• EI
  • Scopus
  • 中国科技期刊卓越行动计划项目资助期刊
  • 北大核心期刊
  • DOAJ
  • EBSCO
  • 中国核心学术期刊RCCSE A+
  • 中国精品科技期刊
  • JST China
  • FSTA
  • 中国农林核心期刊
  • 中国科技核心期刊CSTPCD
  • CA
  • WJCI
  • 食品科学与工程领域高质量科技期刊分级目录第一方阵T1
中国精品科技期刊2020

基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究

王傲, 杨柯, 吕曼, 史雅凝, 辛志宏

王傲, 杨柯, 吕曼, 史雅凝, 辛志宏. 基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究[J]. 食品工业科技, 2017, (12): 164-170. DOI: 10.13386/j.issn1002-0306.2017.12.030
引用本文: 王傲, 杨柯, 吕曼, 史雅凝, 辛志宏. 基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究[J]. 食品工业科技, 2017, (12): 164-170. DOI: 10.13386/j.issn1002-0306.2017.12.030
WANG Ao, YANG Ke, LV Man, SHI Ya-ning, XIN Zhi-hong. Screening and identification of an epiphytic bacterium producing lipopeptides based on NRPS gene[J]. Science and Technology of Food Industry, 2017, (12): 164-170. DOI: 10.13386/j.issn1002-0306.2017.12.030
Citation: WANG Ao, YANG Ke, LV Man, SHI Ya-ning, XIN Zhi-hong. Screening and identification of an epiphytic bacterium producing lipopeptides based on NRPS gene[J]. Science and Technology of Food Industry, 2017, (12): 164-170. DOI: 10.13386/j.issn1002-0306.2017.12.030

基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究

基金项目: 

2016年农产品质量安全风险评估项目(GJFP201601205);

详细信息
    作者简介:

    王傲 (1991-) , 女, 硕士研究生, 研究方向:食品营养与化学, E-mail:2014108056@njau.edu.cn。;

    辛志宏 (1974-) , 男, 博士, 教授, 研究方向:食品营养与安全, E-mail:xzhfood@njau.edu.cn。;

  • 中图分类号: TS255.1

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

  • 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。 
    Abstract: In this study, five epiphytic bacteria were isolated from " summer black" grapes using plate streaking and slant culture methods. After extraction of DNA, 16 S r DNA from the five strains were amplified 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. After construting the NRPS phylogenetic 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 physiochemical analysis and spectroscopic techniques.The study highlights new research methods and theoretical basis for target-screening strains which produced cyclic lipopeptide.
  • [1] 林文翰.红树附生微生物的化学成分研究[C].国家“863”计划资源环境技术领域海洋生物高技术论坛, 2005.
    [2]

    Andrews JH, Harris RF.The ecology and biogeography of microorganisms on plant surfaces[J].Annual Review of Phytopathology, 2000, 38 (1) :145-180.

    [3]

    Cueto M, Jensen PR, Kauffman C, et al.Pestalone, a new antibiotic produced by a marine fungus in response to bacterial challenge[J].Journal of Natural Products, 2001, 64 (11) :1444-1446.

    [4]

    Shigemori H, Bae M A, Yazawa K, et al.Alteramide A, a new tetracyclic alkaloid from a bacterium Alteromonas sp.associated with the marine sponge Halichondria okadai[J].J Org Chem, 1992, 57 (15) :4317-4320.

    [5]

    Hornschuh M, Grotha R, Kutschera U.Epiphytic Bacteria Associated with the Bryophyte Funaria hygrometrica:Effects of Methylobacterium, Strains on Protonema Development[J].Plant Biology, 2002, 4 (4) :682-687.

    [6] 张学君, 徐盈.苹果表面微生物数量及其与两种主要病菌的关系[J].果树学报, 1995 (4) :232-236.
    [7] 单洪友.夏黑葡萄特征特性及栽培技术[J].农技服务, 2007, 24 (9) :95.
    [8] 施南芳, 楼宇涛, 吾建祥.不同品种葡萄贮藏特性研究[J].农技服务, 2014 (2) :119.
    [9] 张俊楠, 彭洁, 刘天行, 等.基于PKSⅠ基因海芦笋内生真菌及其次级代谢产物筛选鉴定[J].食品科学, 2016, 37 (1) :114-119.
    [10]

    Bruner S D, Weber T, Kohli R M, et al.Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain Srf TE[J].Structure, 2002, 10 (3) :301-310.

    [11] 王世媛.非核糖体肽合成酶 (NRPSs) 作用机理与应用的研究进展[J].微生物学报, 2007, 47 (4) :734-737.
    [12]

    W G Weisburg, S M Barns, D A Pelletier, et al.16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology, 1991, 173 (2) :697-703.

    [13]

    Gontang E A, Gaudêncio S P, Fenical W, et al.Sequencebased analysis of secondary-metabolite biosynthesis in marine actinobacteria[J].Applied&Environmental Microbiology, 2010, 76 (8) :2487-2499.

    [14] 唐晓敏.红树林土壤微生物卤代酶基因和宏基因组非核糖体肽合酶基因的克隆和表达[D].厦门:厦门大学, 2008.
    [15]

    Liu X-Y, Yang S-Z, Mu B-Z.Production and characterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121[J].Process Biochemistry, 2009, 44 (10) :1144-1151.

    [16]

    Hideji Itokawa, Toshiko Miyashita, Hiroshi Moita, et al.Structure and Conformational Studies of[Ile7]and[Leu7]Surfactins from Bacillus subtilis natto[J].Chem Pharm Bull, 1994, 42 (3) :604-607.

    [17] 刘丽霞.Surfactin合成酶相关基因研究[D].南京:南京农业大学, 2012.
    [18]

    Thimon L PF, Maget Dana R, Roux B G M.Interactions of bioactive lipopeptides, iturin A and surfaction from Bacillus subtilis[J].Biotechnol Appl Biochem, 1992, 16 (2) :144-151.

    [19] 王大威, 刘永建, 林忠平, 等.一株产生脂肽的枯草芽孢杆菌的分离鉴定及脂肽对原油的作用[J].微生物学报, 2008, 48 (3) :304-311.
计量
  • 文章访问数:  156
  • HTML全文浏览量:  21
  • PDF下载量:  179
  • 被引次数: 0
出版历程
  • 收稿日期:  2016-12-11

目录

    /

    返回文章
    返回
    x 关闭 永久关闭