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Infectious clone recombinant vector of melon aphid-borne yellows virus

A technology of recombinant vectors and yellowing viruses, applied in the direction of viruses/bacteriophages, viruses, viral peptides, etc., can solve the problems of mechanical inoculation, limitation of pathogenicity and host disease resistance research, etc., to improve credibility, Guarantee the effect of uniqueness and stability

Active Publication Date: 2021-03-16
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Application Information

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Problems solved by technology

[0002] Melon aphid-borne yellowing virus (MABYV) is mainly confined to the phloem cells of the host, cannot be inoculated by mechanical means, and can only be transmitted by aphids in a persistent, non-proliferative manner, which greatly limits their pathogenicity and host resistance. research on disease

Method used

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  • Infectious clone recombinant vector of melon aphid-borne yellows virus
  • Infectious clone recombinant vector of melon aphid-borne yellows virus
  • Infectious clone recombinant vector of melon aphid-borne yellows virus

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Embodiment Construction

[0055] A method for constructing an infectious cloning recombinant vector of a melon aphid-transmitted yellowing virus, comprising the following steps:

[0056] (1) Extraction of plant total RNA

[0057] Extract plant total RNA from gourd leaves infected with melon aphid-borne yellowing virus. For specific operations, refer to the instructions of the RNAsimple Total RNA Extraction Kit (TIANGEN, DP419);

[0058] (2) Synthesis of cDNA

[0059] Use the total plant RNA extracted in (1) as a template and Random Primer as a primer to synthesize cDNA, as follows: first add 1 μL Random Primer (50 μmol / L), 1 μL dNTP (10 mmol / L each), 1 μL RNA and 7 μL RNase free dH 2 O, mix well, place the system at 65°C for 5min and place it on ice immediately; then add 4μL 5×PrimeScriptII RT Buffer, 1μL PrimeScript II Reverse Transcriptase (200U / μL), 0.5μL RNase Inhibitor (40U / μL) to the system ) and 4.5 μL RNase free dH 2 O, mix well, put the system at 30°C for 10min, 42°C for 1h, and 70°C for 1...

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Abstract

The invention provides an infectious clone recombinant vector of melon aphid-borne yellows virus, and relates to the field of biology. The recombinant vector contains a full-length genome sequence ofan MABYV virus, the MABYV virus can be a virus capable of infecting bottle gourds, melons, watermelons and / or cucumbers, for example, and as shown in SEQ ID NO.1, clone of the full-length genome sequence is shown in SEQ ID NO.1. The invention overcomes the defects in the prior art, and finds that the MABYV genome gene is difficult to clone to plasmids in one step, and the inventor designs a strategy of combining homologous recombination and fixed-point insertion to succeed. The clone virus genome vector of the invention is used to infect a plant, the plant is finally identified to be infectedand attacked by observing for several weeks. The recombinant vector of the invention can be used for related research on pathogenicity of melon aphid-borne yellows virus, and can also be used for identifying resistance of melon aphid-borne yellows virus of melon crops, so that research on related pathogenesis to the virus and disease resistance mechanism of melons is promoted.

Description

technical field [0001] The present invention relates to the field of biology. Background technique [0002] Melon aphid-borne yellowing virus (MABYV) is mainly confined to the phloem cells of the host, cannot be inoculated by mechanical means, and can only be transmitted by aphids in a persistent, non-proliferative manner, which greatly limits their pathogenicity and host resistance. disease research. Contents of the invention [0003] The present invention overcomes the deficiencies of the prior art, and finds that: the full-length sequence of the MABYV genome is difficult to clone into a plasmid in one step, and the inventor designed a strategy combining homologous recombination and site-directed insertion to succeed; after the viral genome vector of the present invention is introduced into a plant, The experiment continued for several weeks before it was identified that it could infect plants and make them diseased. [0004] In the first aspect, the present invention ...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/40C12N15/64A01H5/00A01H6/34
CPCC07K14/005C12N15/8203C12N15/8283C12N2770/00022
Inventor 刘莉铭古勤生彭斌康保珊吴会杰
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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