CN101988122B - Genotyping detection method of drug resistance to Sanmate of Fusarium graminearum - Google Patents
Genotyping detection method of drug resistance to Sanmate of Fusarium graminearum Download PDFInfo
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Abstract
The invention belongs to a Sanmate resistance genotype molecular detection and SNP typing method of Fusarium graminearum, which can be used for drug resistance monitoring and drug resistance genotype diagnosis to Sanmate and other benzimidazole type bactericides of the Fusarium graminearum which can cause wheat scab. The detection method comprises the following three major steps: (1) extracting DNA of a nuclear genome of a strain to be detected; (2) applying three groups of primer pairs to carry out PCR reaction; and (3) identifying the genotype of the strain with the resistance to the Sanmate according to a PCR product electrophoretogram. By adopting the Tetra-primer ARMS PCR (Tetra-primer amplification refractorymutation system-PCR) technology, the strain with the drug resistance of the Fusarium graminearum and the genotype thereof can be fast and accurately detected, and the level of the drug resistance can be further judged. The detection accuracy is above 95%.
Description
One, technical field
The invention belongs to a kind of Molecular Detection and SNP classifying method of Fusarium graminearum carbendazim resistance strain gene type, can be used for diagnosis and the development trend monitoring of resistance cause of disease colony of benzimidazole germicide (derosal) resistance Fusarium graminearum, carry out the Forecasting of Epiphytotics of Headblight of Wheat of different Detection of insecticide resistances; And can carry out the SNP somatotype, the theoretical foundation of molecular level is provided for resistance management.
Two, technical background
Wheat scab is a kind of worldwide disease that is caused by Fusarium graminearum Fusarium graminearum Schwabe, has a strong impact on the yield and quality of wheat.Adopt for a long time benzimidazole germicide or carry out chemical prevention take this class medicament as main built agent.Benzimidazole germicide is efficient as a class, the wide spectrum systemic fungicide is used on producing, solved the environmental toxicity problem of protective fungicide, has improved the ability that the mankind control this disease.Benzimidazole germicide comprises derosal, F-1991, thiabendazole, thiophanate etc.These sterilant have identical antimicrobial spectrum and antibacterial mechanisms, and they also have identical resistance mechanism, have each other the quadrature transreactance property of medicine.Due to the height specialization of this class medicament, action site is single, adds that frequency of administration is high, the resistance dominant race will occur in many plant pathogenic fungis colony after using for many years, makes chemical control lose effect fully.Through effort in recent years, Agricultural University Of Nanjing's sterilant laboratory study finds that fusarium graminearum is mainly that the sudden change of the 167th, 198 of this genes encodings, 200 amino acids codons can cause that all this bacterium is to the generation of carbendazim resistance because Fusarium graminearum β 2-microtubule protein gene (FGSG_06611.3) sudden change causes to the drug-fast strain of derosal.Known coded 167 amino acids codon mutations are major causes that pathogenic bacteria produces field drug-fastness, account for more than 97% of all mutation type total amounts, performance medium level resistance; The 198 amino acids codon mutations of encoding account for 0.5% left and right of drug resistance gene type, cash high-level resistance; Detection of insecticide resistance and 167 sudden changes of 200 amino acids codon mutations of encoding are similar.
The contriver studies have shown that because of pathogenic bacteria in natural enormous amount, in Fusarium graminearum colony the ratio of resistance individuality reach 3%～5% and the envrionment conditions of suitable morbidity under, it is popular that the resistance wheat scab can occur, be the chemical control failure.Traditional resistance detection method be according to the medicament various dose, the retarding effect of mycelial growth is differentiated drug-fast, length consuming time, the workload of measuring Detection of insecticide resistance is larger, and is can't early prediction resistance disease popular.The present invention is on the basis of resistance Mechanism Study, according to the dependency of β 2-microtubule protein gene point mutation type and Detection of insecticide resistance, use Tetra-primer ARMS round pcr and detect Fusarium graminearum to the resistance of the benzimidazole germicides such as derosal.Tetra-primer ARMS round pcr is more accurate than allele-specific PCR, and outer primer is as positive control, the inner primer different genotype product that can increase specifically.Therefore, the outstanding advantages that has of the present invention is: (1) accuracy is high.By inside and outside two pairs of primer amplification DNA specific fragments, can prevent possible false positive.(2) can diagnose the gene mutation type of drug-fast strain, the judgement Detection of insecticide resistance is for resistance management provides foundation; (3) the resistance detection is consuming time short, only needs 6 hours.
