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Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance

A technology of Phytophthora sojae and tubulin, applied in the field of molecular biology, can solve the problems of long detection period, many human resources and materials, and large workload, and achieve the effect of delaying further development.

Inactive Publication Date: 2013-02-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional biological method refers to the determination of the EC of the agent on the pathogenic bacteria. 50 In order to distinguish sensitive and resistant strains, multiple agents are required to be treated and repeated many times. The detection cycle is long, the workload is heavy, and the human resources and materials are consumed. Moreover, the detection sensitivity of this method is low, and the mutation frequency of drug-resistant strains is required. Above 1%

Method used

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  • Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance
  • Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance
  • Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the sensitivity determination of Phytophthora sojae to benzilamide

[0029] 8 sensitive strains Ps4, Ps6, Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2; 8 resistant strains RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14 -1 and RZ15-1.

[0030] Carrot culture medium: 200g carrots, squeeze the juice, filter through 4 layers of gauze, distill water to 1L, and sterilize with damp heat at 121°C for 20 minutes.

[0031] 1. The experimental steps are as follows:

[0032] 1) Mix benzamid with dimethyl sulfoxide (dimethyl sulfoxide DMSO) to make 10 5 μg / mL stock solution. Dilute benzamid into concentration gradients of 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL and 700 μg / mL, and use 1‰ DMSO as a blank control to determine 8 strains of Phytophthora sojae Sensitivity of susceptible strains. For the sensitivity determination of 8 Phytophthora soybean resistant strains, 1‰DMSO was used as a blank control, and it was measured at 10 5 Inhibition rate at μg / m...

Embodiment 2

[0041] Example 2, Discovery of β-tubulin gene and its corresponding protein mutation site in Phytophthora soybean

[0042] 1. Strains: resistant strains RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1, sensitive strains Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2.

[0043] 2. Method:

[0044] 1) Strain cultivation: use carrot culture medium (200 g of carrots, juice squeezed, filtered with 4 layers of gauze, distilled water to make up to 1 L, 121° C. moist heat sterilization for 20 minutes, after moist heat sterilization for later use) to cultivate Phytophthora soybean at 25° C. The pre-cultured Phytophthora sojae sensitive strains were inoculated on PDA medium covered with cellophane, cultured in the dark at 25°C for 5 days, the mycelia were collected, frozen in liquid nitrogen and stored at -80°C for genomic DNA extraction.

[0045] 2) Genomic DNA of resistant strains and sensitive strains were extracted respectively.

[0046] 3) PCR amplification of the full se...

Embodiment 3

[0054] Embodiment 3, the method for detecting mutation site in Phytophthora soybean

[0055] 1. Primer design

[0056] The strains are the strains PsJMS2 and RZ2-2 used in Example 1.

[0057] The primers are shown in Table 2. Allele specific-PCR primers were designed according to the sequence of the mutant β-tubulin gene. The last base at the 3'-end of the forward primer RZ-FT was consistent with the mutated base. In order to increase the For specificity, a mismatched base was introduced at the second base at the 3'-end of the forward primer (primers RZ-FA, RZ-FC and RZ-FG). The reverse primer is SRZ-A.

[0058] Table 2PCR detects the primers used for Phytophthora sojae strains showing resistance to benzamid

[0059]

[0060]

[0061] The specific reaction system is the same as in Example 2. The primers were subjected to temperature gradient PCR with different annealing temperatures to determine the annealing temperature with high specificity. The reaction program w...

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Abstract

The invention relates to the cloning of a phytophthora sojae beta-microtubulin gene, the detection of the mutation site of the gene and the use of the gene in monitoring the zoxamide resistance, and particularly discloses a molecular detection method for zoxamide resistance caused by mutation of site-716 in the phytophthora sojae beta-microtubulin and special primers thereof. The primer pair consists of DNA molecules represented by SEQID NO:1 and SEQID NO:2, and the polymerase chain reaction (PCR) annealing temperature is 67DEG C. The invention provides a molecular diagnosis method for zoxamide resistance of phytophthora sojae, which has the characteristics of high sensitivity, simplicity, speed and stability, can be used for detecting the zoxamide resistance development trend of field phytophthora sojae with high pass, so that an early warning for medicine resistance can be given. The method has a great significance for control of medicine resistance in pathogenic bacteria.

Description

technical field [0001] The invention relates to the cloning of the β-tubulin gene of Phytophthora sojae and its application in the monitoring of the fungicide benzamid resistance, and specifically discloses a rapid identification of nucleotide point mutations in the β-tubulin gene of Phytophthora sojae Its molecular detection method and special primers for benzamid resistance. It belongs to the technical field of molecular biology. Background technique [0002] Soybean blight is a dangerous and devastating oomycete disease caused by Phytophthora sojae Kaufmann & Gerdemann. It can occur in all growth stages of soybeans, and the susceptible varieties generally lose 25% to 50% of their yield, and the high-susceptibility varieties can reach more than 90%, or even no harvest; and the grain protein content is significantly reduced, and the use value is affected. Since the disease was first discovered in northeastern Indiana, USA in 1948, it has rapidly spread to more than 20 cou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02C12N15/11C12N15/31C07K14/37
Inventor 刘西莉蔡萌陈磊毕扬林东李波涛李健强刘鹏飞
Owner CHINA AGRI UNIV
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