北京鸭肝线粒体DNA的克隆

 本文采用限制内切酶HindⅢ切割经Sepharose 4 B柱纯化的北京鸭肝线粒体DNA,得到五个片段,其大小分别为:A——6.56kb,B——3.12kb,C——3.12kb,D——2.40kb,E——1.40kb。以质粒pWR33为载体,在HindⅢ切点处插入线粒体DNA的HindⅢ酶切片段,将体外重组质粒转化到大肠杆菌HB101内,经筛选、分析:如菌落原位杂交,限制性内切分析和Southern吸印法分析,首次得到了线粒体DNA的HindⅢB、C、D、E四个片段的克隆子。另外还得到了一个与片段A同源的1.60kb大小的片段。

Cloning of Peking Duck Liver Mitochondrial DNA in Escherichia coli

To construct the genomic library of Peking duck liver mtDNA, the mtDNA was purified by Sepharose 4B gel filtration and cloned in E.coli, using the plasmid vector pWR33. The products of mtDNA Hind Ⅲ digestion (the size of five fragments is: A-6.56kb, B-3.12kb,C-3.12kb, D-2.40kb, E-1.40kb) were ligated' with HindⅢ-cut pWR33.The ampicillin-resistant white colonies obtained upon transformation of E.coli Jm83 or HB101 were screened by the following procedures,namely colony hybridization in situ, Agarose gel electrophoresis analysis and Southern blot analysis. In this way the clones of mtDNA HinⅢ fragments, fragment B (3.12kb), fragmagment C (3.12kb), fragment D(2.40kb)and fragment E(1.40kb) were obtained. Forthermore, one fragment about 1.60kb was also cloned which was proved to be homogeneous with the fragment A.Further study about cloning fragment A is to be done.