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Infectious cloning recombinant vector of melon aphid-borne yellowing virus

A recombinant vector, yellow virus technology, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of restricting pathogenicity and host disease resistance research, incapable of mechanical inoculation, etc., to improve reliability, Guaranteed uniqueness and stability

Active Publication Date: 2022-07-22
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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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 cloning recombinant vector of melon aphid-borne yellowing virus
  • Infectious cloning recombinant vector of melon aphid-borne yellowing virus
  • Infectious cloning recombinant vector of melon aphid-borne yellowing virus

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Embodiment Construction

[0055] A method for constructing a muskmelon aphid-borne yellowing virus infectious cloning recombinant vector, comprising the following steps:

[0056] (1) Extraction of plant total RNA

[0057] Extract total plant RNA from gourd leaves infected with melon aphid-borne yellowing virus, and the specific operation refers to the instructions of RNAsimple total RNA extraction kit (TIANGEN, DP419);

[0058] (2) cDNA synthesis

[0059] The plant total RNA extracted in (1) was used as the template and Random Primer was used as the primer to synthesize cDNA, as follows: firstly 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 5 min and then place it on ice immediately; then add 4 μL 5×PrimeScript II RT Buffer, 1 μL PrimeScript II Reverse Transcriptase (200U / μL), 0.5 μL RNase Inhibitor (40U / μL) to the system ) and 4.5μL RNase free dH 2 O, mix well, incubate the system at 30 °C for 10 min, 42 ...

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Abstract

The invention provides an infectious cloning recombinant vector of melon aphid-borne yellowing virus, and relates to the field of biology. The recombinant vector contains the full-length genome sequence of MABYV virus, and the MABYV virus can be a virus that can infect gourd, melon, watermelon and / or cucumber, such as, the clone of the full-length genome sequence is such as SEQ ID NO. 1 shown. The present invention overcomes the deficiencies of the prior art, and finds that it is difficult to clone the MABYV genome gene into a plasmid in one step, and the inventor succeeds in designing a strategy combining homologous recombination and site-directed insertion; Plants for several weeks are eventually identified as capable of infecting the plant, causing it to become ill. The invention can be used for the related research on the pathogenicity of the melon aphid-borne yellowing virus, and can also be used for the resistance identification of the melon crop melon aphid-borne yellowing virus, which promotes the research on the virus-related pathogenic mechanism and the melon disease resistance mechanism.

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 mechanically inoculated, and can only be transmitted by aphids in a persistent, non-proliferative manner, which greatly limits their pathogenicity and host resistance. Disease research. SUMMARY OF THE INVENTION [0003] The present invention overcomes the deficiencies of the prior art, and finds that it is difficult to clone the full-length sequence of the MABYV genome into a plasmid in one step, and the inventor succeeds in designing a strategy combining homologous recombination and site-directed insertion; 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 cause disease. [0004] In the first aspect, the present invention provides ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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