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Method for obtaining dunaliella salina CHYB gene-modified tobacco

A Dunaliella, tobacco technology, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement and other directions, can solve the problems of large research difficulties, long cycle, limited effect, etc., to achieve enhanced salt tolerance, proline content Increase, little effect of relative conductivity change

Inactive Publication Date: 2015-09-30
NINGBO UNIV
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Problems solved by technology

Research on improving plant salt tolerance through traditional breeding methods is difficult, takes a long time, and has limited effects

Method used

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  • Method for obtaining dunaliella salina CHYB gene-modified tobacco
  • Method for obtaining dunaliella salina CHYB gene-modified tobacco
  • Method for obtaining dunaliella salina CHYB gene-modified tobacco

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Embodiment 1

[0028] Plant material and strain plasmids

[0029] The seeds of Nicotiana Tabacum L. were donated by Dr. Chaoyang Hu from Shanghai Jiaotong University, and the aseptic seedlings were cultivated in this laboratory. Soak tobacco seeds in 2.5% sodium hypochlorite solution for 5 minutes, wash them once with sterile water, then soak them with 75% ethanol for 1 minute, wash them with sterile water three times, inoculate them on MS medium, and culture them at 25°C for 35 days to obtain sterile seedlings .

[0030] The pMD-19T cloning vector, DH5α competent cells, cDNA synthesis kits, PCR reagents and restriction enzymes were all purchased from Dalian TAKARA Company; the plant expression vectors of Agrobacterium tumefaciens LBA4404 and pCAMBIA3301 were preserved by our laboratory ; The plant RNA extraction kit was purchased from Omega Corporation of the United States; the primer synthesis and DNA sequencing were completed by Shanghai Sangon Bioengineering Co., Ltd.; the rest of the r...

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Abstract

The invention discloses a method for obtaining a dunaliella salina CHYB gene-modified tobacco. According to the method, firstly, tobacco seeds are dipped in the sodium hypochlorite solution and then are cleaned in sterile water. Secondly, the tobacco seeds are dipped in ethyl alcohol, then flushed in sterile water, inoculated in an Murashige and Skoog medium and cultivated to obtain aseptic seedlings. After that, the construction of a plant expression vector Pcambia3301-chyb and the genetic transformation of agrobacterium tumefaciens are conducted. Finally, the transformation of tobacco is conducted based on the leaf-mediated method. According to the method, the dunaliella salina CHYB gene is successfully transferred into tobacco through the agrobacterium tumefaciens-mediated leaf-mediated process. In this way, obtained transgenic tobacco plants are higher in height, leaf area and chlorophyll content compared with plants in a control group. After the stress treatment in 200 mmol / L NaCl, the chyb-modified transgenic tobacco can still survive and grow. Meanwhile, the relative electrical conductivity of tobacco leaves changes slightly, and the proline content in tobacco leaves increases. The salt tolerance of the chyb-modified transgenic tobacco is improved.

Description

technical field [0001] The invention belongs to the field of tobacco cultivation, in particular to a method for obtaining Dunaliella salina CHYB gene tobacco. Background technique [0002] Soil salinization is a worldwide problem and one of the main factors causing crop yield decline. my country's saline-alkali land is widely distributed and has many types. According to statistics, the total area exceeds 3.6×10 7 hm 2 (Yang Jinsong, 2008). When plants are under salt stress, they will increase their salt resistance by inducing the expression of related genes or accumulating osmotic adjustment substances. Excessive salinity will inhibit the metabolism and growth of plants or even die. The study of improving plant salt tolerance through traditional breeding methods is difficult, takes a long time, and has limited effects. At present, scholars at home and abroad have studied the screening, cloning and transformation of salt-tolerant genes in saline-alkali-tolerant plants (Ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 许峰龚一富周静马颖瑞刘浩王何瑜
Owner NINGBO UNIV
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