嗜热脂肪芽孢杆菌启动子克隆载体pFDC4和表达型载体pFDC11的构建

Construction of Promoter Probe Vector pFDC4 and Gene Expression Vector pFDC11 with High Transformation Efficiency in Bacillus stearothermophilus

以pPL703的衍生质粒pPGV5为载体,从嗜热脂肪芽孢杆菌CU21总DNA的Sau 3A酶切产物中得到1个0.54kb的启动子片段,它能促进载体上的无启动子的cat-86基因在嗜热脂肪芽孢杆菌及枯草芽孢杆菌中表达。这一片段以正、反向插入pPGV5载体,都能使重组质粒转化CU21原生质体的效率提高10^(?)至10(?)倍。Southern杂交实验表明,这一启动子片段与Imanaka等报道的来自CU21中的隐蔽性质粒pBS02的能提高转化效率的1.6kb Eco RI片段是同源的。利用所得到的0.54kb Sau 3A片段构建了新的启动子克隆载体pFDC4和表达型载体pFDC11,二者都能以很高的效率转化CU21原生质体。

A 0.5kb fragment from Sau3A -digested total DNA of Bacillus stearothermophilus CU21 was cloned with the vector pPGVS, a derivative of pPL703. The insertion of this fragment can activate the expression of the promoteless cat-86 gene on the cloning vector in both B. stearothermophilus and Bacillus subtilis hosts. When the 0.54 k,b fragment is present in pPGV5 in either orientation, the transformation efficiency of the plasmid is increased about 103 to 104 fold in CU21 protoplasts. Southern hybridization showed this 0.54 kb fragment was homologous with a 1.6kb fragment, which was shown by Imanaka et al. (1984, J. Gen. Microkiol. 130, 1399-1408) to originate in a cryptic plasmid resident in CU21 and to enhance the transformat on efficiency of another plasmid. With this 0.54kb fragment a new promoter probe vector pFDC4 and a gene expression vector pFDC11 were constructed. Both can transform the CU21 recipient with high efficiency.