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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 investigate the function of TasA gene of Bacillus CQBS03 strain of citrus canker, the full-length DNA sequence encoding the TasA gene was amplified from CQBS03 genomic DNA by PCR and the prokaryotic expression vector pEASY-E1 / The fusion expression protein of TasA gene was expressed in Escherichia coli. The inhibitory effect of the fusion protein on Xanthomonas citri citri was tested by paper disc method. The results showed that the TasA gene of CQBS03 strain contained a complete open reading frame of 786 bp (GenBank accession number JQ309841), encoding 261 amino acid residues; this sequence was identical to one known from Bacillus amyloliquefaciens B. amyloliquefaciens The similarity of homologous TasA gene sequence FJ713580 was 99.75%. The prokaryotic expression product was analyzed by SDS-PAGE, and the fusion protein was detected at about 31 kD. The purified fusion protein had significant inhibitory effect on Citrus canker, and the diameter of inhibition zone reached 11.5 mm after 72 h. Studies have shown that TasA gene is one of the functional genes that inhibits citrus canker by the strain of Bacillus Calmette-Guerin against CQBS03, and the gene has no inhibitory effect on the prokaryotic expression host, which has a good development and utilization prospect.