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Detection method of Phytophthora nicotianae cellulose synthase q1077h mutation site

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

Active Publication Date: 2022-07-08
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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  • Detection method of Phytophthora nicotianae cellulose synthase q1077h mutation site
  • Detection method of Phytophthora nicotianae cellulose synthase q1077h mutation site
  • Detection method of Phytophthora nicotianae cellulose synthase q1077h mutation site

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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 inventor believes that it is necessary to study the cellulose synthase related genes in Phytophthora nicotianae. CesA3 Whether there are similar mutation sites in sensitive and mutant strains will be further studied. In this example, the process of determining the relevant mutation sites is briefly described as follows.

[0065] (1) Design primers

[0066] According to the cellulose synthase gene of the existing pathogenic bacteria (Phytophthora sojae, Phytophthora infestans, Phytophthora acornis) and the genome sequence of P. nicotianae, combined with the homologous alignment results, PCR amplification was designed according to the conserved sequence fragments to obtain the Phytophthora tabaci. mold cellulose synthase-related gene CesA3 The primer sequences are as follows:

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

[00...

Embodiment 2

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

[0087] (1) Primer design

[0088] Based on the aforementioned single-base mutation sequence differences and the needs of the AS-PCR method, for the Q1077H mutation site, the inventors designed the primer sequences as follows:

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

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

[0091] It should be explained that, 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 the subsequent PCR amplification program was adjusted to facilitate detection. The mutation site of G3231T;

[0092] For the V1109L mutation site, the inventors designed the primer sequences as follows:

[0093] Pn33...

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Abstract

The present application belongs to the technical field of tobacco genetic engineering, and specifically relates to a patent application for a method for detecting the Q1077H mutation site in Phytophthora nicotianae cellulose synthase CesA3. Compared with the wild type, after the cDNA sequence of Phytophthora nicotianae cellulose synthase CesA3 gene was mutated from G to T at the 3231st nucleotide from the 5' end, the resistance of Phytophthora nicotianae to dimethomorph can be determined by the sensitive mutation. Correspondingly, amino acid 1077 from the N-terminus of Phytophthora nicotianae cellulose synthase CesA3 was mutated from wild-type glutamine to histidine. Based on the principle of AS-PCR technology, the inventors designed relevant primers for rapid detection and identification of Phytophthora nicotianae with resistance to dimorpholine after mutation, which can be used to adjust the prevention and control strategies of tobacco black shank, as well as delay and control The further development of drug resistance and the research and development of related new chemical drugs lay a certain technical foundation.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2600/106C12Q2531/113C12Q2565/125
Inventor 牟文君宋纪真陈若星李晓闯王得强胡利伟过伟民奚家勤翟振郭建华王爱国
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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