N gene-specific primer pair, detection method and kit for detecting tobacco resistance to TMV

A specific, primer pair technology, applied in the fields of biochemistry and molecular biology, can solve the problems of low efficiency of variety resistance judgment, affecting the conclusion of variety resistance judgment, etc., to achieve high primer amplification efficiency, intuitive result judgment, and primers. sequence-specific effects

Active Publication Date: 2015-09-16
TOBACCO RES INST CHIN AGRI SCI ACAD
View PDF2 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In traditional TMV-resistant breeding, in order to identify whether the cultivated varieties are TMV-resistant, field identification methods are usually adopted, that is, it is necessary to inoculate a single plant of the breeding material and create suitable disease conditions. Therefore, if the inoculation is unsuccessful or If the conditions are not suitable, it will affect the judgment conclusion of variety resistance, and has the disadvantage of low efficiency of variety resistance judgment

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • N gene-specific primer pair, detection method and kit for detecting tobacco resistance to TMV
  • N gene-specific primer pair, detection method and kit for detecting tobacco resistance to TMV
  • N gene-specific primer pair, detection method and kit for detecting tobacco resistance to TMV

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0034] (1) The primer sequence designed and synthesized by the present invention according to the sequence characteristics of the N gene and the characteristics of the functional domain is as follows:

[0035] Upstream primer NPF: 5'-CACTTTTGGCGTACTTCCTT-3'

[0036] Downstream primer NPR: 5'-GAGTTGTACTGAACTACTACTC-3'.

[0037] (2) Preparation of materials: Plant the tobacco seeds of Kuban Sansheng and K326 in special tobacco seedling trays. When the tobacco seedlings grow to the 5-6 leaf stage, take 100 mg of leaves, freeze them with liquid nitrogen, and store them at -80°C for DNA extraction. Among them, Kuban Sansheng is a TMV-resistant tobacco variety containing N gene; K326 is a TMV-sensitive tobacco variety without N gene.

[0038] (3) DNA extraction: The DNA extraction of the present invention is not affected by the growth and development stage of the tobacco plant, and the specific sampling time can be arranged according to the test requirements. The DNA extraction m...

example 2

[0044] (1) 68 tobacco varieties (lines) were selected, and the variety names are shown in Table 1.

[0045] (2) These 68 tobacco seeds were planted in special tobacco seedling trays. When the tobacco seedlings grew to the 5-6 leaf stage, 100 mg of leaves were taken, quick-frozen in liquid nitrogen, and stored at -80°C for DNA extraction. At the same time, these materials were inoculated with TMV bacterial liquid by friction method, and the TMV resistance identification was carried out.

[0046] (3) Genomic DNA of the above-mentioned tobacco varieties was extracted with a plant genomic DNA extraction kit purchased from Beijing Quanshijin Biotechnology Co., Ltd. See the instruction manual for specific operation steps.

[0047] (4) Using the DNA of the tobacco variety (line) to be tested as a template, PCR amplification is performed using NPR / NPF primers. The PCR reaction system and reaction conditions were the same as in Comparative Example 1.

[0048] (5) The detection of PC...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides an N gene specific primer pair, a method and a kit for detecting resistance of tobacco to a TMV. The specific primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:2. The method comprises the following steps: designing a pair of specific primers in a tobacco N gene sequence specific zone; with the DNA of a to-be-detected tobacco variety as a template, performing PCR amplification on the N gene specific primer pair; and detecting a PCR amplification product by electrophoresis and judging whether the PCR amplification product contains a 865bp characteristic band, wherein if so, the tobacco has resistance to TMV, and otherwise, the tobacco does not have N gene mediated resistance to TMV. The method has the advantages of reliable result, high detection speed and the like and is simple to operate, the breeding process of TMV-resisting breeding materials can be greatly accelerated, the breeding period is shortened and the breeding efficiency is enhanced.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and in particular relates to an N gene-specific primer pair, a detection method and a kit for detecting tobacco resistance to TMV. Background technique [0002] Tobacco mosaic virus (TMV, tobacco mosaic virus) is a common and serious disease in tobacco growing areas all over the world. After the onset, the yield of tobacco leaves decreases and the quality deteriorates, which brings great harm to the production of tobacco leaves. At present, there is no effective medicament for the prevention and treatment of TMV, and measures such as early prevention and comprehensive prevention and control can only be taken. Due to the shortage of cultivated land resources and the impact of continuous cropping, it is particularly urgent and important to cultivate tobacco varieties resistant to TMV in production. [0003] The N gene originated from the wild species of Nicotiana glutinosa, and i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 张玉罗成刚杨爱国张伟蒋彩虹常爱霞冯全福钱玉梅程立锐耿锐梅任民
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap