双抗性质粒pSC33、pSC48的构建及其特性研究

截短的短小芽孢杆菌质粒pCJ3与去除了复制功能的金黄色葡萄球菌质粒PUB110经EcoRI酶切,DNA连接酶连接后组建T_o~r及K_m~r的双抗性的重组质粒pSC33和和PSC48。根据电泳迁移率估算pSC33及pSC48的大小分别为6.7及6.27Kb。具有BamHⅠ、AVaⅠ、XbaⅠ及BgLⅡ等限制酶的单切点,其中BgLⅡ切点位于卡那霉素抗性基因内。pSC33及pSC48能转化枯草杆菌各种突变体的感受态细胞,转化率比亲本质粒高一个数量级,也能转化枯草杆菌的原生质体。pSC33及pSC48在枯草杆菌BR151中表现稳定,以PSC48和载体克隆了滑鼠蛇肝线粒体DNA片段。

The Construction and Characterization of Chimeric Plasmids pSC33, PSC48

Chimeric plasmids pSC33, pSC4g were constructed from the transcated pl-asmid pCJ3 (pAL32) from B.pumilusA3 and the S. aureus plasmid pUB110 origin of replication has been removed. The chimeric plasmids have both Kmr and Tcr markers and molecular sizes of 6.7 and6.27kb respectively. They have four unique sites for BamH Ⅰ, Ava Ⅰ, XbaIand BglⅡ. Among them, BglⅡ site is on Kmr gene. The transformation frequencies of pSC33 and pSC48 for the competent cells of different B.subtilis strains are 6×103-7.3×104 transformants/μgDNA which is one magnitude higher than this parental plasmids pAl32 and pUB110. Their transformation frequencies for protoplasts are 1.0×103 to 5.3×105 transformants/μgDNA.They are stable in B.subtilis after ten generations without any plasmid elimination. Their segregation rates are 2.4% and 5.2% respectively .we have cloned the mitochondrial DNA fragments from Ptyas mucosus by Kmr insertion inactivation with pSC48 as vector.