CN105154432B - A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea - Google Patents

A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea Download PDF

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CN105154432B
CN105154432B CN201510593450.0A CN201510593450A CN105154432B CN 105154432 B CN105154432 B CN 105154432B CN 201510593450 A CN201510593450 A CN 201510593450A CN 105154432 B CN105154432 B CN 105154432B
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seq
pathogen
botrytis cinerea
pcr
expand
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CN105154432A (en
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马雪梅
张鑫
吕宝北
赵鹏翔
谢飞
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Beijing University of Technology
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Beijing University of Technology
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Abstract

A kind of method for being used to expand four genes of the pathogen of Botrytis cinerea and its multiple PCR primer group are related to biology field.The present invention is on the basis of known the pathogen of Botrytis cinerea is to the drug resistance mutant site of the various sterilization agent such as benzimidazole, Boscalid class, iprodione and fenhexamid, and a kind of of foundation is used to expand β tub, the PCR primer group of tetra- kinds of genetic fragments of BsOS1, erg27, SdhB simultaneously.The present invention can be expanded directly using the pathogen of Botrytis cinerea genome of extraction as template, obtain four genetic fragments of enrichment with the primer sets of design to template.The present invention is that four drug-resistant mutation genes of the pathogen of Botrytis cinerea are carried out while expanded using multiple PCR method first, and the detection for later stage anti-medicine site greatly reduces workload.The present invention is expanded four genetic fragments by the optimization to PCR reaction systems in same PCR reactions.

