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Infectious clone of grape berry necrosis virus and its construction method

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

Active Publication Date: 2022-01-21
FRUIT TREE INST OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • 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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  • Infectious clone of grape berry necrosis virus and its construction method
  • Infectious clone of grape berry necrosis virus and its construction method
  • Infectious clone of grape berry necrosis virus and its construction method

Examples

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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 infective clone of grape berry necrosis virus and a construction method thereof. The invasive cloning vector is a seamless cloning method of segmentally amplified grape berry necrosis virus genome fragments connected to pCB301‑2x35S‑HDV containing double 35S promoters and ribozyme HDV‑RZ RZ ‑NOS carrier. The vector was transformed into Agrobacterium EHA105 competent cells by freeze-thaw method, and its infectivity was verified by inoculating Western tobacco and grapes with Agrobacterium. The GINV invasive clone constructed by the invention can be stably and highly expressed in the Agrobacterium EHA105 strain, and can successfully infect western tobacco and grape hosts. The acquisition of GINV invasive clones makes the research of GINV easy to operate at the DNA level and provides a powerful tool for subsequent research on the 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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Patent Type & Authority Patents(China)
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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