芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

Cloning antagonistic protein TasA gene in Bacillus CQBS03 and its prokaryotic expression

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框(GenBank登录号为JQ309841),编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。

In order to study the function of TasA gene of biocontrol bacteria Bacillus CQBS03 which was isolated from citrus leaf and had bactcriostasis activity to Xanthomonas citri cirri, the full-length DNA se- quence encoding TasA gene was amplified from Bacillus CQBS03 genomic DNA by PCR. After that, we constructed the prokaryotic expression vector pEASY-E1/TasA and expressed in Escherichia coli BL21. The antagonism of the expression fused protein to X. cirri citri was tested with filter paper-petri dish meth- od. The sequencing result showed that full-length DNA sequence of TasA gene included a complete ORF of 786 base pairs, （ GenBank accession number JQ309841 ） , which encoded 261 amino acid residues The TasA gene of CQBS03 had a high sequence similarity with TasA gene of Bacillus amyloliquefaciens （as high as 99.75% ）. A 31 kD fused protein was obtained from mutant E. coli BI21-pEASY-E1/TasA cells. Fused protein of TasA gene purified by Ni-NTA chromatography revealed obvious antibacterial ac- tivities to X. citri citri, and the diameter of antagonism zone reached 11.5 mm after 72 hours. The resultsproved that the TasA gene was one of the most efficient antagonism function genes of bacteria CQBS03, and it can be used in citrus canker biocontrol.