Description

A kind of method for being used to expand four genes of the pathogen of Botrytis cinerea and its multiplex PCR draw Thing group
Technical field
The present invention relates to biology field, and in particular to method that is a kind of while expanding the pathogen of Botrytis cinerea and for The primer sets of the resistant mutation gene of four kinds of bactericide.
Background technology
Gray mold be in open country, a kind of fungal disease that protecting field is common and preventing and treating is extremely difficult, its pathogenic bacteria is ash Botrytis (Botrytis Cinerea), belong to Deuteromycotina.The host range of the pathogen of Botrytis cinerea is quite varied, including 200 Common vegetables and fruit, very harmful in agricultural production in various plants, such as the life such as tomato, cucumber, grape.Mesh The preceding prevention and controls for gray mold include cultural control, biological control and chemical prevention etc., wherein still based on chemical prevention. Conventional bactericide includes benzimidazole, dicarboximide class, N- carbanilic acids esters, pyrimidine fungicides etc..
The pathogen of Botrytis cinerea has the characteristics that breeding is fast, hereditary variation is big and grade of fit is high, and various sterilization agent is generated A certain degree of resistance, or even generate multiple resistance.The main reason for being developed immunity to drugs to bactericide is pharmaceutically-active target Point mutation occurs for locus gene so that bactericide can not play the effect of its script.Choosing of the bacterial strain in field in various sterilization agent Select under pressure, it is most likely that produce a variety of cross resistances, therefore dashed forward for a kind of specific resistance of bactericide of the pathogen of Botrytis cinerea Become the needs that detection has been insufficient for agricultural production, detection is very while realizing it to various sterilization agent resistant mutation Significant.
The reason for the pathogen of Botrytis cinerea produces resistance to benzimidazole germicide and N- pyrimidine fungicides bactericide be β- The mutation such as E198A, E198V, E198K, F200Y occurs for microtubule protein gene (β-tub)；Dicarboximide fungicide is produced The reason for resistance be histidine kinase gene (BcOS1) occur I365S, I365R, I365N, Q369P, V368F+Q369H, Q369P+N373S, N373S etc. are mutated；The reason for producing resistance to fenhexamid bactericide is chlC4 gene (erg27) mutation such as F412S, F412I, F412V occurs；The reason for producing resistance to Boscalid bactericide is butanedioic acid dehydrogenation The mutation such as H272Y, H272R, H272L, P225T, P225F, P225L, N230I occurs for enzyme iron-sulfur protein gene (SdhB).This hair Bright is on the basis of known the pathogen of Botrytis cinerea is to mutational sites such as the resistances to the action of a drug of various sterilization agent, and design primer sets expand simultaneously Tetra- β-tub, BcOS1, erg27, SdhB genetic fragments, can be as the mould of the high-flux detection methods such as follow-up genetic chip Plate, so as to realize the pathogen of Botrytis cinerea to being detected while four kinds of bactericide resistant mutations.
The content of the invention
The present invention is more to benzimidazole, Boscalid class, iprodione and fenhexamid etc. in known the pathogen of Botrytis cinerea On the basis of the drug resistance mutant site of kind bactericide, one kind of foundation is used to expand β-tub, BcOS1, erg27, SdhB simultaneously The PCR primer group of four kinds of genetic fragments.The present invention can be directly using the pathogen of Botrytis cinerea genome of extraction as template, with design Primer sets template is expanded, obtain four genetic fragments of enrichment.The present invention is first using multiple PCR method to ash Four drug-resistant mutation genes of botrytis expand simultaneously, and the detection for later stage anti-medicine site greatly reduces work Amount.
The present invention is devised for expanding the pathogen of Botrytis cinerea β-tub, BcOS1, erg27, SdhB tetra- genetic fragments are drawn Thing group, by the optimization to PCR reaction systems, four genetic fragments are expanded in same PCR reactions.
The advantages of present invention is prominent be：(1) expand first in same PCR reactions the pathogen of Botrytis cinerea β-tub, BcOS1, Tetra- genetic fragments of erg27, SdhB, the resistance mutation sites to most bactericide are contained in four genetic fragments, are examined for the later stage Survey the pathogen of Botrytis cinerea reduces workload to the resistance to the action of a drug of various sterilization agent.(2) non-specific amplification in reacting is few, to follow-up Detection do not influence.
A kind of method for expanding four genes of the pathogen of Botrytis cinerea, it is characterised in that altogether in two steps：
The first step：Extract the pathogen of Botrytis cinerea genome
Second step：Enter performing PCR reaction：Using four pairs of primers, be respectively used for amplifying the pathogen of Botrytis cinerea β-tub, BcOS1, Erg27, SdhB gene, four pairs of primers are respectively
SEQ ID NO:1CGATACCGTTGTCGAGCCAT
SEQ ID NO:2GGTTGCTGAGCTTCAAGGTT
SEQ ID NO:3ACACCGACCCAGCACCAGA
SEQ ID NO:4TTAGCAATAACCGCCCAAA
SEQ ID NO:5CACCCGCGTAGCAAGAGATG
SEQ ID NO:6TGAGTCAGGTCTCCCTTTGC
SEQ ID NO:7ATCATGCCCTTGAATTTTGTG
SEQ ID NO:8CTTTGCCTCCCCTTTCTTCTC
Contain the template that the genomic DNA of the testing sample of extraction reacts as PCR, reactant in PCR reaction mixtures System is as follows,
PCR response procedures：94℃5min；94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec；72℃10min.
More specifically the object of the present invention is achieved like this：
Primer sets that are a kind of while expanding the anti-medicine phases correlation gene of four the pathogen of Botrytis cinerea, its system include four pairs of multiplex PCRs Amplimer, it is respectively used for amplifying tetra- the pathogen of Botrytis cinerea β-tub, BcOS1, erg27, SdhB genetic fragments；PCR reaction groups Point, purchased from invitrogen companies, including Taq enzyme, dNTPs, PCR buffer solutions (are free of Mg2+), MgCl2.Using UNIQ-10 posts Formula fungal genomic DNA extraction agent box, purchased from Sangon Biotech (Shanghai) Co., Ltd., extract the base of testing sample Because of a group DNA.
Primer sequence used is respectively：SEQ ID NO:1CGATACCGTTGTCGAGCCAT, SEQ ID NO: 2GGTTGCTGAGCTTCAAGGTT, for expanding β-tublin genes；SEQ ID NO:3ACACCGACCCAGCACCAGA, SEQ ID NO:4TTAGCAATAACCGCCCAAA, for expanding SdhB gene；SEQ ID NO:5CACCCGCGTAGCAAGAGATG, SEQ ID NO:6TGAGTCAGGTCTCCCTTTGC, for expanding BcOS1 genes；SEQ ID NO: 7ATCATGCCCTTGAATTTTGTG, SEQ ID NO:8CTTTGCCTCCCCTTTCTTCTC, for expanding Erg27 genes.
Experimental group and control group PCR reactions are carried out, the testing sample containing extraction wherein in experimental group PCR reaction mixtures The template reacted as PCR of genomic DNA, reaction system is as follows,
Distilled water (the ddH of equivalent is added in control group PCR reaction mixtures2O) the template as PCR reactions, reactant System is as follows：
PCR response procedures：94℃5min；94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec, 35cycles；72℃ 10min.After PCR terminates, 5 μ L reaction solutions are taken to carry out Ago-Gel (2%) electrophoresis, testing result.
Brief description of the drawings
Fig. 1 electrophoresis detection result figures of the embodiment of the present invention.
Embodiment
The multiplexed PCR amplification of embodiment 1, bacterial strain sample
It is limited purchased from raw work bioengineering (Shanghai) share using UNIQ-10 pillar fungal genomic DNA extraction agent boxes Company, extract the genomic DNA of 5 plants of bacterial strains to be detected.PCR reactive components, purchased from invitrogen companies, including Taq enzyme, DNTPs, PCR buffer solution (are free of Mg2+), MgCl2。
Experimental group and control group PCR reactions are carried out, the testing sample containing extraction wherein in experimental group PCR reaction mixtures The template reacted as PCR of genomic DNA, reaction system is as follows,
Distilled water (the ddH of equivalent is added in control group PCR reaction mixtures2O) the template as PCR reactions, reactant System is as follows：
PCR response procedures：94℃5min；94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec；72℃10min.
After PCR terminates, 5 μ L reaction solutions are taken to carry out electricity in Ago-Gel (2%, i.e. 0.6g solutes are dissolved in 30ml solvents) Swimming, testing result.As shown in figure 1, loading wells M is DNA marker, loading wells 1-5 is detection sample, and loading wells 6 is negative right According to group.According to electrophoresis result, primer of the invention can Successful amplification go out tetra- β-tub, BcOS1, erg27, SdhB gene pieces Section.

