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Plant virus inoculation method

A plant virus and virus inoculation technology, which is applied in the field of plant protection and genetic engineering, can solve the problem that the inoculation method cannot meet the application of large-scale inoculation in the field, and achieves the effects of low cost, simple and convenient use, and high efficiency.

Inactive Publication Date: 2013-06-12
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Aiming at the problem that the existing inoculation methods cannot meet the needs of large-scale inoculation applications in the field, the present invention provides a method for large-scale inoculation of viruses in the field using Agrobacterium, which includes the following steps: transforming the infectious clone of the virus into the agrobacterium Bacillus, after cultivation, the bacteria are collected by centrifugation, and after appropriate dilution, the bacteria are sprayed on the surface of the leaves; Agrobacterium can infect plants through stomata and wounds, thereby bringing the virus into plant cells

Method used

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Examples

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Effect test

Embodiment 1

[0021] Example 1: Expression of exogenous protein by Agrobacterium spraying (taking GFP as an example)

[0022] 1. The virus-infectious clone is transformed into Agrobacterium, and the liquid nitrogen freeze-thaw method is adopted.

[0023] Take out 1 tube of Agrobacterium GV3101 competent cells (100-200 μl) from the -80°C refrigerator and thaw on ice. After thawing, add 100-300ng virus-infectious clone pCamTVBMV-GFP. Freeze in liquid nitrogen for 1-5 minutes. Immediately after taking it out, put it in a 37°C water bath for 5 minutes.

[0024] Add 800 μl LB culture medium without antibiotics, and incubate on a shaker at 28°C for 3h. Collect the bacteria by centrifugation at 5000r / min, keep about 200 μl of culture solution in the centrifuge tube and resuspend the bacteria.

[0025] Spread the bacteria evenly on the solid medium plate containing the corresponding antibiotics on the ultra-clean bench, and incubate at 28°C for about 2 days until colonies appear. Colonies were...

Embodiment 2

[0032] Embodiment 2: the attenuated strain of virus by Agrobacterium spray inoculation

[0033] 1. The virus-infectious clone is transformed into Agrobacterium, and the liquid nitrogen freeze-thaw method is adopted.

[0034] Take out 1 tube of Agrobacterium GV3101 competent cells (100-200 μl) from the -80°C refrigerator and thaw on ice. After thawing, add 100-300ng of the plasmid of TVBMV attenuated strain D198K. Freeze in liquid nitrogen for 1-5 minutes. Immediately after taking it out, put it in a 37°C water bath for 5 minutes.

[0035] Add 800 μl LB culture medium without antibiotics, and incubate on a shaker at 28°C for 3h.

[0036] Collect the bacteria by centrifugation at 5000r / min, keep about 200 μl of culture solution in the centrifuge tube and resuspend the bacteria.

[0037] Spread the bacteria evenly on the solid medium plate containing the corresponding antibiotics on the ultra-clean bench, and incubate at 28°C for about 2 days until colonies appear. Colonies ...

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Abstract

The invention provides a novel plant virus inoculation method, and in particular provides a field large-scale virus inoculation method by using a bacterium, mainly targeting at infectious clone of a plant virus which can be replicated in agrobacterium. The principle of the method is as follows: infectious clone of the plant virus is transformed into agrobacterium, and the virus can be brought into a plant cell by using the characteristic of natural wound and wound plant infection of the agrobacterium. The method is simple to operate, low in cost, high in efficiency, and suitable for field large-scale application, and can be used for the inoculation of a weak virus strain of the virus to protect a plant from the infection by a strong virus strain, and heterologous protein with high added value can be expressed in the plant using a virus vector.

Description

Technical field [0001] The present invention belongs to the field of plant protection and genetic engineering, and provides a new method of plant virus vaccination. Specifically, it is a method that uses a large -scale vaccination of viruses in the field in the field. Background technique [0002] Through genetic engineering means, the agglomerate CDNA cloning of plant virus can be constructed.Using the infected CDNA cloning, you can study the pathogenic mechanism of the virus, and then the cloning can be transformed into a weak toxic vaccine, which can be protected by cross -protection.The cloned transformation is an expression carrier to produce high -value exogenous proteins such as animal vaccine and antiviral protein.At present, there are more and more weak strains and expression vectors developed and developed. [0003] The bottleneck of limiting the use of weak strains and the application of the expression carrier is large -scale vaccination technology.According to the dif...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 李向东高瑞丛倩倩许斐斐郭兆奎李现道竺晓平
Owner SHANDONG AGRICULTURAL UNIVERSITY
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