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Construction method for CCYV infectious vector

The invention relates to a technology of a chlorotic yellow virus and a construction method, which is applied in the field of construction of an infective vector of melon chloro yellow virus, can solve the problems of restricting the research of virus gene function, incapable of mechanical friction inoculation, etc., and achieves poor stability and replication. The effect of enhanced quantity and small vector genome

Inactive Publication Date: 2015-04-29
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

Like other viruses of the Trichovirus genus, CCYV is a vascular-transmitted virus and cannot be inoculated by mechanical friction, which greatly limits the study of the gene function of the virus

Method used

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  • Construction method for CCYV infectious vector

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Embodiment Construction

[0032] The invention uses the melon chlorosis and yellowing virus as the material to construct the infectious vector, and the melon chlorotic and yellowing virus genome has two components, which are RNA1 and RNA2 respectively. In the present invention, two fragments of RNA1 and two fragments of RNA2 are sequentially constructed on the plant expression vector pCB301M, and the virus genome enters the plant cells through the method of Agrobacterium infiltration and begins to replicate automatically, thereby realizing efficient infection of plants by the virus.

[0033] In June 2012, cucumber leaves were picked in the greenhouse of Zhengzhou Vegetable Research Institute, Henan Province.

[0034] Construction of Infectious Vectors of Cucurbit Chlorosis Virus RNA1 and RNA2

[0035] a Extraction of plant total RNA

[0036] (1) Take 0.1 g of fresh leaves from the cucumber diseased plants preserved in our laboratory, add liquid nitrogen and grind them into powder, transfer them into a...

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Abstract

The invention relates to a construction method for a CCYV (Cucurbit Chlorotic Yellows Virus) infectious vector. The construction method comprises the steps as follows: construction for infectious cloning is performed on RNA 1 and RNA 2 components of a genome by adopting the CCYV as material, the 8.6 Kb RNA 1 and the 8 Kb RNA 2 are constructed to an efficient plant expression vector pCB301M respectively, and then recombinant vectors are converted to enter an agrobacterium strain with a calcium chloride conversion method, so that an agrobacterium mediated virus vector which has high infection on the host plant is obtained. According to the invention, a mature method and a mature system are provided for interaction research between the structure and functions of the viral genome and between the structure of the viral genome and the host and for research on expression of heterologous protein and functions of the plant genome.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing an infectious vector of melon chlorosis and yellowing virus. Background technique [0002] Cucurbit chlorotic yellow virus ( Cucurbit chlorotic yellows virus, CCYV) belongs to the Cycloviridae ( Closteroviridae ), Trichovirus ( Crinivirus ), the virion is linear, the length is between 650-900 nm, the genome is composed of two positive-sense single-stranded RNAs, namely RNA1 and RAN2, and the RNA1 genome is about 8.6 Kb, encoding 4 open reading frames, which are A base transferase (MTR), RNA-dependent RNA polymerase (RdRp), P6 and P22, the RNA2 genome is about 8 Kb, encoding 8 open reading frames, among which heat shock protein homologue (Hsp70h), P59, coat protein (CP ), repeated coat protein (CPm) and P26 are relatively conserved within the genus. Like other viruses of the genus Trichovirus, CCYV is a vascular-transmitted virus that cannot be i...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/66
Inventor 施艳王振跃孙虎孙炳剑潘正茂范志业
Owner HENAN AGRICULTURAL UNIVERSITY
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