Claims (2)

  1. A kind of 1. method for expanding the anti-medicine phases correlation gene of the pathogen of Botrytis cinerea four, it is characterised in that altogether in two steps：
    The first step：Extract the pathogen of Botrytis cinerea genome
    Second step：Enter performing PCR reaction：Using four pairs of primers, be respectively used for amplifying the pathogen of Botrytis cinerea β-tub, BcOS1, erg27, SdhB gene, four pairs of primers are respectively
    SEQ ID NO:1 CGATACCGTTGTCGAGCCAT
    SEQ ID NO:2 GGTTGCTGAGCTTCAAGGTT
    SEQ ID NO:3 ACACCGACCCAGCACCAGA
    SEQ ID NO:4 TTAGCAATAACCGCCCAAA
    SEQ ID NO:5 CACCCGCGTAGCAAGAGATG
    SEQ ID NO:6 TGAGTCAGGTCTCCCTTTGC
    SEQ ID NO:7 ATCATGCCCTTGAATTTTGTG
    SEQ ID NO:8 CTTTGCCTCCCCTTTCTTCTC
    Contain the template that the genomic DNA of the testing sample of extraction reacts as PCR in PCR reaction mixtures, reaction system is such as Under,
    PCR response procedures：94℃5min；94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec；72℃10min.
  2. A kind of 2. multiple PCR primer group for being used to expand the anti-medicine phases correlation gene of the pathogen of Botrytis cinerea four, corresponding to the grey grape of amplification Spore bacterium β-tub, BcOS1, erg27, SdhB gene, four pairs of primers are respectively
    SEQ ID NO:1 CGATACCGTTGTCGAGCCAT
    SEQ ID NO:2 GGTTGCTGAGCTTCAAGGTT
    SEQ ID NO:3 ACACCGACCCAGCACCAGA
    SEQ ID NO:4 TTAGCAATAACCGCCCAAA
    SEQ ID NO:5 CACCCGCGTAGCAAGAGATG
    SEQ ID NO:6 TGAGTCAGGTCTCCCTTTGC
    SEQ ID NO:7 ATCATGCCCTTGAATTTTGTG
    SEQ ID NO:8 CTTTGCCTCCCCTTTCTTCTC。
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MX2017003228A (en) * 2014-09-11 2017-06-19 Agrofresh Inc Methods for pathogen detection and disease management on meats, plants, or plant parts.
CN106119364A (en) * 2016-07-02 2016-11-16 北京工业大学 A kind of based on suspension chip system for the multiple detection method of Botrytis cinerea drug resistance related locus
CN108018374A (en) * 2017-12-29 2018-05-11 华中农业大学 For detecting drug-fast kit of the Botrytis cinerea to benzimidazole germicide
CN109338004B (en) * 2018-11-12 2021-06-22 上海市农业科学院 Primer combination, kit and method for detecting resistance of botrytis cinerea to boscalid
CN115786560B (en) * 2022-08-12 2024-03-22 上海市农业科学院 Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of Botrytis cinerea

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