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A kind of construction method of pvmv infective 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, constraints on the relationship between RNA viruses and hosts, and research on pathogenic mechanisms

Active Publication Date: 2021-05-11
HAINAN UNIV
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  • Abstract
  • Description
  • Claims
  • 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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  • A kind of construction method of pvmv infective full-length cDNA clone
  • A kind of construction method of pvmv infective full-length cDNA clone
  • A kind of construction method of pvmv infective full-length cDNA clone

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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 PVMV infective full-length cDNA clone. The method of the present invention comprises the following steps: using the degeneracy of codons, replacing A at position 2363 of the full-length cDNA sequence corresponding to the genomic RNA of sweet pepper vein mottle virus with C to obtain SEQ ID No.1; according to SEQ ID No. The recognition sites of two single restriction endonucleases AatII and BamH I existing in .1 divide SEQ ID No.1 into three sections; prepare three fragments and clone them into the same plant expression vector to obtain The recombinant expression vector containing SEQ ID No.1 is the full-length cDNA clone of sweet pepper vein mottle virus infectivity. By using the method, PVMV infective clones can be constructed quickly and efficiently at low cost, and the invention provides a theoretical basis for preventing and controlling 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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Patent Type & Authority Patents(China)
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 UNIV
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