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Method for identifying tobacco resistance by use of tomato spotted wilt virus NSm gene

A technology of tomato spotted wilt virus and avirulent gene, applied in the direction of virus, viral peptide, genetic engineering, etc., can solve the problems that susceptible varieties cannot be 100% diseased, resistance identification of resistant isolated populations cannot be satisfied, and achieve accelerated identification. and selection effects

Active Publication Date: 2017-11-28
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of TSWV virus sources to inoculate susceptible varieties requires fine and skilled operation, and a slight improper operation may easily cause the phenomenon that 100% of the susceptible varieties cannot be infected, resulting in the resistance identification of resistant segregation populations failing to meet the requirements of genetic analysis.

Method used

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  • Method for identifying tobacco resistance by use of tomato spotted wilt virus NSm gene
  • Method for identifying tobacco resistance by use of tomato spotted wilt virus NSm gene
  • Method for identifying tobacco resistance by use of tomato spotted wilt virus NSm gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Determination of avirulent genes that stimulate RTSW disease-resistant sites to produce allergic reactions

[0046] 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

[0070] Example 2 Comparison of resistance identification using traditional methods and transient infiltration of Agrobacterium using expression vector pK2-35S-NSm for tobacco resistance identification

[0071] Polalta×K326 BC6F1S refers to the population obtained from the hybridization of Polalta×K326 BC5F1 resistance identified as susceptible (ie rtsw / rtsw genotype) and K326, which should be all susceptible.

[0072] Polalta×K326 BC6F1R refers to Polalta×K326 BC5F1 resistance identified as disease resistance, self-seed to Polalta×K326 BC5F3 to determine the resistance of a single plant (that is, RTSW / RTSW genotype) and the population obtained after K326 hybridization , should be disease resistant for all.

[0073] 1. Using traditional methods for resistance identification

[0074] Pick N. alata (PI42334), Polalta, Nicotiana benthamiana ( N. benthamiana )and N. tabacun CV. 5 plants of K326, 15 plants of Polalta×K326 BC6F1S and 15 plants of Polalta×K326 BC6F1R (potted plan...

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Abstract

The invention relates to a method for identifying tobacco resistance by use of a tomato spotted wilt virus NSm gene and belongs to the technical field of plant protection. The NSm gene contains a nucleotide sequence shown in SEQ ID No.1. The NSm gene is an avirulence gene which stimulates tobacco containing a resistant locus RTSW to produce disease resistance response. An agrobacterium tumefaciens transient expression system is adopted for transient expression of the NSm gene in tobacco leaves, and whether test tobacco contains the resistant locus RTSW is judged by detecting whether the test tobacco shows an allergic reaction. The resistance of the tobacco to the tomato spotted wilt virus disease can be rapidly and accurately identified with the method, and the method can be applied to disease-resistant breeding as well as positioning and cloning of disease-resistant genes.

Description

technical field [0001] The invention belongs to the technical field of plant protection, and further belongs to the technical field of identification of tobacco virus disease resistance, in particular to a method for identifying tobacco resistance by using NSm gene of tomato spotted wilt virus. Background technique [0002] Tomato spotted wilt virus ( Orthotospovirus ) virus is a type of plant virus with the widest host range and the most serious occurrence. Its representative species, Tomato Spotted Wilt Virus (TSWV), has posed a greater threat to tobacco leaf production in Yunnan tobacco-growing areas. According to the test results of samples collected from various places (states, cities) and counties in Yunnan in recent years, TSWV is distributed on tobacco in Yunnan province and tends to expand and intensify. What is even more worrying is that none of the main flue-cured tobacco varieties in Yunnan Province are resistant to TSWV. Field investigations found that the exis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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方敦煌肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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