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Grape berry necrosis virus infectious clone and construction method thereof

A technology of necrotic virus and construction method, applied in the direction of virus, viral peptide, virus/bacteriophage, etc., can solve the problem of unproven direct relationship between GINV and achieve high-efficiency expression

Active Publication Date: 2019-01-25
FRUIT TREE INST OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the construction of an infectious full-length cDNA clone of a hairyvirus that infects grapes, and the direct relationship between GINV and the occurrence of the disease has not yet been proved

Method used

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  • Grape berry necrosis virus infectious clone and construction method thereof
  • Grape berry necrosis virus infectious clone and construction method thereof
  • Grape berry necrosis virus infectious clone and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1, Construction of Infectious Clones of Necrosis Virus in Grape Berries and Verification of Infectiousness

[0042] 1. Construction of Infectious Clones of Necrosis Virus in Grape Berries

[0043] 1. Synthesis of cDNA

[0044] (1) Extraction of RNA

[0045] Gather the Beida grape leaf that infects grape berry necrosis virus (GINV), carry out RNA extraction according to the literature: the people such as Fan Xudong, Phytopathology Acta, 2014,44(5):455-460 The column type extraction method that reports, and by The concentration of RNA was measured by ultra-micro-volume ultraviolet-visible spectrophotometer (SDS-11).

[0046] (2) cDNA synthesis by reverse transcription

[0047] Reverse transcription was carried out in a 1.5mL sterilized centrifuge tube, and 7.0μL of sterilized pure water, oligodT 18 (Synthesized by Shanghai Sangon Bioengineering Technology Service Company) 1.0 μL, total RNA 2.0 μL, centrifuge and mix well, bathe in 95°C water for 5 min, place o...

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Abstract

The invention discloses an infectious clone of grape berry necrosis virus and a construction method thereof. The infectious cloning vector is obtained by connecting segmented amplified grape berry necrosis virus (GBNV) genomic fragment to pCB301-2x35S-HDVRZ-NOS carrier containing double 35S promoter and ribozyme HDV-RZ in a seamlessly clone. The vector was transfected into competent cells of Agrobacterium tumefaciens EHA105 by freeze-thaw method, and its infectivity was tested by inoculating western tobacco and grape. The GINV infectious clone constructed by the invention can be stably and efficiently expressed in Agrobacterium tumefaciens EHA105 strain, and can successfully infect western tobacco and grape hosts. The cloning of GINV infectious clones makes the study of GINV easy to operate at DNA level, and provides a powerful tool for the further study of biological characteristics and pathogenicity of GINV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an infectious clone of necrosis virus in grape berries and a construction method thereof. Background technique [0002] In 1984, necrosis in grape berries (called mosaic disease at the earliest) was discovered for the first time on Kyoho grapes in Japan. The disease caused serious harm to the vineyards in Yamanashi Prefecture, Japan. Some sensitive varieties (such as Kyoho, Pioneer, Lichuan Seedless, and Kangbaier Zaosheng) are infected with the disease, the plant growth is weakened, the germination is delayed in spring, and the necrosis inside the stem, short nodes, mosaic leaves, small fruit grains, and discoloration outside the fruit appear. and fruit necrosis. After the diseased plants were rubbed inoculated with Quinoa, a linear virus with a size of 740×12nm was found and named as Grapevine berry inner necrosis virus (GINV). GINV is one of the members of the Tricho...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/34C12N1/21C12R1/01
CPCC07K14/005C12N2710/00021
Inventor 范旭东董雅凤张尊平任芳胡国君
Owner FRUIT TREE INST OF CHINESE ACAD OF AGRI SCI
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