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[目的]克隆解淀粉芽孢杆菌TF28抗菌蛋白基因Tas A并进行原核表达和抑菌活性研究。[方法]采用PCR方法扩增抗菌蛋白基因Tas A,连接p ET22b载体,导入E.coli BL21(DE3)菌株,进行IPTG低温诱导表达,用His柱纯化表达产物,采用纸片方法测定其抑菌活性。[结果]从解淀粉芽孢杆菌TF28中克隆了抗菌蛋白基因Tas A,以p ET22b为表达载体构建高效表达抗菌蛋白Tas A的基因工程菌株,该菌株在0.05 mmol/L IPTG 15℃诱导4 h,Tas A蛋白表达率为34.2%,经His柱纯化后获得SDS-PAGE电泳一条带的纯化Tas A蛋白,其含量为67.8 mg/L,收率为90.8%。该蛋白抑制番茄灰霉病和叶霉病、玉米茎基腐病和水稻稻曲病菌生长。[结论]实现抗菌蛋白基因Tas A的原核表达,表达率34.2%,表达蛋白具有广谱抑菌活性,在植病生防方面具有应用潜力。
[Objective] The research aimed to clone Tas A of Bacillus amyloliquefaciens TF28 and study its prokaryotic expression and antibacterial activity. [Method] The antimicrobial protein gene Tas A was amplified by PCR and ligated into p ET22b vector. The recombinant plasmid was introduced into E. coli BL21 (DE3) strain and induced by low temperature induction with IPTG. The expressed product was purified with His column and tested for antibacterial activity active. [Result] The gene of Tas A was cloned from Bacillus amyloliquefaciens TF28, and the genetically engineered strain highly expressing the antimicrobial protein Tas A was constructed using p ET22b as the expression vector. The strain was induced by 0.05 mmol / L IPTG at 15 ℃ for 4 h, The expression of Tas A protein was 34.2%. After purified by His column, the purified protein A was purified by SDS-PAGE. The content of purified protein was 67.8 mg / L and the yield was 90.8%. The protein inhibits Botrytis cinerea and leaf mold, corn stem rot and rice false smut growth. [Conclusion] The prokaryotic expression of the antimicrobial protein Tas A was achieved, the expression rate was 34.2%. The expressed protein had broad spectrum antibacterial activity and had potential application in the biocontrol of plant diseases.