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Construction and application of tobacco vein banding mosaic virus infectious clone

A Mosaic Virus, Vein Banding Technology

Inactive Publication Date: 2013-04-10
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TVBMV is mainly transmitted by aphids in a non-persistent manner in the field, and TVBMV and potato virus X (PVX) have a synergistic effect, once combined infection will cause severe symptoms and cause greater losses

Method used

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  • Construction and application of tobacco vein banding mosaic virus infectious clone
  • Construction and application of tobacco vein banding mosaic virus infectious clone
  • Construction and application of tobacco vein banding mosaic virus infectious clone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Purification of Tobacco Vein Mosaic Virus Particles

[0038] About 200 g of diseased leaves were weighed and left overnight at 4°C. Add 200mL of 0.2mol / L phosphate buffer (pH=8.0, 0.15% mercaptoethanol, 0.01mol / L EDTA) to homogenate, and centrifuge at 8000r / min at 4°C for 15-20 minutes. Add 1% TritonX-100 to the supernatant and stir at 4°C for 3 hours, add PEG600040g / L and NaCl (0.2M final concentration) and stir for 1.5 hours, then centrifuge at 8000r / min at 4°C for 15-20 minutes. Precipitate with 0.2mol / L phosphate buffer (pH=8.0, 1%TritonX-100) 1 / 10 of the original volume and gently shake overnight at 4°C. Centrifuge at low speed to remove insoluble impurities. The supernatant was centrifuged at 40,000 r / min for 1 hour, and the pellet was resuspended with 8 mL of 0.2 mol / L phosphate buffer, and kept overnight at 4°C. Carefully place 2mL of the virus suspension on the top layer of 30% sucrose (prepared in 0.05M phosphate buffer), and centrifuge at 90,000...

Embodiment 2

[0039] Embodiment 2: the extraction of virus RNA

[0040] Take 200 μL of virus extract and add an equal volume of extraction buffer (40mmol / L Tris-Cl, pH9.0, 2mmol / L EDTA, 2% SDS), vortex, add RNase inhibitor, 65°C for 5 minutes. Add an equal volume of phenol, vortex, 60°C for 1 minute, and centrifuge at 13000r / min for 5 minutes. Add an equal volume of phenol / chloroform (1:1, pH=5.2) to the supernatant, and centrifuge at 13000r / min for 5 minutes. The supernatant was precipitated with absolute ethanol and centrifuged at 13000r / min for 5 minutes. The precipitate was redissolved with 50 μL DEPC water and stored at -80°C for later use.

Embodiment 3

[0041] Example 3: Construction of Tobacco Vein Mosaic Virus Infectious Clones

[0042] Using the viral RNA obtained in Example 2 as a template, reverse transcription was performed with random primers. According to the existing restriction enzyme map of the whole genome of tobacco vein mosaic virus, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5' untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplified HC- The fragment between the pro gene Nru I restriction site and the 6K2Xho I restriction site, the inventor named the fragment pHC-6K2; PCR amplification of the 6K2Xho I restriction site to the poly(A) tail Fragment, the inventor named the fragment p6K2-polyA ( figure 1 ).

[0043] The reverse transcriptase used for cDNA synthesis was Moloney m...

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Abstract

The invention relates to the field of plant virus gene engineering and provides a construction and an application of a tobacco vein banding mosaic virus infectious clone. A genome of a tobacco vein banding mosaic virus HN39 isolate is cloned to the downstream part of a 35S promoter in a genetic recombination mode, and the infectious clone with strong infectivity is obtained. A plant expression vector can be constructed by utilizing the infectious clone and used for stably and efficiently expressing extrinsic protein in a host plant body, and the infectious clone can be also modified into a low virulent strain for protecting a plant from being infected with a high virulent strain.

Description

technical field [0001] The present invention relates to the field of plant virus genetic engineering, in particular, the present invention relates to the construction method and application of the invasive clone of tobacco vein banding mosaic virus (Tobacco vein banding mosaic virus, TVBMV) HN39 isolate of the genus Potatovirus. Background technique [0002] Infectious cDNA cloning of plant viruses is an important tool for studying the pathogenic mechanism of viruses. After obtaining the invasive cDNA clone, the reverse genetics method can be used to analyze the role of viral genes in the process of infection and pathogenicity, and then attenuated strains can be obtained to protect plants from the harm of virulent strains. Infectious cDNA clones of plant viruses can also be transformed into expression vectors for the production of antiviral proteins, medical veterinary vaccines and antibodies. The use of viral vectors to express foreign proteins in plants has the advantages...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/82A01H5/00
Inventor 李向东高瑞田延平刘焕庭
Owner SHANDONG AGRICULTURAL UNIVERSITY
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