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Method for detecting gene nucleotide mutation site of rice ustilaginoidea virens cyp51

A technology of rice smut bacteria and mutation sites, applied in the field of molecular biology, can solve the problems of heavy workload, low sensitivity, and long time-consuming, and achieve the effect of heavy workload, high accuracy, and long cycle

Inactive Publication Date: 2017-06-27
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional determination methods all need to prepare the culture medium containing the drug and calculate the effective inhibitory medium concentration, which takes a long time, a large workload and low sensitivity.

Method used

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  • Method for detecting gene nucleotide mutation site of rice ustilaginoidea virens cyp51
  • Method for detecting gene nucleotide mutation site of rice ustilaginoidea virens cyp51
  • Method for detecting gene nucleotide mutation site of rice ustilaginoidea virens cyp51

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Discovery of cyp51 gene and CYP51 protein mutation sites in rice smut.

[0034] 1. Strains: The resistant strains are F10-338C and F10-338D, and the sensitive strains are F10-338, JY13107, NH13018, SX13035, and NH13052.

[0035] 2. Method:

[0036] 1) Strain culture: culture the strain on PSA medium at 28°C for 5-7 days, take 7-10 bacterial cakes and shake them in PSB liquid medium for 5 days, filter the mycelium balls, and remove the cultured solution, freeze-dried in a freeze dryer and stored at -20°C.

[0037] 2) Genomic DNA of resistant strains and sensitive strains were extracted respectively.

[0038] 3) Genomic RNA of resistant strains and sensitive strains were extracted respectively.

[0039] 4) RNA reverse transcription.

[0040] 5) PCR amplification of lanosterol 14α-demethylase cyp51 gene

[0041] Table 1 Cloning primers of lanosterol 14α-demethylase cyp51 gene

[0042]

[0043] The primers used are S-4 / A-3 in Table 1.

[0044] PCR react...

Embodiment 2

[0058] Embodiment 2, the detection of mutation site in rice smut bacterium

[0059] Experiment 1. Primer design

[0060] Primers are listed in Table 2.

[0061] Table 2. Primers used in AS-PCR detection of resistance of rice smut to tebuconazole

[0062]

[0063] In this method, the last base of the downstream primer is consistent with the mutant (Table 2, primer Uv137AR), and the penultimate base introduces a mismatched base (Table 2, primers Uv137BR, Uv137CR, Uv137DR), and combined with different annealing Amplification at a higher temperature increases the specificity of the primers. The reaction conditions are as follows:

[0064] The PCR reaction system is as follows: 25 µL reaction system includes: 0.5 µL Taq DNA polymerase (2.5 U / µL), 2.5 µL 10×PCR buffer (containing Mg 2+ ), 2.0 μL 10mM dNTPs, 0.5 μL each primer, 1 μL template DNA, 18 μL ddH 2 O.

[0065] PCR reaction conditions: pre-denaturation at 94°C for 3min, 98°C for 10s, 44-56°C for 30s, 68°C for 45s, 3...

Embodiment 3

[0073] Embodiment 3, the rice false smut bacteria detection of resistance to tebuconazole

[0074] The PCR amplification system and reaction conditions were consistent with Experiment 1 in Example 2, and the annealing temperature was 51.6°C.

[0075] The rice curve balls to be tested are: rice curve balls produced by inoculating rice plants with resistant strains F10-338C and F10-338D respectively; rice curve balls produced by inoculating rice plants with sensitive strains F10-338, JY13107, NH13018 and SX13035 respectively. The rice curved balls were quickly ground with liquid nitrogen, and then the DNA molecules in the rice curved balls were extracted using a DNA extraction kit.

[0076] The result is as Figure 4 shown. The results showed that the primers Uv137F1 / Uv137DR could only amplify a specific 391 bp band (lane 1, 2) from the rice curve balls produced by inoculating rice plants with a strain resistant to tebuconazole, but not from a sensitive strain. Specific 391 b...

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Abstract

The invention provides a method for detecting a gene nucleotide mutation site of rice ustilaginoidea virens cyp51, belongs to the technical field of molecular biology and relates to a primer for detecting the gene nucleotide mutation site of the rice ustilaginoidea virens cyp51. Sequences of the primer are shown as SEQID No.1 and SEQID No.2. The method for detecting a tebuconazole resistance mutation site of rice ustilaginoidea virens has the advantages of being high in sensitivity, simple, rapid, good in stability and wide in applicability, can effectively distinguish sensitive strains and drug-resistant strains and is used for detecting tebuconazole resistance occurrence and development trend of the rice ustilaginoidea virens. The molecular detection method has the important significance on early warning of drug resistance of the rice ustilaginoidea virens, formulation of a reasonable disease management scheme and effective control of drug resistance development of diseases.

Description

technical field [0001] The invention relates to a method for detecting the nucleotide mutation site of rice smut cyp51 gene. It belongs to the technical field of molecular biology. Background technique [0002] Rice false smut (False smut of rice) is commonly known as blue powdery mildew, black ball disease, and grain flower disease. It is a fungal disease on the ear of rice. ". Rice false smut mainly occurs on the spikes of rice at the mature stage. The sexual state of the pathogen (Villosiclava virens) belongs to the genus Ergot of the Ascomycota phylum Ascomycetes, and the asexual state (Ustilaginoidea virens) belongs to the genus Chlorosclerotinia of the half-knowledge phylum Coelospora. Since the 1980s, with the vigorous promotion of hybrid rice, upright and dense-panicle high-yielding rice varieties in my country and the improvement of farmland fertility, rice false smut has risen from a minor disease to a major disease in my country's rice production. The diseased ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
CPCC12Q1/6895C12Q2600/156
Inventor 石妞妞杜宜新陈福如阮宏椿杨秀娟甘林代玉立
Owner INST OF PLANT PROTECTION FAAS
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