Three, summary of the invention
Technical problem the object of the present invention is to provide a kind of quick diagnosis and molecular detecting method of Fusarium graminearum carbendazim resistance strain gene type.The present invention is on the basis of and three kinds of point mutation types of known existence machine-processed to carbendazim-resistance the research Fusarium graminearum, first the Tetra-primer ARMS round pcr of research and development.The transgenation mechanism possible according to resistant strain, use three groups of primers to detect quickly and accurately fusarium graminearum carbendazim resistance subpopulation, Detection accuracy reaches more than 95%, and is popular in time taking scientific and effective measure to control this season resistance disease, has practical value.
Technical scheme β 2-microtubule protein gene (FGSG_06611.3) is as the detection gene of Fasarium graminearum for resisting carbendazim, and the codon origination point sudden change of its 167th, the 198th of coding, the 200th amino acids is as the foundation (table 1) of Molecular Detection.
The genotyping detection method of Fusarium graminearum carbendazim resistance gene type was divided into for three steps:
The first step: adopt conventional phenol chloroform isoamyl alcohol method, extract respectively the nuclear DNA of testing sample.
Second step: use three groups of primer pairs, carry out the PCR reaction, obtain three groups of PCR products.
The primer that is used for the detection of Tetra-primer ARMS round pcr has specificity, and this primer pair can be to β
2167,198 of-tubulins, 200 site mutations carry out specific recognition (table 1).
1. Fusarium graminearum β increases
2-microtubule protein gene segment comprises the Auele Specific Primer pair of 167 bit codons:
F-out167 5′GACCACCTTCAGGGTTTCCAGCTGACGC3′
R-out167 5′GATCGGCGTACGAAGGATCGGCGATCTT3′
F-in167T 5′GTTCCCCGATCGCATGATGGCCACTTT3′
R-in167A 5′GAAACCTTGGGCGAGGGCATAACGGGAT3′
2. Fusarium graminearum β increases
2-microtubule protein gene segment comprises the Auele Specific Primer pair of 198 bit codons:
F-out198 5′AGCTCGTTGAGGAAGCCATTGATGTT3′
R-out198 5′CATGTTAACAGCGAGCTTTCGCAGAT3′
F-in198A 5′GAACCAGCTCGTCGAGAACTCTGCCA3′
R-in198G 5′GAGCCTCGTTATCGATACAGAAGGTATC3′
3. Fusarium graminearum β increases
2-microtubule protein gene segment comprises the Auele Specific Primer pair of 200 bit codons:
F-out200 5′ACAACTGGGCCAAGGGTCATTACACC3′
R-out200 5′AAGTCGGGGGAACGGAATCATGTTAAC3′
F-in200T 5′CCAGCTCGTCGAGAACTCTGACGAGACTTT3′
R-in200A 5′TCGTACAGAGCCTCGTTATCGATACGGT3′
PCR reaction system and amplification program:
(1) comprise the gene fragment amplification of 167 bit codons:
Reaction system:
Amplification condition:
(2) comprise the gene fragment amplification of 198 bit codons:
Reaction system:
Amplification condition:
(3) comprise the gene fragment amplification of 200 bit codons:
Reaction system;
Amplification condition:
The 3rd step: with the PCR product electrophoresis that obtains, can precise Identification strain gene type according to electrophoretogram, and judge the bacterial strain Detection of insecticide resistance with this.
Molecular Detection and the SNP classifying method of beneficial effect Fusarium graminearum carbendazim resistance of the present invention strain gene type, compared with prior art:
1. at present domestic and international research unit all adopts the mycelial growth method to detect the Fusarium graminearum resistance, takes at least 6 days but the method relates to sampling, separation and Culture needs 3～5 days and indoor a large amount of insecticide sensitivity determination experiment, and the cycle is longer.Not yet there is at present ripe resisting carbendazim fusarium graminearum molecular detection technology to use.Therefore, the research of Tetra-primer ARMS round pcr is used and has been filled up this blank, can detect early stage in morbidity, gains time in time taking rational control strategy.
