泡桐内生枯草芽孢杆菌JDB-1草菌素的抑菌活性及其spaS基因的克隆

journal6 ›› 2011, Vol. 32 ›› Issue (4): 92-98.

泡桐内生枯草芽孢杆菌JDB-1草菌素的抑菌活性及其spaS基因的克隆

（吉首大学生物资源与环境科学学院，湖南吉首 416000）

出版日期:2011-07-25 发布日期:2012-04-07
通讯作者: 恩特马克·布拉提白（1961-），男，新疆伊犁人，吉首大学生物资源与环境科学学院教授，博士，主要从事分子生物学与基因工程研究.
基金资助:
湖南省大学生研究性学习和创新性实验计划项目(2009105)

Antimicrobial Activity of Subtilin from Bacillus Subtilis JDB-1 and Cloning spaS Gene

(College of Biology and Invironment Science,Jishou University,Jishou 416000,Hunan China)

Online:2011-07-25 Published:2012-04-07

摘要/Abstract

摘要：用硫酸铵沉淀法从泡桐内生枯草芽孢杆菌JDB-1发酵液中粗提枯草菌素，采用纸片扩散法和琼脂糖扩散法分别检测枯草菌素对细菌和真菌的抑菌活性，采用PCR从菌株JDB-1基因组DNA中扩增出枯草菌素基因spaS，并克隆到pMD18-T载体，测定spaS基因的核苷酸序列.结果表明硫酸铵粗提的枯草菌素对大肠杆菌K12、恶臭假单胞杆菌AS1.1003、赤霉菌JSD-2、赤霉菌JSD-7和白色念珠菌ATCC10123等细菌和和真菌具有较强的抑制活性.DNA测序结果显示spaS基因大小为171 bp，与已报道不同菌株spaS基因核苷酸序列的同源性为95％.

关键词： 枯草芽孢杆菌, 枯草菌素, 抑菌活性, 基因克隆, 序列测定

Abstract: The bacterial subtilin was purified from the fermentation broth of Bacillus subtilis JDB-1 by using the ammonium sulfate precipitation,and its antimicrobial activity against five tested microorganisms was investigated by using the agar diffusion test or the paper disc test.The spaS gene encoding subtilin was amplified from genomic DNA of Bacillus subtilis JDB-1 by PCR，and the PCR products cloned into the pMD18-T vector and subsequently introduced the competent cells of E. coli DH5α.The recombinant plasmid pMD18-spaS was identified by PCR and then sequenced.The results showed that the purified subtilin displayed antimicrobial activities against E. coli K12,Pseudomonas putida AS1.1003,Gibberella moniliformis JSD-8 and Candida albicans ATCC10123,respectively.The sequence analysis showed that the coding region spaS gene of Bacillus subtilis JDB-1 was 171bp in length,and the nucleotide sequence similarity of the spaS genes between the JDB-1 strain and the previously reported other bacterial strains was 95%.

Key words: bacillus subtilis, subtilin, antimicrobial activity, cloning, sequencing

吴瑞方, 龚凤娟, 杨镒蔓, 恩特马克.布拉提白. 泡桐内生枯草芽孢杆菌JDB-1草菌素的抑菌活性及其spaS基因的克隆[J]. journal6, 2011, 32(4): 92-98.

WU Rui-Fang, GONG Feng-Juan, YANG Yi-Man, EnTe-Ma-Ke-.Bu-La-Ti-Bai . Antimicrobial Activity of Subtilin from Bacillus Subtilis JDB-1 and Cloning spaS Gene[J]. journal6, 2011, 32(4): 92-98.

参考文献

[1] TORSTEN S.Bacillus Subtilis Antibiotics:Structures,Syntheses and Specific Functions [J].Molecular Microbiology,2005,56(4):845-857.

[2] 王红革.地衣芽孢杆菌α2淀粉酶基因在枯草芽孢杆菌中的诱导表达 [J].微生物学报,1997,37(2):101-106.

[3] 冯清平,薛林贵.复合诱变原生质体选育耐热碱菌 [J].微生物学报,1996,36(6):453-459.

[4] MAURIELLOE M F,DUC L H,ISTICATO R,et al.Display of Heterologous Antigens on the Bacillus Subtilis Spore Coat Using CotC as a Fusion Partner [J].Vaccine,2004,22(9-10):1 177 -1 187.

[5] KUNST F,OGASAWARA N,MOSZER L,et al.The Complete Genome Sequence of the Gram-Positive Bacterium Bacillus Subtilis [J].Nature,1997,390(6657):249-256.

[6] JOHNSON A B.Bacitracin:A New Antibiotic Produced by a Member of the B. Subtilis Group [J].Science,1945,102(2650):376-377

[7] KLEIN C,KALETTA C,SCHNELL N,et al.Analysis of Genes Involved in Biosynthesis of the Lantibiotic Subtilin [J].Applied and Enviromental Microbiology,1992,58(1):132-142.

[8] 惠明,窦丽娜,田青,等.枯草芽孢杆菌的应用研究进展 [J].安徽农业科学,2008,36(27):11 623-11 627.

[9] SUTYAK K E,WIRAWAN R E,AROTCHEVA A A,et al.Isolation of the Bacillus Subtilis Antimicrobial Peptide Subtilosin from the Dairy Product-Derived Bacillus Amyloliquefaciens [J].Applied and Enviromental Microbiology,2008,104(4):1 067-1 074.

[10] KLEIN C,ENTIAN K D.Genes Involved in Self-Protection Against the Lantibiotic Subtilin Produced by Bacillus Subtilis ATCC 6633 [J].Applied and Enviromental Microbiology,1994,60(8):2 793-2 801.

[11] 李晶,杨谦.生防枯草芽孢杆菌的研究进展 [J].安徽农业科学,2008,36(1):106-132.

[12] 陈兰,张小平,Lindstrom K.药用植物内生芽孢杆菌的多样性和系统发育研究 [J].微生物学报,2008,48(4):432-438.

[13] 施碧红,罗秀针,郑虹,等.枯草菌素产生菌芽孢杆菌FBl23的发酵条件优化及其16S rRNA序列分析 [J].工业微生物,2008,38(4):14-18.