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Recombinant plant rhabdovirus vector and construction method thereof

A plant rhabdovirus and vector technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, plant genetic improvement, etc. To solve problems such as genetics, to achieve the effect of good repeatability, broad application prospects and simple operation

Active Publication Date: 2015-10-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Third, safety issues. Viruses are one of the important pathogens of plants. Although the virus vector has been modified so that it cannot cause severe symptoms or be transmitted by the vector, there are still certain environmental safety hazards in the release of the virus vector. Therefore, plant viruses The application of expression vectors is greatly limited (Gleba et al., Curr Opin Biotechnol, 2007, 18:134-141)
[0006] However, there is no report on the reverse genetics of plant negative-strand RNA viruses in the world, and it is impossible to carry out genetic manipulation of this type of virus

Method used

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  • Recombinant plant rhabdovirus vector and construction method thereof
  • Recombinant plant rhabdovirus vector and construction method thereof
  • Recombinant plant rhabdovirus vector and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1. SYNV cDNA transcription vector construction

[0045] The SYNV genome is a single-stranded negative-sense RNA with a length of about 13.7 kb. Total RNA was extracted from leaves of Nicotiana benthamiana infected with SYNV, and used as a template for reverse transcription PCR to obtain full-length genome cDNA. The full-length cDNA was cloned into the plant expression vector pCB301 to obtain the SYNV cDNA transcription vector, named pSYNV, and pSYNV was introduced into Agrobacterium GV3101 by electroporation.

[0046] The construction process of infecting pSYNV transcription vector comprises the following steps:

[0047] 1) Extract total RNA from SYNV-infected Nicotiana benthamiana leaves:

[0048] Take 0.1 g leaves, add liquid nitrogen and grind into powder; quickly transfer the powder to a pre-cooled 1.5 mL centrifuge tube, and quickly add pre-cooled 1 mL TRIzol extract; mix up and down, fully lyse, and place at room temperature for 5 min; 0.2 mL of chlorof...

Embodiment 2

[0055] Example 2. RNP core protein expression vector construction

[0056]The ribonucleoprotein RNP core of SYNV includes three proteins, N, P and L. These three proteins are inserted into the same expression vector at the same time to obtain pGD-NPL. In this expression vector, the three proteins are driven by different 35S promoters and NOS transcription terminator control, that is to say, the same expression vector contains three expression cassettes, expressing N, P and L respectively ( figure 2 .A).

[0057] The above four expression vectors were electroporated into Agrobacterium GV3101 respectively.

Embodiment 3

[0058] Example 3. Viral RNA silencing suppressor protein expression vector construction

[0059] Three RNA silencing suppressors encoded by other viruses: tobacco etch virus (Tobacco etch virus, TEV) Hc-Pro gene, tomato bushy stunt virus (TBSV) P19 gene and barley stripe mosaic virus ( Barley stripe mosaic virus, BSMV) γb gene. These three silencing suppressors were respectively inserted into pGD vectors to obtain three expression vectors: pGD-Hc-Pro, pGD-P19 and pGD-γb ( figure 2 .B).

[0060] The above three silencing suppressor expression vectors were introduced into Agrobacterium GV3101 by electroporation respectively.

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Abstract

The invention discloses a recombinant plant rhabdovirus vector and a construction method thereof. The recombinant plant rhabdovirus vector comprises a modified plant rhabdovirus genome, a transcription unit which is induced newly and a heteroduplex nucleotide sequence, wherein the transcription unit is inserted into the plant rhabdovirus genome to express the heteroduplex nucleotide sequence, the heteroduplex nucleotide sequence is replaced into a glycoprotein transcription unit of a recombinant plant rhabdovirus; the recombinant plant rhabdovirus has copying and infecting capacity, and one antigenic polypeptide or other protein is coded by the heteroduplex nucleotide sequence. The virus expression vector is the first one which can express a negative-sense RNA virus vector of foreign protein on plants, and the recombinant plant rhabdovirus vector has the advantages that the expression quantity is high, the expression stability is good, a relative long foreign gene segment can be inserted, and inserted foreign gene segment is not prone to loosing; the recombinant plant rhabdovirus vector can be used for expressing of multiple kinds of foreign protein and can also be used for preparing animal vaccines, and wide application values and application prospects are achieved.

Description

technical field [0001] The present invention relates to the fields of plant genetic engineering and virus molecular biology. Specifically, the present invention relates to a plant negative-sense RNA rhabdovirus-a method for constructing a recombinant virus expression vector of chicory yellow net virus and a method for expressing foreign genes Methods. Background technique [0002] With the development of plant genetic engineering, the use of plants as bioreactors to produce foreign proteins has attracted more and more attention. In recent years, studies have confirmed that plant systems have the ability to express active mammalian proteins, and the product quality, cost and In terms of security, obvious advantages have been shown. There are two main ways to express exogenous genes in plants: one is to integrate exogenous genes into plant genomes for stable expression by transgenic methods, and the other is to insert exogenous genes into viral genomes and use plant virus...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/66
Inventor 李正和马晓楠王强钱莎莎孙凯周雪平
Owner ZHEJIANG UNIV
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