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Infectious clone vector of pepper mild mottle virus, agrobacterium tumefaciens strain, method for preparing same and application of infectious clone vector

A technology of mottled virus and cloning vector, which is applied in the field of genetic engineering, can solve the economic loss of peppers and other problems, and achieve the effect of simple operation and good repeatability

Inactive Publication Date: 2016-01-06
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the occurrence of PMMoV has been detected in Xinjiang, Beijing, Shandong, Hebei, Ningxia and other regions in my country, and it shows a trend of expansion and spread, causing significant economic losses to pepper production.

Method used

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  • Infectious clone vector of pepper mild mottle virus, agrobacterium tumefaciens strain, method for preparing same and application of infectious clone vector
  • Infectious clone vector of pepper mild mottle virus, agrobacterium tumefaciens strain, method for preparing same and application of infectious clone vector
  • Infectious clone vector of pepper mild mottle virus, agrobacterium tumefaciens strain, method for preparing same and application of infectious clone vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning and determination of the complete genome sequence of pepper light mottle virus

[0043] 1. Extraction of plant total RNA

[0044] The total RNA of diseased leaves was extracted by the Trizol method, and the specific operation was as follows: About 100㎎ fresh pepper diseased leaf samples were ground into powder in liquid nitrogen, and then 1ml Trizol was quickly added. Transfer to a 2ml sterile centrifuge tube, shake and mix vigorously to fully lyse, and place at room temperature for 5 minutes. Centrifuge at 132000rpm for 5min at 4°C. Take the supernatant, add 1 / 5 volume of chloroform, vibrate vigorously for 15 seconds, and place on ice for 2-3 minutes. Centrifuge at 132000rpm for 30min at 4°C. The bottom layer is a chloroform phase, the middle layer is a protein phase, and the upper layer is a colorless aqueous phase containing RNA. Transfer the supernatant to a new round of centrifuge tubes (be careful not to inhale the protein layer), add 1 / 5 vo...

Embodiment 2

[0047] Embodiment 2: Construction of capsicum mild mottle virus infectious clone pCB-PMMoV

[0048] According to the complete sequence obtained in Example 1, primers pCass-CH-PMMoV(+), pCass-CH-PMMoV(-), PMMoV3369(+) and primer PMMoV3369(-) were designed. Using the cDNA synthesized by 3'-RACE in Example 1 as a template, use the primer pair pCass-CH-PMMoV(+) / PMMoV3369(-) and the primer pair PMMoV3369(+) / pCass-CH-PMMoV(-) respectively Two segments amplify the full sequence of PMMOV. Using ClonExpress TM The MultiS Recombination Cloning Kit (Nanjing Nuoweizan Biotechnology Co., Ltd.) recombined the purified amplified target fragment with the pCB301-CH vector digested by StuI and SmaI, and used 10 μL system reaction, in which 5×CEMultiSBuffer 2 μL, StuI and SmaI double digestion linearized cloning vector pCB301-CH120ng, fragment p35SPMMoV1-336968ng, fragment pPMMoV3369-RZ60ng, Exnase TM MultiS1μl, and finally adjust the system to 10μL with ddH2O. Gently mix the components by p...

Embodiment 3

[0049] Embodiment 3: Construction of the capsicum mild mottle virus invasive clone pCB-PMMoV-GFP carrying exogenous gene GFP

[0050] Insert the coat protein gene promoter and exogenous GFP gene of tobacco light green mosaic virus (Tobaccomildgreenmosaicvirus, TMGMV) into the obtained PMMoV invasive cloning vector pCB-PMMoV, and obtain an infectious gene that can express foreign genes. PMMoV invasive cloning vector pCB-PMMoV-GFP. The obtained PMMoV invasive cloning vector pCB-PMMoV template, primer PMMoV-5348NcoI (+), SeqIDNo: 7 and PMMoV-MP-TMVGFP (-), SeqIDNo: 8 is a primer, amplifying a 330bp band; p35S-GFP as template, PMMoV-MP-TMVGFP(+), SeqIDNo: 9 and TMGMV-PMMoVCP(-), SeqIDNo: 10 for amplification of TMGMV promoter and exogenous GFP gene, the product size is about 1200bp. The PMMoV invasive cloning vector pCB-PMMoV is used as a template, TMGMV-PMMoVCP(+), SeqIDNo:11 and PCASS-CH-PMMoV(-), SeqIDNo:6 are primers, and the amplification includes the PMMoVCP segment and the...

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Abstract

The invention relates to the field of genetic engineering, in particular to preparation of an infectious clone vector of a Pepper mild mottle virus (PMMoV) of RNA (ribonucleic acid) plant viruses and tobacco mosaic viruses, pathogenesis analysis on an infectiously cloned agrobacterium tumefaciens strain with the pepper mild mottle virus and exogenous proteins infectiously cloned and expressed by the aid of the PMMoV. The preparation, the pathogenesis analysis and exogenous proteins have the advantages that genomes of Shanghai bay isolate of the pepper mild mottle virus are constructed onto efficient plant expression vectors pCB301-CH by the aid of a genetic recombination process, the recombinant vectors are transferred into the agrobacterium tumefaciens strain by means of electroporation, and accordingly the virus vector with infection ability can be obtained; the plant expression vectors can be constructed by means of infectious cloning, and the exogenous proteins can be efficiently and stably expressed in host plants; mature systems can be provided for studying structures and functions of the virus genomes and studying interaction between the virus and hosts.

Description

technical field [0001] The present invention relates to the field of genetic engineering. Specifically, the present invention relates to the preparation of an RNA plant virus-Peppermildmottle virus (Peppermildmottlevirus, PMMoV) invasive cloning vector of the genus Tobacco Mosaic Virus, which carries the infectivity of Peppermildmottle virus. Pathogenicity analysis of cloned Agrobacterium strains and expression of foreign proteins using PMMoV invasive clones. Background technique [0002] Pepper light mottle virus belongs to the genus Tobamovirus (genus Tobamovirus) of the family Tymoviridae. Its virus particles are rod-shaped. The virus genome is about 6.4kb, including the 5' and 3' non-coding regions and Four open reading frames encode replication-associated proteins of 126kDa and 183kDa, mobile proteins of 28.3kDa and coat proteins of 17.1kDa, respectively. The virus seriously threatens the production of solanaceous crops such as sweet peppers, peppers, and tomatoes. Whe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N1/21C12N15/66C12R1/01
Inventor 燕飞郑红英韩科雷肖彩利彭杰军鲁宇文林林陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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