2. traditional resistance detection method is commonly used differentiates that dosage detects, and can only distinguish drug-fast strain and sensitive strain, can not determine Detection of insecticide resistance.This technology can be carried out the SNP somatotype to being positioned at the 167th of β 2-microtubule protein gene, the 198th, the 200th bit codon, accurately judges Detection of insecticide resistance, can provide theoretical foundation for the resistance management technology of formulating science.
Four, description of drawings
Fig. 1 Tetra-primerARMS-PCR detects β
2The electrophorogram of-microtubule protein gene the 167th bit codon mutation gene type
Fig. 2 Tetra-primerARMS-PCR detects β
2The electrophorogram of-microtubule protein gene the 198th bit codon mutation gene type
Fig. 3 Tetra-primerARMS-PCR detects β
2The electrophorogram of-microtubule protein gene the 200th bit codon mutation gene type
Five, embodiment
The genotype strain (167) of embodiment 1, detection the 167th bit codon origination point sudden change
Extract bacterial strain ZF-43, ZF-21, ZF-52, the R9 genomic dna comprises the primer amplified pair of 167 bit codon DNA fragmentations with the Fusarium graminearum β 2-microtubule protein gene that increases:
F-out167 5′GACCACCTTCAGGGTTTCCAGCTGACGC3′
R-out167 5′GATCGGCGTACGAAGGATCGGCGATCTT3′
F-in167T 5′GTTCCCCGATCGCATGATGGCCACTTT3′
R-in167A 5′GAAACCTTGGGCGAGGGCATAACGGGAT3′
Contain DNA profiling 2 μ l in 25 μ lPCR systems, 2.5 μ l10 * buffer, 2 μ l dNTP (2.5mM each), 1.5 μ lMgCl2 (25mM), each 0.2 μ l of F-out167/R-out167 (10 μ M), F-in167T (10 μ M) 0.5 μ l, R-in167A (10 μ M) 0.8 μ l, (Dalian is precious biological, Takara) for TaqDNA polysaccharase 1U.Reaction is carried out on PTC-200 (Bio-rad, Inc.), reaction conditions: 94 ℃ of 5min; 94 ℃ of 15s, 66 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 5min; 4 ℃ of preservations.Get 8 μ lPCR products electrophoresis on 2% agarose TBE glue, ultraviolet visualization is taken pictures and is obtained electrophoretogram (Fig. 1).
Detected result is consistent with expection, all bacterial strains all obtain a 292bp with reference to fragment, sensitive strain (ZF-43, ZF-21) amplification obtains a 201bp specific fragment, in antibiotic strain (ZF-52, R9) amplification obtain the specific fragment of a 146bp.
The genotype strain (198) of embodiment 2, detection the 198th bit codon origination point sudden change
Extract bacterial strain ZF43, ZF-21, J2, the ZF43-6 genomic dna comprises the primer amplified pair of 198 bit codon DNA fragmentations with the Fusarium graminearum β 2-microtubule protein gene that increases:
F-out198 5′AGCTCGTTGAGGAAGCCATTGATGTT3′
R-out198 5′CATGTTAACAGCGAGCTTTCGCAGAT3′
F-in198A 5′GAACCAGCTCGTCGAGAACTCTGCCA3′
R-in198G 5′GAGCCTCGTTATCGATACAGAAGGTATC3′
Contain DNA profiling 2 μ l in 25 μ lPCR systems, 2.5 μ l10 * buffer, 2 μ ldNTP (2.5mM each), 1.5 μ lMgCl2 (25mM), each 0.4 μ l of F-out198/R-out198 (10 μ M), F-in198A (10 μ M) 0.15 μ l, R-in198G0.4 μ l, (Dalian is precious biological, Takara) for TaqDNA polysaccharase 1U.Reaction is carried out on PTC-200 (Bio-rad, Inc.), reaction conditions: 94 ℃ of 5min; 94 ℃ of 15s, 65 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 5min; 4 ℃ of preservations.Get 8 μ l PCR products electrophoresis on 2% agarose TBE glue, ultraviolet visualization is taken pictures and is obtained electrophoretogram (Fig. 2).
Detected result is consistent with expection, all bacterial strains all obtain a 440bp with reference to fragment, sensitive strain (ZF-43, ZF-21) amplification obtains a 288bp specific fragment, high resistance bacterial strain (J2, ZF43-6) amplification obtains the specific fragment of a 206bp.
