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Method for detecting phytophora capsic CesA3 gene nucleotide point mutation and drug resistance of same to CAA bactericides

A technology of Phytophthora capsicum and drug resistance, applied in the field of molecular biology, can solve the problems of large workload, low detection sensitivity, long detection period and the like

Active Publication Date: 2014-05-28
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary biological methods need to determine the EC of the agent to the pathogenic bacteria 50 To compare the differences between sensitive and resistant strains, the test requires multiple treatments and multiple repetitions, so the detection cycle is long, the workload is heavy, and more human resources and materials are consumed
Moreover, the detection sensitivity of the above conventional methods is low, and the mutation frequency of drug-resistant strains can only be detected when it is above 1%.

Method used

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  • Method for detecting phytophora capsic CesA3 gene nucleotide point mutation and drug resistance of same to CAA bactericides
  • Method for detecting phytophora capsic CesA3 gene nucleotide point mutation and drug resistance of same to CAA bactericides
  • Method for detecting phytophora capsic CesA3 gene nucleotide point mutation and drug resistance of same to CAA bactericides

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1, discovery of CesA3 gene and CesA3 protein mutation site in Phytophthora capsici

[0033]1. Strains: resistant strains are R1-1, R1-2, R1-3, R1-5, R1-7, R1-9, R2-1, R2-2, R2-3, R2-4 and R2- 5. The sensitive strains are PCAS1, PCAS2, nm13, PG13 and SY12.

[0034] 2. Method:

[0035] 1) Templates: Genomic DNA of resistant strains and sensitive strains were used as templates respectively.

[0036] 2) PCR amplification of cellulose synthase CesA3 gene

[0037] Primer: C3F1 (5'-TTACTTACCGTACTCCGATCGCACG-3')

[0038] C3R1 (5'-GCATGAACTCCAAAACACAACAACAGC-3');

[0039] The PCR reaction of the target fragment length less than 2kb uses Promega reagents. The 25 μL reaction system includes 1 μL template DNA (30-50ng), 0.5 μL 10mM dNTP, 2.5 μL 10× amplification buffer (containing 20mM Mg 2+ ), 0.3 μL Taq DNA polymerase (5U / μL), 0.3 μL of each primer (10 μM), 20.1 μL ddH2O, and the reaction system was multiplied or decreased according to the needs of subsequent expe...

Embodiment 2

[0046] Embodiment 2, the method for detecting mutation site in Phytophthora capsici

[0047] 1. Primer design

[0048] The strains are the strains PCAS1 and R2-5 used in Example 1.

[0049] Allele specific-PCR primers were designed according to the sequence of the mutant cesA3 gene. The forward primer was PC05F1, and the last base at the 3'-end of the reverse primer PCR05AR was complementary to the mutated base (Table 1). To increase the specificity of the primers, a mismatched base was introduced at the second base at the 3'-end of the reverse primer (Table 1, primers PCR05BR, PCR05CR, and PCR05DR).

[0050] Primers are listed in Table 1.

[0051] Table 1 Cloning of cellulose synthase cesA3 gene and primers for drug resistance detection.

[0052]

[0053] The specific reaction system is the same as that in Example 1, and the primers are subjected to temperature gradient PCR with different annealing temperatures to determine the annealing temperature with high specificit...

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Abstract

The invention relates to clone of phytophora capsic cellulose synthase related genes CesA3 and application of the clone in resistance detection of CAA bactericides, in particular to a method for detecting drug resistance molecules of phytophora capsic to the CAA bactericides and a special primer and belongs to the technical field of molecular biology. The primer consists of deoxyribonucleic acid (DNA) molecules represented by SEG ID NO:1 and SEG ID NO:2. The method for detecting drug resistance produced phytophora capsic point mutation to the CAA bactericides has the advantages of being high in sensitivity, simple, quick and stable in stality, can be used for quickly and sensitively detecting occurrence and development trends of resistance of the phytophora capsic in fields to the CAA bactericides and early warning resistance diseases, guiding and timely adjusting disease control strategies, and has great significance on effective control of development of the resistance diseases.

Description

technical field [0001] The invention relates to the cloning of Phytophthora capsici cellulose synthase-related gene CesA3 and its application in the monitoring of CAAs fungicide resistance, specifically disclosing a rapid identification of nucleotide point mutations of Phytophthora capsici CesA3 gene and its effect on CAAs Molecular detection methods and special primers for fungicide resistance. It belongs to the technical field of molecular biology. Background technique [0002] Capsicum blight is a worldwide important disease, which was first discovered in the United States in 1918 and has now spread to pepper growing areas all over the world. The pathogen of pepper blight is Phytophthora capsici Leonian, a diploid, filamentous, heterothallic oomycete. Phytophthora capsici has a wide range of hosts and can infect more than 50 species of plants in Solanaceae, Cucurbitaceae, Leguminosae and Pinaceae. There are reports of plant diseases caused by Phytophthora capsici in fi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N9/58C12N15/57C12R1/645
Inventor 刘西莉卢晓红陈磊毕扬庞智黎蔡萌王治文
Owner CHINA AGRI UNIV
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