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Construction method of pepper veinal mottle virus (PVMV) infectious full-length cDNA clone

A full-length, single technology, applied in the field of plant genetic engineering, can solve problems such as genetic manipulation difficulties, restricting the relationship between RNA viruses and hosts, and research on pathogenic mechanisms, achieving low-cost effects

Active Publication Date: 2019-11-08
HAINAN UNIVERSITY
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  • Application Information

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Problems solved by technology

However, for RNA viruses, it is quite difficult to genetically manipulate them at the RNA level, which largely restricts the relationship between RNA viruses and their hosts and the study of pathogenic mechanisms.

Method used

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  • Construction method of pepper veinal mottle virus (PVMV) infectious full-length cDNA clone
  • Construction method of pepper veinal mottle virus (PVMV) infectious full-length cDNA clone
  • Construction method of pepper veinal mottle virus (PVMV) infectious full-length cDNA clone

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Experimental program
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Embodiment 1

[0032]Example 1. Construction and biological activity detection of PVMV infective full-length cDNA clone

[0033] 1. Materials and methods

[0034] 1. Test materials

[0035] Plants to be tested: field yellow lantern pepper plants with PVMV virus, healthy yellow lantern pepper and Nicotiana benthamiana inoculated in the laboratory.

[0036] Main reagents: Polysaccharides&Polyphenolics-rich RNAprep Pure Kit (Polysaccharides&Polyphenolics-rich RNAprep Pure), Rapid Plasmid Small Extraction Kit and 2×Taq PCR Mastermix are all products of Tiangen Biotechnology (Beijing) Company; restriction enzymes are NEB company (NEW ENGLAND Biolabs) products; RT-PCR amplification reagents and T4DNA ligase mainly from Thermo Fisher Technology (China) Co., Ltd. (Thermo Fisher); CyclePure Kit purification kit and Gel Extraction Kit purification reagents The box is a product of OMEGA; the clone competent cell Trans5αChemically Competent Cell comes from Beijing Quanshijin Biotechnology Co., Ltd., a...

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Abstract

The invention discloses a method for constructing a pepper veinal mottle virus (PVMV) infectious full-length cDNA clone. The method comprises the following steps: using degeneracy of codons, replacinga 2363th A of a full-length cDNA sequence corresponding to genomic RNA of pepper veinal mottle virus with C, and obtaining SEQ ID No.1; according to recognition sites for two single restriction endonucleases, AatII and BamH I, dividing the SEQ ID No.1 into three segments; and preparing and obtaining the three fragments and cloning into a same plant expression vector to obtain a recombinant expression vector of the SEQ ID No.1, that is, the pepper veinal mottle virus (PVMV) infectious full-length cDNA clone. The method can construct the PVMV infective clone quickly and efficiently at low costand provides a theoretical basis for prevention and treatment of related diseases in the future.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for constructing PVMV infective full-length cDNA clones. Background technique [0002] At present, the relationship between virus and host and its pathogenic mechanism are hot topics in the field of virology research, and the construction of infectious clones of viruses is a powerful tool for research. Infectious clones refer to cDNA clones of viruses that are infectious or in vitro transcripts that are infectious (Boyer et al., 1994). Since molecular cloning is based on the level of DNA, the infectious clones of DNA viruses were first prepared. However, for RNA viruses, it is quite difficult to genetically manipulate them at the RNA level, which largely restricts the research on the relationship between RNA viruses and their hosts and the pathogenic mechanism. Until the early 1990s, the discovery and commercialization of reverse transcriptase facilitated our r...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/82C12N1/21C12R1/01
CPCC07K14/055C12N15/8201C12N2770/34022
Inventor 崔红光胡巍耀秦丽杨柯
Owner HAINAN UNIVERSITY
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