苏云金芽胞杆菌质粒p26复制功能区的克隆  

Cloning of a replicon of plasmid p26 from Bacillus thuringiensis strain

作　　者：季思思[1] 朱义广[1] 彭东海[1] 阮丽芳[1] 孙明[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2011年第5期558-562,共5页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(3087066)

摘　　要：利用特异引物从基因组BAC文库筛选到1个35kb的片段,对该片段测序比对分析发现可能含有p26的复制区。为了验证该片段中是否含有p26的复制区,首先,将大肠杆菌和苏云金芽胞杆菌穿梭载体pHT304用EcoRⅠ酶切切掉苏云金芽胞杆菌复制子后自连,获得复制子克隆载体pHT304E;随后,将12kbEcoRⅠ片段亚克隆到载体pHT304E上电转化无晶体突变株BMB171,获得具有抗性的转化子,证明该片段具有复制功能。缺失实验证明最小复制功能区包含2个必需的ORF。经过40代无抗培养,最小复制区复制稳定性达到90%以上。In this study,a 35-kb fragment was screened from the genome BAC library of a Bacillus thuringiensis strain with special primers.Sequence comparison of this fragment indicated that it contained a replicon of the plasmid p26.To test whether this fragment contain a replicon or not,a vector pHT304E that does not replicate in B.thuringiensis was constructed through deleting a 2.1-kb EcoRV fragment of pHT304 which containing the replication protein of B.thuringiensis.A 12-kb EcoRⅠ fragment cloned with the probe of replication vector pHT304E revealed that it was capable of supporting the replication in Bacillus strain.The replication region of p26 was still stable after 40 generations under the nonselective condition.

关 键 词：苏云金芽胞杆菌 质粒 特异引物 细菌人工染色体文库 克隆 复制子 缺失突变 

分 类 号：Q754[生物学—分子生物学]