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Screening method for tobacco antiviral regulatory genes and application of screening method

A technology for regulating genes and screening methods, applied in the fields of application, genetic engineering, botanical equipment and methods, etc., can solve the problem of no obvious phenotype of a single member gene, achieve good market application prospects, comprehensive screening, and rapid acquisition Effect

Inactive Publication Date: 2019-04-12
GUIZHOU TOBACCO SCI RES INST
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AI Technical Summary

Problems solved by technology

Many plant genes exist in the form of gene families, and gene members within a family often have similar functions, so mutations to a single member gene in a gene family often have no obvious phenotype

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Screening and identification of antiviral regulatory genes of Ben's cigarette

[0042] This example provides a set of screening and identification technology systems for the antiviral regulatory genes of Nicotiana benthamiana based on cDNA library VIGS technology. The main steps include:

[0043] (1) Construction of VIGS-cDNA library of Nicotiana benthamiana

[0044] With the VIGS vector pTRVG as the target vector, the Nicotiana benthamiana cDNA library was constructed and homogenized to obtain the Nicotiana benthamiana VIGS-cDNA library that can be directly screened and analyzed for gene silencing in the tobacco. Specific steps are as follows:

[0045] The TRIzol Reagent kit was used to extract the total RNA of Nicotiana benthamiana. Reuse MAG mRNAisolation Kit for extracting Nicotiana benthamiana mRNA. Follow the steps below to construct a normalized Uncut cDNA primary library:

[0046] CloneMiner II cDNA Library Construction Kit and Trimmer-2 cDNA normalization ...

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Abstract

The invention provides a screening method for tobacco antiviral regulatory genes and an application of the screening method. The screening method comprises the following steps: firstly constructing atobacco cDNA library by using a virus-induced gene silencing (VIGS) vector as a target vector, and performing homogenization treatment to obtain a VIGS-cDNA library capable of being directly used forgene silencing screening analysis; transforming VIGS-cDNA library plasmid into agrobacteria to obtain an agrobacterium library; performing spread plate culture on the agrobacterium library to obtain single colonies, performing VIGS silencing analysis on each single colony, and performing screening to obtain antiviral-related colonies; and performing sequencing analysis on genes corresponding to the screened antiviral-related colonies, performing homology analysis on the obtained sequences, and performing function prediction to obtain the antiviral regulatory genes. The method provided by the invention has the advantages of simple operation, no need to construct transgenic plants or mutants, short time consuming, comprehensive screening and high efficiency, is particularly suitable for screening of mutant lethal genes, and has broad application prospects.

Description

Technical field [0001] The invention belongs to the technical field of plant disease resistance regulatory gene screening and application technology, and relates to a tobacco antiviral regulatory gene screening method and application. Background technique [0002] A plant cDNA library refers to a collection of all cDNAs of a plant species. These cDNAs are usually inserted into a vector, transformed into E. coli, and mixed and stored. After the build is completed, it can be selected and used at any time. In addition, by selecting and using different library construction vectors, different plant functional genomics studies can be carried out. For example, a library constructed with plant expression vectors can be used for transient overexpression analysis in plants to screen for regulatory genes with specific target functions. Therefore, the utilization of plant cDNA library is the basic research method of plant functional genomics, and it is also one of the main ways to screen,...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6806C12N15/82A01H5/00A01H6/82C12Q1/70C12Q1/18
CPCC12N15/8283C12Q1/18C12Q1/6806C40B50/06
Inventor 任学良蔡新忠郭玉双王仁刚耿召良
Owner GUIZHOU TOBACCO SCI RES INST
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