Method for preparing broad-spectrum plant virus-resistant transgenic plant and application

A technology for anti-plant virus and transgenic plants, applied in the field of preparation of transgenic plants

Inactive Publication Date: 2012-06-13
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for preparing broad-spectrum plant virus-resistant transgenic plant and application
  • Method for preparing broad-spectrum plant virus-resistant transgenic plant and application
  • Method for preparing broad-spectrum plant virus-resistant transgenic plant and application

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Embodiment 1

[0031] A method for preparing a transgenic plant with broad-spectrum resistance to plant viruses, the steps of which are:

[0032] .Construction of transformation vector and plant transformation:

[0033] Construction of the transformation vector (construction of the transformation vector and plant transformation) primers were designed according to the aiiA gene sequence of Bacillus thuringiensis (B. In addition to Bgl II and BstE II restriction sites and protective bases, the total DNA of Bacillus thuringiensis (isolated by our laboratory) was extracted, and the aiiA gene was cloned by PCR (Wang et al., Science in China Series C: Life Sciences, 2007, 50 (3): 385-39), after double digestion, it was connected to the expression vector pA8 (purchased from CAMBIA company pCAMBIA 1305.1 vector, referred to as pA8 vector) after double digestion with Bgl II and BstE II , named pA8-aiiA plasmid. The empty vector pA8 and the constructed pA8K-aiiA plasmid were respectively transformed...

Embodiment 2

[0037] An application of a transgenic plant with broad-spectrum resistance to plant viruses in tobacco, the application process is as follows:

[0038] Resistance of transformed tobacco to different plant viruses:

[0039] When the regenerated tobacco plants had 4-5 leaves, the selection PCR and RT-PCR tests were all positive ( figure 1 ) with the same size as the test plants. Add appropriate amount of buffer (K 2 HPO 4 1g / 100mL, Na 2 SO 3 0.1g / 100mL) and an appropriate amount of quartz sand are ground into a homogenate, and the virus juice is dipped with a brush to inoculate different plant viruses on the surface of the plant leaves by friction inoculation, including tobacco mosaic virus (TMV), potato X and Y virus ( PVX and PVY), GFP-labeled TMV virus (GFP-TMV for short) and Plum pox potyvirus virus (GFP-PPV for short) (wherein TMV, PVX, and PVY viruses are preserved by the applicant's unit - Wuhan Institute of Virology, Chinese Academy of Sciences Provided by the ce...

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Abstract

The invention discloses a method for preparing a broad-spectrum plant virus-resistant transgenic plant and application. The method comprises the following steps of: a, constructing a transformation vector, namely performing restriction enzyme digestion on an aiiA gene of Bacillus thuringiensis by using restriction enzyme, connecting the gene to a plant binary transformation vector pA8, and transferring the connected plant binary transformation vector pA8-aiiA into a strain EHA105 of Agrobacterium tumefaciens by a freeze-thawing method; and b, performing genetic transformation of plants, namely transforming selenium tobacco and Nicotiana benthamiana respectively by a leaf disc transformation method, screening on a Murashige and Skoog (MS) culture medium containing hygromycin to obtain transformed seedlings, and obtaining intact regenerated plants through a rooting culture medium, wherein the regenerated plants comprise a pA8 regenerated plant for transforming an empty vector and a regenerated plant for transforming the pA8-aiiA. The method is feasible and easy to operate, and can be widely applied to the control of viral diseases of economical crops because the broad-spectrum resistance to different plant viruses is strengthened.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for preparing transgenic plants with broad-spectrum resistance to plant viruses. At the same time, it also relates to the application of a transgenic plant with broad-spectrum resistance to plant viruses. Specifically, the present invention relates to the transformation of the gene aiiA encoding the quorum-sensing signal molecule degradation protein in Bacillus thuringiensis to obtain a transgene with broad-spectrum resistance to plant viruses by transforming tobacco with Agrobacterium Tobacco has good antiviral effects on different types of important plant viruses such as tobacco mosaic virus, potato virus X, and potato virus Y. The technical method of the invention can be widely applied to the prevention and treatment of viral diseases of economic crops. Background technique [0002] In nature, many plants are infected by viruses and cause viral diseases. ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/31A01H4/00A01H5/00
Inventor 陈士云张勇杨宝玉
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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