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Method for structuring Chinese wheat mosaic virus infectivity clone and application of method

A technology of wheat mosaic virus and virus genome, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of hindering replication, understanding of pathogenic mechanism of assembly, and limiting identification of wheat resistance, etc. problem, to achieve the effect that is beneficial to wheat resistance and screening

Pending Publication Date: 2016-05-04
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

The full-length cDNA cloning of the virus is an important technical means for in-depth understanding of the biological characteristics of the virus. The lack of CWMV invasive clones not only seriously hinders people's understanding of the mechanisms of CWMV infection, replication, assembly, and pathogenicity, but also limits the experimental Identification of resistance in indoor wheat
Since the complete genome sequence of CWMV was determined in our laboratory, we have been trying to construct its full-length cDNA invasive clone, but all failed

Method used

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  • Method for structuring Chinese wheat mosaic virus infectivity clone and application of method
  • Method for structuring Chinese wheat mosaic virus infectivity clone and application of method
  • Method for structuring Chinese wheat mosaic virus infectivity clone and application of method

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Embodiment

[0021] Embodiment: A simple and fast method for constructing Chinese wheat mosaic virus invasive clone and its infection activity analysis to wheat, specific steps:

[0022] Primer design: A total of 4 primers were designed based on the RNA1 and RNA2 sequences of the Chinese wheat mosaic virus genome fragments determined by our laboratory. for amplifying RNA2;

[0023] Total RNA extraction of virus genome: take the extract containing 50-100 μl of CWMV particles, use Trizol reagent (Invitrogen) to extract total RNA of virus genome according to the reagent instructions, transfer the virus extract into a 1.5ml new centrifuge tube, add 1ml Trizol reagent (Invitrogen), After shaking and mixing, add 0.2ml chloroform, let stand for 3-5min after mixing, centrifuge at 12000g for 10min, take the supernatant to a new centrifuge tube and add an equal volume of isopropanol, mix and centrifuge at 12000g for 10min at 4°C, discard The supernatant and the pellet were washed twice with 70% eth...

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Abstract

The invention relates to a method for easily and quickly structuring a Chinese wheat mosaic virus (CWMV) whole-length cDNA infectivity clone. One-step method RT-PCR amplification is carried out with a purified viral genome RNA as a template by applying one set of viral genome specific primers, wherein the number of the primers is four, and the primers are P1F, P1R, P2F and P2R respectively; an amplification product is purified and then connected to a carrier. It is shown through biological activity analysis that the CWMV whole-length cDNA clone obtained through the method can successfully infect wheat and other plants and can be replicated, subjected to system movement and assembled in plant bodies, and thus it is indicated that the clone has the infection activity and can be used for research of related virus function genomes and analysis of plant disease resistance.

Description

technical field [0001] The invention relates to a method for constructing a full-length cDNA infectious clone of an important crop virus and its application field, in particular to a method for constructing an infectious clone of Chinese wheat mosaic virus and its application. Background technique [0002] Soil-borne viral diseases of cereal crops occur widely in the world and have been difficult to control. At the end of the last century, our laboratory first discovered a soil-borne virus that caused wheat mosaic virus in Shandong, my country. Molecular identification showed that the soil-borne virus was a new species of plant virus, named Chinese wheat mosaic virus (Chinese wheat mosaic virus, CWMV). The new species of the virus has been classified into the genus Furovirus of the family Virgaviridae. CWMV can be transmitted by the obligate root-parasitic Polymyxagraminis and can survive for several years in the dormant spores of the fungal vector. Therefore, it is difficu...

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Application Information

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IPC IPC(8): C12N15/82C12N15/66
CPCC12N15/8203C12N15/66
Inventor 张恒木羊健张芬李静陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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