The genotype strain (200) of embodiment 3, detection the 200th bit codon origination point sudden change
Extract bacterial strain ZF43, ZF-21, NT-7, the t1 genomic dna comprises the DNA fragmentation primer amplified pair of 200 bit codons with the Fusarium graminearum β 2-microtubule protein gene that increases:
F-out200 5′ACAACTGGGCCAAGGGTCATTACACC3′
R-out200 5′AAGTCGGGGGAACGGAATCATGTTAAC3′
F-in200T 5′CCAGCTCGTCGAGAACTCTGACGAGACTTT3′
R-in200A 5′TCGTACAGAGCCTCGTTATCGATACGGT3′
Contain DNA profiling 2 μ l in 25 μ lPCR systems, 2.5 μ l10 * buffer, 2 μ l dNTP (2.5mM each), 1.5 μ lMgCl2 (25mM), each 0.8 μ l of F-out200/R-out200 (10 μ M), each 0.5 μ l of F-in200T/R-in200A (10 μ M), (Dalian is precious biological, Takara) for TaqDNA polysaccharase 1U.Reaction is carried out on PTC-200 (Bio-rad, Inc.), reaction conditions: 94 ℃ of 5min; 94 ℃ of 15s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 5min; 4 ℃ of preservations.Get 8 μ lPCR products electrophoresis on 2% agarose TBE glue, ultraviolet visualization is taken pictures and is obtained electrophoretogram (Fig. 3).
Detected result is consistent with expection, all bacterial strains all obtain a 494bp with reference to fragment, sensitive strain (ZF-43, ZF-21) amplification obtains a 221bp specific fragment, in antibiotic strain (NT-7, t1) amplification obtain the specific fragment of a 331bp.
Table 1 Fusarium graminearum source, to carbendazim resistance phenotype and resistance phenotype and genotypic relation
* ZF-43-6 is the induction of resistance bacterial strain.
Claims (1)
1. one kind is used for the primer pair that Tetra-primer ARMS round pcr detects Fusarium graminearum (Fusarium graminearum) carbendazim-resistance strain gene type, it is characterized in that: described primer pair can carry out specific recognition to 167 of β 2-tubulins, 198,200 site mutations, and its sequence is:
1. the Fusarium graminearum β 2-microtubule protein gene segment that increases comprises the Auele Specific Primer pair of 167 bit codons:
F-out167 5′GACCACCTTCAGGGTTTCCAGCTGACGC3′
R-out167 5′GATCGGCGTACGAAGGATCGGCGATCTT3′
F-in167T 5′GTTCCCCGATCGCATGATGGCCACTTT3′
R-in167A 5′GAAACCTTGGGCGAGGGCATAACGGGAT3′
2. the Fusarium graminearum β 2-microtubule protein gene segment that increases comprises the Auele Specific Primer pair of 198 bit codons:
F-out198 5′AGCTCGTTGAGGAAGCCATTGATGTT3′
R-out198 5′CATGTTAACAGCGAGCTTTCGCAGAT3′
F-in198A 5′GAACCAGCTCGTCGAGAACTCTGCCA3′
R-in198G 5′GAGCCTCGTTATCGATACAGAAGGTATC3′
3. the Fusarium graminearum β 2-microtubule protein gene segment that increases comprises the Auele Specific Primer pair of 200 bit codons:
F-out200 5′ACAACTGGGCCAAGGGTCATTACACC3′
R-out200 5′AAGTCGGGGGAACGGAATCATGTTAAC3′
F-in200T 5℃CAGCTCGTCGAGAACTCTGACGAGACTTT3′
R-in200A 5′TCGTACAGAGCCTCGTTATCGATACGGT3′。
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| CN103409533B (en) * | 2013-08-15 | 2016-01-13 | 浙江大学 | Detect primer and the method for the F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences |
| CN103436628B (en) * | 2013-09-23 | 2014-10-29 | 南京农业大学 | Method for rapidly detecting moderately-resistant strain in fusarium graminearum to carbendazim |
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| CN104911252A (en) * | 2015-01-25 | 2015-09-16 | 江苏发士达生物科技有限公司 | Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof |
| CN104911252B (en) * | 2015-01-25 | 2016-02-17 | 江苏发士达生物科技有限公司 | For detecting the primer pair for the treatment of AIDS medicine 3TC and FTC resistant mutational site and probe and application thereof |
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