嗜碱芽孢杆菌PB92碱性蛋白酶基因启动子的克隆及应用

Cloning,characterization and application of the promoter region of the alkaline protease gene in Bacillus alcalophillus PB92

利用TAIL-PCR方法从嗜碱芽孢杆菌PB92基因组中扩增出碱性蛋白酶基因上游启动子活性片段,测序分析后登录GenBank(EU130686)。此序列具有多个启动子特征区域,且在-538～-370 bp和-275～-128 bp两个区域存在反向读码框。对启动子片段进行功能缺失的分析结果表明,TSS上游105 bp片段具有明显启动子活性,但以长为414 bp～619 bp时活性更为显著。此外,对PB92碱性蛋白酶的信号肽分析结果表明,此信号肽具有典型分泌型(Sec-type)信号肽结构。将PB92碱性蛋白酶基因启动子和信号肽序列克隆入pBE2,构建成表达载体pBEAC,并以其为载体实现了植物甜蛋白monellin基因在枯草芽孢杆菌1A751中的高效表达。

Promoter function fragment of alkaline protease gene （GenBank accession number： EU130686） was cloned from Bacillus alcalophillus PB92 genome by TAIL-PCR. Sequenced and analyzed revealed that it contains several typical promoter characterized regions. Two reverse translation frames were located in -538--370 bp and-275-128 bp. Deletion analysis of the sequence demonstrated that 414 bp to 619 bp upstream of the TSS showed predominant promoter activity, and a 105 bp length sequence can serve as this function. Additionally, a representative Sec-type signal peptide structure was detected in PB92 AprE signal peptide. By cloning the PaprE and alkaline protease signal peptide gene into pBE2, an expression vector pBEAC was constructed, and a plant sweet protein monellin gene was highly expressed in B. subtilis 1A751.