苏云金芽孢杆菌伴胞晶体产生菌BZ-1的分离筛选及鉴定  

Isolation,screening and identification of parasporal crystal-producing Bacillus thuringiensis strain BZ-1

作　　者：孔芳[1] 高勇[1] 朱霁晖 薛正莲[1] 杨超英[1] 

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000

出　　处：《中国生物制品学杂志》2014年第9期1153-1157,1163,共6页Chinese Journal of Biologicals

基　　金：2012年国家大学生创新创业训练计划项目(201210363090);中国博士后科学基金(2013M541735);安徽省教育厅项目(AH201310363325)资助

摘　　要：目的从被污染的蝴蝶兰初代培养基中分离筛选苏云金芽孢杆菌伴胞晶体产生菌,并进行鉴定。方法分离自被污染的蝴蝶兰初代培养基的苏云金芽孢杆菌经富集后,筛选伴胞晶体产生菌BZ-1,显微镜观察进行鉴定;采用PCR法分析菌株BZ-1的16S rDNA和脂肽抗生素合成相关基因,SDS-PAGE分析菌株的伴胞晶体;制备菌株发酵粗提液,提取活性物质,分别置于不同温度(50、60、80、100℃)30 min、用不同的蛋白酶(蛋白酶K、胰蛋白酶)于37℃处理30 min,以枯草芽孢杆菌作为指示菌检测菌株的抑菌活性;并检测菌株对小菜蛾的毒力。结果菌株BZ-1能产生芽孢,并在芽孢旁形成多个无规则伴胞晶体;该菌株的16S rDNA扩增产物测序结果经与GenBank中登录的16S rDNA核苷酸序列进行BLAST比对,与B.anthracis str.Ames(NR_074453)、B.cereus(NR_074540)和B.thuringiensis Bt407(NR_102506)菌株相似度达99%,表明菌株BZ-1属于芽孢杆菌属,结合扫描电镜观察结果,可初步确定该菌株为苏云金芽孢杆菌;该菌株可扩增出脂肽抗生素合成相关基因sfp、mycB、ituA、fenB;该菌株从培养2 d芽孢形成后开始分泌相对分子质量约130 000的晶体蛋白,4 d时蛋白表达量增加,6 d后蛋白表达量基本达到稳定;该菌株具有一定的热稳定性,对蛋白酶K具有一定的耐受性;该菌株于30℃培养6 d,对小菜蛾表现出较高的毒力。结论从被污染的蝴蝶兰初代培养基中分离筛选的苏云金芽孢杆菌伴胞晶体产生菌BZ-1所产的抗菌物质主要抑制革兰阳性细菌,具有较高的毒力,可作为原始菌株发酵进行田间生物防治,也可为构建杀虫基因工程菌以及培育抗虫转基因植物提供高毒力的候选基因,具有较好的应用前景。Objective To isolate and identify a parasporal crystal-producing strain of Bacillus thuringiensis from the contaminated initial medium of Phalaenopsis seedlings.Methods The Bacillus thuringiensis was isolated and enriched,based on which s parasporal crystal-producing strain BZ-1 was screened and identified by microscopy.The 16 S rDNA and lipopeptide antibiotic synthesis related genes were analyzed by PCR,while the parasporal crystal by SDS-PAGE.Fermentation supernatant of the strain was prepared,from which active substance was extracted and treated at various temperatures（50,60,80 and 100 ℃）with various proteases（proteinase K and trypsin）for 30 min respectively,then determined for bacteriostatic activity using Bacillus subtilis as an indicator,and analyzed for virulence to Plutella xylostella.Results Strain BZ-1 produced germ as well as blunt diamond-shaped or inconstant crystals.The sequence of PCR product of 16 S rDNA of the strain was compared with those in GenBank by BLAST software,and the result showed homologies of 99% to those of B.anthracis str.Ames（NR074453）,B.cereus（NR074540）and B.thuringiensis Bt407（NR102506）,indicating that strain BZ-1 belonged to Bacillus.The strain was preliminarily identified as Bacillus thuringiensis by sequencing combined with scanning electron microscopy.Lipopeptide antibiotic synthesis related genes sfp,mycB,ituA and fenB were amplified from strain BZ-1.Crystal protein with a relative molecular mass of about 130 000started to be secreted by the strain formation of germ 2 d after culture,of which the expression level increased 4 d and was stable 6 d after culture.Strain BZ-1 showed a certain heat stability and a certain resistance to proteinase K.After culture at 30 ℃ for 6 d,the strain showed strong virulence to Plutella xylostella.Conclusion The antibacterial substance produced by parasporal crystal-producing strain BZ-1 of Bacillus thuringiensis isolated from the contaminated initial medium of Phalaenopsis seedlings mainly inhibited Gram positive bacte

关 键 词：苏云金芽孢杆菌 伴胞晶体 脂肽抗生素 抑菌活性 毒力 

分 类 号：S182[农业科学—农业基础科学]