, gene gun bombardment parameters are arranged as following: a DNA concentration is 1 to 3 mug / mul, bombardment times is 2 to 3, the target distance is 3 to 6cm, bombardment pressure is 1100 to 1350psi, and a vacuum degree is26 to 28mmHg; in the gene gun method, co-culture time is 36 to 48h after gene gun bombardment is finished; in the gene gun method, a utilized precipitator is calcium chloride or spermidine when a microcarrier and the plasmid containing the target genes are utilized to prepare an embedded microcarrier. The method disclosed by the invention can improve a tripterygium wilfordii genetic transformation rate and has significance in researching tripterygium wilfordii genetic transformation.

Description

technical field [0001] The invention relates to a genetic transformation method of Tripterygium wilfordii mediated by a gene gun. Background technique [0002] Tripterygium wilfordii Hook.f. is a plant of Euonymus family. It is a woody vine, also known as chrysanthemum wilfordii Hook.f., which is mainly distributed in the south and southwest of the Yangtze River Basin in my country. As a traditional Chinese medicine, it tastes pungent, bitter, and cold in nature. It has the functions of promoting blood circulation and removing blood stasis, clearing away heat and detoxification, reducing swelling and dissipating stagnation, killing insects and stopping bleeding. Clinically, it is widely used in the treatment of autoimmune diseases such as rheumatoid arthritis, glomerulonephritis, lupus erythematosus and various skin diseases, and has become one of the hot research natural medicines at home and abroad. [0003] So far, most of the natural compounds isolated and identified fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/89C12N15/29C07K14/415A01H5/00A01H6/00
CPCC07K14/415C12N15/8243C12N15/895
Inventor 黄璐琦高伟赵瑜君郭娟
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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