Method and special primer pair for rapidly identifying drug resistance of phytophthora sojae to dimethomorph

A technology of Phytophthora soybean and primer pairs, which is applied in the field of molecular biology, can solve the problems of long detection cycle, many human resources and materials, and low detection sensitivity, and achieve the effect of delaying further development

Active Publication Date: 2016-08-17
CHINA AGRI UNIV
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Problems solved by technology

These methods are based on the determination of the EC of the agent against the pathogenic bacteria 50 In order to distinguish sensitive and resistant strains, multiple agents are usually required to be treated and repeated many times, so this method has a long detection cycle, a large workload, and consumes more human resources and materials; and this method has low detection sensitivity. , requiring the mutation frequency of drug-resistant strains to be above 1%

Method used

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  • Method and special primer pair for rapidly identifying drug resistance of phytophthora sojae to dimethomorph
  • Method and special primer pair for rapidly identifying drug resistance of phytophthora sojae to dimethomorph
  • Method and special primer pair for rapidly identifying drug resistance of phytophthora sojae to dimethomorph

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, Phytophthora sojae is sensitive to dimethomorph

[0026] Three sensitive strains Ps6, Ps13 and PsJMS2; two resistant strains RX9-1 and RX9-2.

[0027] 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 20min.

[0028] 1. The experimental steps are as follows:

[0029] 1) Dimethomorph was prepared into 5×10 with dimethyl sulfoxide (DMSO) 4 μg / ml stock solution. For the sensitivity determination of three Phytophthora soybean strains, dimethomorph was diluted to a concentration gradient of 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL and 200 μg / mL . For the sensitivity determination of two Phytophthora sojae resistant strains, dimethomorph was serially diluted to 5×10 3 μg / mL, 1×10 4 μg / mL, 1.5×10 4 μg / mL, 2×10 4 μg / mL and 2.5×10 4 Concentration gradient of μg / mL.

[0030] 2) In the order from low to high, use a pipette to draw 480 ...

Embodiment 2

[0038] Example 2. Discovery of cellulose synthase-related gene CesA3 and its corresponding protein mutation site in Phytophthora soybean

[0039] 1. Strains: resistant strains RX9-1 and RX9-2, sensitive strains Ps6, Ps13 and PsJMS2.

[0040] 2. Method:

[0041] 1) Template: Genomic DNA and cDNA of resistant strains and sensitive strains were used as templates respectively.

[0042] 2) PCR amplification of the full sequence of the cellulose synthase-related gene CesA3

[0043] Primer (Table 2): Ps CesA3F (5'-GTTCGTCGTTGCCTTCACC-3')

[0044] Ps CesA3R(5'-TACTGCCTTCCCGTTTCCT-3')

[0045] Reagents from Beijing Quanshijin Biotechnology Co., Ltd. were used. The 50 μL reaction system included 1 μL template DNA (30-50 ng), 4 μL 10 mM dNTP, 5 μL 10× amplification buffer (containing 20 mM Mg 2+ ), 1 μL Taq DNA polymerase (5U / μL), 1 μL of each primer (10 μM), 37 μL ddH 2 O, the reaction system is multiplied or decreased according to the needs of subsequent experiments. The reaction...

Embodiment 3

[0050] Embodiment 3, the method for detecting mutation site in Phytophthora soybean

[0051] 1. Primer design

[0052] The strains are the strains Ps6, RX2-1 and RX9-1 used in Example 1.

[0053] AS-PCR primers were designed according to the sequence of mutant RX9-1 cellulose synthase-related gene CesA3. The primer pair Ps3070F-Ps3070A / T / C / G.R was used to detect the mutation site of G3070C. Wherein, the last base of the 3'-end of the reverse primer Ps3070A.R is the mutated base, and the forward primer is Ps3070F (Table 2). In order 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 2, primers Ps3070T / C / G.R).

[0054] Table 2 detects the PCR primers used for Phytophthora sojae strains showing resistance to dimethomorph

[0055]

[0056] The specific reaction system is the same as that in Example 2, and temperature gradient PCR with different annealing temperatures is performed on the ...

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Abstract

The invention relates to clone of a phytophthora sojae cellulose synthase related gene CesA3 and application thereof in resistance detection and monitoring of dimethomorph, in particular relates to a molecular detection method and a special primer pair for identifying drug resistance of phytophthora sojae to dimethomorph, and belongs to the technical field of molecular biology. The primer pair consists of DNA molecules shown in SEQ ID NO:1 and SEQ ID NO:2, and the PCR annealing temperature of the primer pair is 61 DEG C. A method for detecting point mutation of phytophthora sojae with drug resistance to dimethomorph, which is provided by the invention, has the characteristics of high sensitivity, simplicity, convenience, rapidness and good stability, can be adopted to rapidly and sensitively detect the occurrence and development situation of resistance of field phytophthora sojae to dimethomorph, and has important significance for early warming of resistance to pathogenic bacteria, thereby achieving the purposes that disease control and treatment strategies are instructed and adjusted in time, development of drug resistance of the pathogenic bacteria is effectively controlled, and the market service life of a bactericide is prolonged.

Description

technical field [0001] The invention relates to the cloning of Phytophthora soybean cellulose synthase-related gene CesA3 and its application in the detection and monitoring of dimethomorph resistance, and specifically discloses a method for rapidly identifying the nucleotide point mutation of Phytophthora soybean CesA3 gene and its application. Molecular detection method and special primers for resistance to dimethomorph. 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 the northeast of Indiana in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C07K14/37
CPCC07K14/37C12Q1/6895C12Q2600/156
Inventor 刘西莉蔡萌林东苗建强陈磊毕扬宋晰李健强刘鹏飞张文华
Owner CHINA AGRI UNIV
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