摘要
[目的]本文旨在建立一种利用PCR技术鉴别抗菌脂肽类型和筛选抗菌脂肽产生菌的快速、准确、灵敏的方法。[方法]以枯草芽孢杆菌(Bacillus subtilis)、淀粉液化芽孢杆菌(B.amyloliquefaciens)和多黏类芽孢杆菌(Paenibacillus polymyxa)的典型菌株为研究对象,针对几种代表性脂肽的合成酶基因设计特异性引物进行PCR扩增,并通过测序法验证检测结果的正确性,推测芽孢杆菌生物合成抗菌物质的潜在能力。[结果]PCR结果表明:所有检测菌株均具有Surfactin合成酶调控基因;除P.polymyxa JSa-9外,其他菌株均检测到Fengycin合成酶基因的存在;而Iturin合成酶基因仅在B.amyloliquefaciens ES-2中检测得到;B.subtilis fmbj、B.subtilis fmbR、B.subtilis fmbR-2、B.amyloliquefaciens ES-2和P.polymyxa JSa-9-81均具有Bacillomycin D合成酶调控基因;B.subtilis fmbR、PB2、fmbj、PB198、fmb4、fmb2和fmb3菌株中发现了Subtilosin A合成酶基因的存在;而只有P.polymyxa JSa-9和P.polymyxa JSa-9-81检测到Polymyxin合成酶调控基因。[结论]基于PCR快速检测和测序相结合的方法,我们建立了一种抗菌脂肽产生菌快速识别、筛选和抗菌脂肽快速鉴定的方法,该方法具有快速、灵敏度高、特异性强等特点。
[Objectives]The paper aims to establish the rapid identification method of antimicrobial lipopeptides and screening method of antimicrobial lipopeptide-producing strains based on PCR amplification. [Methods]Bacillus subtilis,B. amyloliquefaciens and Paenibacillus polymyxa were both detected for the research. We took the regulatory genes of lipopeptide synthetases as detecting targets to design specific primers for PCR amplification. Meanwhile the gene sequencing ensured that the PCR provided correct results. So we speculated that they might have the potential ability to synthesize the specific lipopeptides. [Results]Based on the PCR ampification results,it confirmed that all of the strains had the genes required for surfactin biosynthesis. Besides P. polymyxa JSa-9,other strains were positive for the genes of Fengycin synthetases in the PCR amplification. As for the genes of Iturin synthetases,only B. amyloliquefaciens ES-2 was positive for the genes in the detection. Meanwhile,we amplified the genes of Bacillomycin D synthetases in B. subtilis fmbj,B. subtilis fmbR,B. subtilis fmbR-2,B. amyloliquefaciens ES-2 and P. polymyxa JSa-9-81. The genes encoding Subtilosin A were detected in many stains,such as B. subtilis fmbR,PB2,fmbj,PB198,fmb4,fmb2 and fmb3. And P. polymyxa JSa-9 and P. polymyxa JSa-9-81 were detected by the target genes of Polymyxin synthetases. [Conclusions]Based on the above means,we established the rapid identification method of antimicrobial lipopeptides and screening method of antimicrobial lipopeptide-producing strains. Therefore the established PCR technique provides a sensitive,specific,fast and reliable method for the study of antimicrobial lipopeptides.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2016年第5期858-864,共7页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31271828)
关键词
抗菌脂肽
芽孢杆菌
调控基因
检测
antimicrobial lipopeptide
Bacillus
encoding gene
detect
