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Method for detecting V1109L mutation site in phytophthora parasitica var. nicotianae cellulose synthase

A technology of V1109L and cellulose synthase, applied in the field of tobacco genetic engineering

Pending Publication Date: 2021-02-23
ZHENGZHOU TOBACCO RES INST OF CNTC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there have been no detailed reports on whether there is a similar mutation in Phytophthora nicotianae cellulose synthase CesA3, and how it affects fungicide resistance.

Method used

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  • Method for detecting V1109L mutation site in phytophthora parasitica var. nicotianae cellulose synthase
  • Method for detecting V1109L mutation site in phytophthora parasitica var. nicotianae cellulose synthase
  • Method for detecting V1109L mutation site in phytophthora parasitica var. nicotianae cellulose synthase

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Experimental program
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Effect test

Embodiment 1

[0064] Based on the obvious differences in the resistance of related strains and the reason for the mutation of related genes in other existing species, the inventors believe that it is necessary to analyze the cellulose synthase related genes in Phytophthora tabacum CesA3 Whether there are similar mutation sites in the sensitive and mutant strains is further studied, and the process of determining the relevant mutation sites is briefly introduced in this example as follows.

[0065] (1) Design primers

[0066] According to the cellulose synthase gene of the existing pathogens (Phytophthora soybean, Phytophthora infestans, Phytophthora oak) and the genome sequence of Phytophthora nicotianae, combined with the results of homologous comparison, PCR amplification was designed according to the conserved sequence fragments to obtain the tobacco Phytophthora mycocellulose synthase related gene CesA3 The primer sequences are as follows:

[0067] A3F11: 5'-ATGGGGCTCACCGGC-3',

[...

Embodiment 2

[0086] Based on the results of the single point mutation in Example 1, and in combination with the requirements of the AS-PCR method, the inventors further designed relevant primers to detect and determine the relevant mutation sites. The specific process is introduced as follows.

[0087] (1) Primer design

[0088] Based on the aforementioned single-base mutation sequence difference and AS-PCR method requirements, the inventor designed the primer sequence for the Q1077H mutation site as follows:

[0089] Pn3231-F1: 5'-GGCTTCTTCGTCATGAGCCAT-3',

[0090] PnA3-R: 5'-CTACGTCGACGCCGTCGAAC-3';

[0091] What needs to be explained is that when designing the primers, in order to improve the specificity of the primers, the penultimate base of the 3' end of Pn3231-F1 was mismatched with the target sequence, and at the same time, it was coordinated with the adjustment of the subsequent PCR amplification program to detect The mutation site G3231T;

[0092] For the V1109L mutation site...

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Abstract

The invention belongs to the technical field of tobacco gene engineering and particularly relates to a method for detecting a V1109L mutation site in a phytophthora parasitica var. nicotianae cellulose synthase CesA3. Compared with a wild type, after a 3325th nucleotide of a cDNA sequence of a phytophthora parasitica var. nicotianae cellulose synthase CesA3 gene mutates from G to C from a 5'end, aresistance of a phytophthora parasitica var. nicotianae to dimethomorph can be mutated from a sensitive type to a high-resistance type. Correspondingly, a 1109th amino acid of the phytophthora nicotianae cellulose synthase CesA3 is mutates from a wild-type valine into leucine from an N end. Related primers are designed on the basis of an AS-PCR technical principle, so that the mutated phytophthora parasitica var. nicotianae with the dimethomorph resistance can be quickly detected and identified, and a certain technical foundation can be laid for regulating a prevention and treatment strategyfor tobacco black shank, delaying and controlling a further development of drug resistance, and promoting research and development of related new chemical drugs.

Description

technical field [0001] This application belongs to the technical field of tobacco genetic engineering, and specifically relates to the patent application for a method for detecting the V1109L mutation site in Phytophthora tobacco cellulose synthase CesA3. Background technique [0002] Tobacco black shank (Tobacco black shank) is a devastating soil-borne disease worldwide, and its pathogen is Phytophthora var. Phytophthora parasitica var nicotianae (Breda de Hann) Tuker, ie Phytophthora nicotiana). In recent years, with the expansion of flue-cured tobacco continuous cropping planting area and the continuous accumulation of diseased and residual biomass in the field, tobacco black shank has become the main root disease of tobacco in my country, causing huge economic losses. According to incomplete statistics, the incidence of tobacco black shank disease in the main smoking areas of Henan is 8% to 15%, and the annual output value loss caused by tobacco black shank is 200 to 3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6895C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/106C12Q2600/156C12Q2531/113C12Q2565/125C12Q2521/107
Inventor 牟文君宋纪真王得强陈若星李晓闯郭建华薛超群王爱国胡利伟过伟民奚家勤翟振
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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