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Method for screening disease-resistant genes by aid of tomato spotted wilt virus NSm (new smoking materials) genes

A technology of tomato spotted wilt virus and disease resistance gene, which is applied in the direction of virus, viral peptide, genetic engineering, etc., to achieve the effect of saving manpower, easy operation and good repeatability

Active Publication Date: 2017-10-17
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but from wild tobacco N. alata medium resistance gene RTSW Has not been cloned, its corresponding TSWV Avr Genes have not been reported

Method used

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  • Method for screening disease-resistant genes by aid of tomato spotted wilt virus NSm (new smoking materials) genes
  • Method for screening disease-resistant genes by aid of tomato spotted wilt virus NSm (new smoking materials) genes
  • Method for screening disease-resistant genes by aid of tomato spotted wilt virus NSm (new smoking materials) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Determination of avirulent genes that stimulate RTSW disease-resistant sites to produce allergic reactions

[0048] The identification of avirulent genes through 5 functional genes of TSWV virus ( NSs , NSm , Gn , Gc and N ) cloning, expression vector construction and transient infiltration of Agrobacterium. During the construction of all vectors, CACC was added to the 5' end of the forward primer to amplify the entire expression cassette fragment, so that the 5' end of the fragment was complementary to the GTGG sequence at the protruding end of the entry vector pENTR / D-TOPO. Under the action of topoisomerase I, the gene is inserted in the correct direction between the two attL recombination sites of the pENTR / D-TOPO vector to form an entry clone with two attL recombination sites; extract and construct the correct entry The cloning plasmid is mixed with the destination vector pK2GW7 at a molar ratio of 1:1-3:1. Under the action of LR Clonase Enzyme Mi...

Embodiment 2

[0072] Example 2 Comparison of Screening Resistance Genes Using Tomato Spotted Wilt Virus NSm Genes and Traditional Methods

[0073] (1) Co-infiltrate Nicotiana benthamiana with the avirulent gene NSm plant expression vector Agrobacterium and candidate disease resistance genes for disease resistance gene screening

[0074]Primers were designed to amplify the candidate gene from N. alata (PI42334), and the expression vector of the candidate gene was constructed and transformed into Agrobacterium. Respectively co-infiltrate Nicotiana benthamiana with the Agrobacterium carrying the non-toxic gene NSm plant expression vector pK2-35S-NSm, if there is a candidate disease resistance gene that can induce allergic necrosis in the leaf infiltration area, then it shows that the gene is disease resistance Gene.

[0075] N'au Transient expression vector pHellsgate8-N ' au in the patent "An N'au gene resistant to tobacco mosaic virus and its cloning method and application, applicants: L...

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Abstract

The invention relates to a method for screening disease-resistant genes by the aid of tomato spotted wilt virus NSm (new smoking material) genes, and belongs to the technical field of plant gene cloning. Nucleotide sequences of the tomato spotted wilt virus NSm genes are shown as SEQ ID No.1. The method includes constructing plant expression vectors of the tomato spotted wilt virus NSm genes; acquiring agrobacterium tumefaciens by means of transformation; jointly infiltrating nicotiana benthamiana in the agrobacterium tumefaciens and candidate disease-resistant genes; observing nicotiana benthamiana leaves to determine whether the nicotiana benthamiana leaves have characters of hypersensitive necrosis or not; authenticating the candidate disease-resistant genes. The method has the advantages that the disease-resistant genes can be quickly authenticated by the method in a high-throughput manner, and accordingly important application value of the method developed on the basis of genes for screening disease-resistant genes of plants is indicated.

Description

technical field [0001] The invention belongs to the technical field of plant gene cloning, and in particular relates to a method for screening disease-resistant genes by utilizing the NSm gene of tomato spotted wilt virus. Background technique [0002] Tomato Spotted Wilt Virus (TSWV) has posed a huge threat to the production of tobacco, tomato, pepper, vegetables and other crops. All the existing main crop varieties can be infected by tomato spotted wilt virus, which becomes a potential threat to the epidemic and outbreak of tomato spotted wilt virus. Due to the characteristics of its transmission medium, thrips, which has a short development period, small size and easy concealment, and easy resistance to insecticides, the existing control measures are difficult to achieve ideal control effects. Breeding disease-resistant crop varieties using disease-resistant genes is the most economical and effective means. [0003] In the past, the identification and screening of disea...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/74C12Q1/18
CPCC07K14/005C12N15/74C12N2760/12022C12Q1/18G01N2333/08
Inventor 黄昌军刘勇于海芹童治军曾建敏高玉龙
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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