基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究  

Screening and identification of an epiphytic bacterium producing lipopeptides based on NRPS gene

作　　者：王傲[1] 杨柯[1] 吕曼[1] 史雅凝 辛志宏[1] 

机构地区：[1]南京农业大学食品科技学院,江苏南京210095

出　　处：《食品工业科技》2017年第12期164-170,共7页Science and Technology of Food Industry

基　　金：2016年农产品质量安全风险评估项目(GJFP201601205)

摘　　要：本研究采用平板稀释法和斜面分离纯化技术,从夏黑葡萄中筛选分离到5株附生细菌,提取菌株的基因组DNA,PCR扩增16S r DNA序列,产物纯化后进行克隆测序,利用MEGA 6.0软件对序列进行同源性比对分析,构建系统进化树后,将5株附生细菌分别鉴定为克雷伯肺炎杆菌(Klebsiella pneumoniae)、枯草芽孢杆菌(Bacillus subtilis)、克考氏菌(Kocuria marina)、嗜气芽孢杆菌(Bacillus aerophilus)、类芽孢杆菌(Paenibacillus agaridevorans)。在此基础上,以非核糖体多肽合成酶基因(non-ribosomal peptide compounds synthetase,NRPS)为靶点,筛选到含有目的条带的菌株PTWA2,经系统发育树预测该菌株能够产生环脂肽类化合物。该菌株发酵产物经有机溶剂萃取、常压硅胶柱层析、Sephedex LH-20等分离手段纯化得到一个单体化合物,采用核磁共振和质谱方法鉴定该化合物为环脂肽Surfactin A。该研究结果为以功能基因定向筛选产生环脂肽化合物的菌株提供了新的研究方法与理论依据。In this study, five epiphytic bacteria were isolated from ＂ summer black＂ grapes using plate streaking and slant culture methods.After extraction of DNA, 16S rDNA from the five strains were anlplified for sequence homology analysis, and the phylogenetic trees were constructed with MEGA 6.0.The results showed that the five strains were characterized as Klebsiella pneumoniae , Bacillus subtilis , Kocuria marina, Bacillus aerophilus , Paenibacillus agaridevorans , respectively. On this basis, the strain PTWA2 was screened with the non-ribosomal peptide compounds synthetase （NRPS）gene.Aftcr constmting the NRPS phylogenetie tree,the results showed that the PTWA2 was capable of producing lipopeptide.As a result, a pure compound was obtained from the fermentation product of the PTWA2 by solvent extraction, silica gel column chromatography and Sephedex LH-20 chromatography and its chemical sturture was identified as surfactin A by physioehemical analysis and spectroscopic techniques.The study highlights new research methods and theoretical basis for target-screening strains which produced cyclic lipopeptide.

关 键 词：附生菌 16S RDNA 序列分析 非核糖体多肽合成酶 系统发育树 结构鉴定 

分 类 号：TS201.2[轻工技术与工程—食品科学]