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Tobacco mosaic virus resistant N'au gene and cloning method and application thereof

A technology of anti-tobacco mosaic and cloning method, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of slow yellowing of upper leaves, narrow genetic background, and lack of breakthrough progress in breeding of flue-cured tobacco resistant to TMV

Active Publication Date: 2016-01-27
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to chain burdens such as low yield and slow yellowing of upper leaves, carrying N Genetic flue-cured tobacco varieties cannot meet the urgent needs of production
and N There is a lack of breakthroughs in TMV-resistant flue-cured tobacco breeding due to the linkage redundancy of closely linked genetically derived chromosomal segments from Tobacco heart leaf, the narrow genetic background of TMV resistance sources that have been used in breeding, and the limitations of conventional breeding methods.

Method used

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  • Tobacco mosaic virus resistant N'au gene and cloning method and application thereof
  • Tobacco mosaic virus resistant N'au gene and cloning method and application thereof
  • Tobacco mosaic virus resistant N'au gene and cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: N. alata (PI42334) against TMV-U1 strain N'au Gene discovery and functional verification

[0059] (1) N. alata (PI42334) resistant TMV-U1 strain, resistance differs from N Gene

[0060] Pick N. sylverstris (PI555569), N. alata (PI42334), Coker176, K326 and other four kinds of tobacco products were planted with 15 plants (potted plants), and when they had 4-5 leaves, they were inoculated with TMV-U1 strain and blank control respectively. Symptoms were surveyed and recorded on days 5, 7, and 14 after inoculation.

[0061] Results on Day 7 of virus inoculation showed: N. alata (PI42334) and Coker176 anti-TMV-U1 strains, N. sylverstris and K326 sensitive TMV-U1 strains (Table 1, figure 1 ). this means N. alata (PI42334) and Coker176 in the presence of resistance genes against TMV-U1 strains.

[0062] The result of table 14 kinds of tobacco varieties being inoculated with TMV-U1 strain

[0063]

[0064] Note: HR is disease resistance respon...

Embodiment 2

[0080] Example 2: N. alata (PI42334) N'au Gene cloning and sequence analysis

[0081] (1) Extraction of total tobacco DNA: Fresh young leaves of tobacco were taken, and the total genomic DNA of tobacco was extracted with QIAGEND Neasy Plant Mini kit. DNA quality was initially detected by UV spectrophotometry (Nanodrop) and agarose gel electrophoresis. DNA samples with acceptable quality were diluted to 100ng / μL with 0.5×TE solution, and stored for later use.

[0082] (2) N'au Gene cloning: with N. alata (PI42334) and N. sylvestris The DNA of (PI555569) was used as a template, and PCR amplification was performed with primers N'-H8-F (5'-ATGGAGATTGGCTTAGCAGT-3') and primers N'-H8-R (5'-TCACAGGCATTCACAATCGA-3'). The total volume of the PCR reaction system is 50 μL, including 4.0 μL of 100 ng / μL DNA sample, 10.0 μL of 5×PCR buffer, 4 μL of dNTPs (2.5 mmol / Leach), and 10 μmol / L of primers N'-H8-F and N'-H8-R 2.0 μL each, PrimeSTARGXLDNA Polymerase 1 μL, ddH 2 O27 μL. Th...

Embodiment 3

[0085] Example 3: N'au The polypeptide sequence encoded by the gene

[0086] according to N'au The nucleotide sequence of the gene was deduced using the molecular biology software MEGA6 N'au The amino acid sequence of the polypeptide encoded by the gene is shown in SEQ ID No.2.

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Abstract

The invention relates to isolation cloning and breeding application of a tobacco mosaic virus (TMV) resistant N'au gene. A nucleotide sequence of the TMV resistant N'au gene is shown as SEQ ID NO.1, and amino acid of polypeptide coded by the TMV resistant N'au gene is shown as SEQ ID NO.2. A non-transgenic TMV-resistant tobacco variety can be obtained by trans-breeding an N'au gene contained in germplasm resources into a TMV-sensitive main planting tobacco variety through a conventional breeding means. The N'au gene in the main planting tobacco variety has a homologous sequence high in nucleotide rate, so that short introgression fragment single plants carrying the N'au gene can be obtained easily through conventional breeding so as to obtain a TMV-resistant variety low in linkage drag. The gene can resist strains U1 and Cg of TMV at the same time. The novel antiviral gene N'au has great application prospect in cultivation of TMV-resistant tobacco.

Description

technical field [0001] The invention belongs to the technical field of plant protection, and further belongs to the technical field of tobacco virus prevention and control, in particular to an anti-tobacco mosaic virus N'au Genes and their cloning methods and applications. Background technique [0002] Tobacco mosaic virus (TMV) is an important tobacco disease in China, and its annual loss ranks at the top of the list of top ten tobacco infectious diseases (Zhu Xianchao, 2002). TMV virus has TMV-Cg strains and TMV-U1 strains and other strains, and TMV-U1 strains are the main harmful strains on tobacco. The main varieties of flue-cured tobacco in China, K326 and Yunyan 87, are not resistant to TMV-U1 strains. Generally, measures such as cultivating non-toxic seedlings, chemical control, and destroying diseased residues in the field are adopted to control the occurrence and prevalence of TMV. However, TMV outbreaks in local fields still occur from time to time, causing large...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C07K14/415
Inventor 刘勇袁欣婕黄昌军李永平于海芹陈学军肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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