苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。

Cloning,expression and sequence analysis of chiA,chiB in Bacillus thuringiensis subsp. colmeri 15A3

Two DNA fragments encoding chitinase A and B were amplified from total genomic DNA of Bacillus thuringiensis subsp. colmeri 15A3, and then ligated with pUCm-T cloning vector. The recombinant plasmids pUCm-chiA and pUCm-chiB were transformed into Escherichia coli XL-Blue respectively. Both chiA and chiB were successfully expressed in E. coli with their natural promoters independent of any chitin. Additionally, the expressed ChiA and ChiB could be secreted from E. coli cells. It was proved that two chitinases were constitutively expressed in strain 15A3. The nucleotide sequence of chiA (GenBank Accession Number: EF103273) with a length of 1426bp included an open reading fram(ORF) of 1083 bp encoding for a protein of 360 amino acids . The deduced amino acid sequence showed that the mature protein ChiA with a predicted molecular mass of 36kDa consisted of a single catalytic domain. The nucleotide sequence of chiB (GenBank Accession Number: EF103273) with a length of 2279 bp included an ORF of 2031bp encoding for a protein of 676 amino acid residues. The deduced amino acid sequence showed that the mature protein ChiB with a predicted molecular mass of 70.6kD was composed of three domains: catalytic domain, chitin-binding domain and fibronectin type III-like domain. The comparison of their upstream sequences informed that there were differences between putative promoters of chiA and chiB.