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Infectious clone vector of melon necrotic spot virus and construction method of infectious clone vector

A technology of viral infectivity and cloning vectors, applied in the field of muskmelon virus-melon necrotic spot virus infectious cloning vectors and its construction, can solve problems such as blindness and difficulty in finding attenuated strains, and achieve positive rate improvement, process saving, avoid cumbersome effects

Active Publication Date: 2016-02-03
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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Problems solved by technology

The discovery of weak strains in nature is extremely difficult and blind

Method used

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  • Infectious clone vector of melon necrotic spot virus and construction method of infectious clone vector
  • Infectious clone vector of melon necrotic spot virus and construction method of infectious clone vector
  • Infectious clone vector of melon necrotic spot virus and construction method of infectious clone vector

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Embodiment 1

[0023] An infectious cloning vector for melon necrotic spot virus, such as figure 2 As shown, it was obtained by recombining the whole genome of melon necrotic spot virus with the plant expression vector pCB301-2x35S-MCS-HDVRZ-NOS with 35S promoter and ribozyme Rz. Plant expression vector pCB301-2x35S-MCS-HDVRZ-NOS with 35S promoter and ribozyme Rz such as figure 1 As shown, it has been published (Yao Min, Zhang Tianqi, Tian Zhichao, Wang Yuanchao, Tao Xiaorong. Agrobacterium-mediated CMV invasive clone and 2b deletion mutant construction, Chinese Agricultural Sciences, 2011,44(14):3060-3068) ; The complete gene sequence of the melon necrotic spot virus is shown in SEQ ID NO:08.

[0024] The above-mentioned method for constructing an infectious cloning vector of melon necrotic spot virus comprises the following steps:

[0025] 1. Extract the crude virus liquid from the pathogenic strain infected with melon necrotic spot virus according to the conventional method, and extra...

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Abstract

Disclosed is an infectious clone vector of a melon necrotic spot virus. The infectious clone vector pCB301-MNSV is obtained by recombination of a melon necrotic spot virus whole genome and a plant expression vector pCB301-2x35S-MCS-HDVRZ-NOS with a 35S promoter and ribozyme Rz. The infectious clone vector pCB301-MNSV is transferred into agrobacterium GV3101 competent cells through a freeze-thaw method, and then a melon seedling is inoculated to detect infectivity of the pCB301-MNSV through an agrobacterium injection method. The infectious clone vector is expressed in agrobacterium strongly, is capable of infecting cucurbit plants such as melons, watermelons and cucumbers but cannot infect tobacco, animals and humans, and accordingly relative safety is achieved and studies on viruses can be operated on the DNA (deoxyribonucleic acid) level easily because of infectious cloning success.

Description

technical field [0001] The invention relates to the field of genetic engineering, and relates to a muskmelon virus-melon necrotic spot virus infectious cloning vector and a construction method thereof. Background technique [0002] MNSV belongs to the carnation mottle virus genus ( Carmovir μs ), the virion is spherical, with a diameter of about 30nm, the genome is a positive-sense single-stranded RNA of about 4.3Kb, the 5' end does not contain a cap structure and the 3' end does not contain a poly(A) structure, encoding 5 open reading frames. It is mainly transmitted naturally by seeds, soil fungus, and Cucumber black-headed leaf beetle, and can also be transmitted by mechanical friction inoculation. Its host range is limited to some plants of Cucurbitaceae, such as watermelon, pumpkin, gourd, cucumber and melon, etc., and it is a systemic infection on melon. Symptoms caused on melons include: necrotic spots or chlorotic spots appear on leaves, petioles and stems, dwarfin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N1/21G01N33/569C12Q1/70C12Q1/68
Inventor 吴会杰古勤生刘莉铭
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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