短小芽胞杆菌抑菌蛋白TasA基因克隆及功能分析  

Cloning and Functional Analyses of TasA,an Antimicrobial Protein Gene from Bacillus pumilus DX01

作　　者：刘通[1] 陈云鹏[1] 李琼洁 顾振芳[1] 

机构地区：[1]上海交通大学农业与生物学院,农业部都市农业(南方)重点实验室,上海200240

出　　处：《上海交通大学学报（农业科学版）》2014年第5期48-52,56,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家自然科学基金面上项目(31071721)

摘　　要：为了研究短小芽胞杆菌Bacillus pumilus DX01菌株的抑菌相关基因的功能,采用PCR方法从DX01基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建原核表达载体pET2-TasA,在大肠杆菌Escherichia coli BL21中表达获得TasA基因的融合表达蛋白,分别利用悬滴法和平板打饼法检测融合蛋白对玉米小斑病菌(Bipolaris maydis)分生孢子萌发和对玉米小斑病菌及水稻稻瘟病菌菌丝体生长的抑制活性,结果表明TasA融合蛋白对这2种植物病原真菌的生长有着显著的抑制作用。TasA基因包含1个780bp的完整开放阅读框(GenBank:KC692521),编码259个氨基酸残基;该序列与B.subtilis菌株PEBS2501(GenBank::FJ713581)、B.amyloliquefaciens HF-01(GenBank:JF781312)和B.subtilis菌株BWST7003(GenBank:AP012496)同源抑菌蛋白基因序列相似性达99%。原核表达产物经SDS-PAGE分析检测到约32.3kDa的融合蛋白;经Ni-NTA柱纯化后的融合蛋白对供试病原菌均有显著的抑制作用。经测定发酵液的TasA融合蛋白产率为28μg/mL,短小芽胞杆菌抑菌蛋白TasA基因具有良好的抑菌效果。In order to explore the function of antiphytopathogen-associated gene isolated from Bacillus pumilus strain DX01,a full length of antimicrobial protein gene, TasA was amplified using DX01 genomic DNA as template by PCR. Its prokaryotic expression vector pET28-TasA was consturcted and introduced into in Escherichia coli BL21 cells and the {used TasA protein was successfully expressed in BL21 cells and purified. The fused TasA protein showed significant antimicrobial activities against Phyricularia grisea and Bipolaris maydis by using spread plate and punching methods, respectively. TasA DNA sequence includes an open reading frame （ORF） of 780 nucleotides which encodes an antimicrobial protein of 259 amino acid residues and there is 99% nucleotide sequence identity with the corresponding genes of B. subtilis PEBS2501 （GenBank accession number FJ713581）, B. amyloliquefaciens HF-01 （JF781312） and B. subtilis BWST7003 （AP012496）. A 32. 3 kDa pET28-TasA was detected through SDS-PAGE on a Ni-NTA resin column exhibited high antip Bipolaris rnaydis. The production efficiency of solution of BL21 transformant, and this ant antiphytopathogenic activity. fused protein expressed in E. coli BL21 habouring plasmid analysis. Moreover, the His-tagged fusion protein purified hytopathogenic activities against Phyricularia grisea and target protein was measured at 28 μg/mL in zymolysis timicrobial protein of B. pumilus showed an obvious

关 键 词：短小芽胞杆菌 TasA基因 抑菌蛋白 抑菌活性 

分 类 号：S476[农业科学—农业昆虫与害虫防治]