Method for high-flux screening of host genes essential for plant virus copying

A plant virus and high-throughput technology, applied in the field of genetic engineering, can solve the problems of low virus infection efficiency and difficult gene expression control, and achieve the effect of saving time and improving efficiency

Inactive Publication Date: 2018-11-06
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problems of low virus infection efficiency and difficult gene expression control in the current invasive clone construction method, the present invention provides a method for large-throughput screening of host genes necessary for plant virus replication, comprising the following steps:

Method used

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  • Method for high-flux screening of host genes essential for plant virus copying
  • Method for high-flux screening of host genes essential for plant virus copying

Examples

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Embodiment 1

[0034] Example 1: A method for large-scale screening of essential host genes for the replication of TUV.

[0035] Step 1. Construction of an inducible turnip mosaic virus (TuMV) invasive expression vector.

[0036] 1) In order to screen the offspring of transgenic crops transformed with an inducible viral vector more intuitively and conveniently, this embodiment uses the TuMV infectious plasmid pCambia-TuMV-GFP with a green fluorescent marker gene GFP tag as a template, and the pCambia-TuMV -The GFP plasmid contains a TuMV genome (TuMV-GFP) with a sequence encoding green fluorescent protein (GFP) inserted between the encoding P1 and HcPro proteins, as shown in SEQ ID NO:11, the infectious plasmid pCambia -TuMV-GFP is described in Cotton S, Grangeon R, Thivierge K, et al., 2009.Turnip mosaic virusRNA replication complex vesicles are mobile,align with microfilaments,and areeach derived from a single viral genome.J Virol 83,10460-71 .

[0037] Without the addition of fluorescen...

Embodiment 2

[0073] Example 2. A method for large-scale screening of host genes essential for the replication of Potato virus X.

[0074] Repeat Example 1, the difference from Example 1 is that the inducible virus-infectious clone described in this example is a potato virus X (Potato virus X, PVX) infectious clone with a GFP tag: with Potato virus X infectious clone with GFP label is template (PVX.GFP-2A-CP), sees shown in SEQ ID NO: 12, and described infectious plasmid is recorded in Cruz SS, Chapman S, Roberts AG, Roberts IM, Prior DAM, Oparka KJ.1996.Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.Proc.Natl.Acad.Sci.USA,93:6286-6290.Use primer 9 and primer 10 to amplify 1-3953nt sequence , denoted as sequence E (including estrogen-inducible promoter partial sequence, total length 3973nt), using primer 11 and primer 12 to amplify the 3933-3987nt sequence, denoted as sequence F (including polyA and T3A terminator partial sequence in pMDC7 vector , tot...

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Abstract

The invention discloses a method for high-flux screening of host genes essential for plant virus copying, belongs to the technical field of gene engineering and aims to solve the problem that at present, an infective cloning construction method is low in virus infection efficiency and make gene expression difficult to control. The method for high-flux screening of host genes essential for plant virus copying specifically comprises the steps that an inducible promoter is utilized to establish inducible virus infection cloning, transgenic plants are used for constructing a mutant library throughagrobacterium tumefaciens-mediated transformation plants, an inducer is utilized to induce virus infection after mutants germinate, the mutants are screened to obtain mutant plants which cannot be induced by viruses, and the host genes essential for plant virus copying are positioned and cloned by cooperating with a map-based cloning and whole genome sequencing method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for large-throughput screening of essential host genes for plant virus replication. Background technique [0002] The RNA of plant positive-sense RNA viruses is infectious. After entering plant cells, it can be translated as mRNA by the ribosomes of the cells to produce the proteins required for virus replication (Flasinski et al., 1996), and then initiate life processes such as virus replication and movement . Plant positive-sense RNA viruses can be engineered by constructing infectious clones for long-term storage (Flasinski et al., 1996, Takahashi et al., 1997). At present, the invasive clones of many plant positive RNA viruses have been reported, and the summary methods can be classified into two categories: 1) Insert the cDNA of the whole genome of the virus into a constitutive promoter, such as the 35S promoter of cauliflower mosaic virus (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8203
Inventor 程晓非武晓云
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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