【摘要】 利用叶绿体基因组在进化过程中高度保守的特点 ,根据烟草、水稻和玉米叶绿体基因组全序列资料 ,设计合成引物 ,PCR扩增并克隆了油菜叶绿体两个重要的功能基因rbcL和atpB(GenBank登录号分别为AF2 67640和AF2 67641) ,并以此作为定点整合外源基因的同源重组片段。以来自叶绿体的强启动子PpsbA和Prrn等驱动PHB合成途径中 3个关键酶基因phbA、phbB和phbC ,分别构建表达盒 ,并将它们按照其在原始菌株中的自然转录顺序phbC phbA phbB相串联 ,最后连同选择标记基因aadA表达盒一起 ,克隆到油菜叶绿体同源片段中 ,构建成phb基因定点整合载体pRCABZ和pRCABF。酶切及Southern杂交结果证明所构建的转化载体符合预期设计。叶绿体转化及后续工作目前正在进行之中。更多还原

【Abstract】 Although the production of PHBs was significantly increased after gene products were targeted to plastids, PHBs were not accumulated to the level which makes the production economically viable. Another probable factor which limits the accumulation of PHBs to the present level is that foreign prokaryotic gene is incompatible with the eukaryotic expression system. So we tried to introduce the genes involved in PHB biosynthetic pathway from cyanobacteria directly to the plastid genome. Since chloroplast genomes were highly conservative in evolution. PCR primers to be used in amplifying chloroplast DNA fragments of Brassica napus were designed basing on the corresponding sequences in the chloroplast genomes of Nicotiana tobacum, Oryza sativa, and Zea mays. Two important chloroplast genes, rbcL and atpB (Fig.2B,C), were amplified and cloned from Brassica napus. These two genes were then used as targeting sequences to direct foreign genes into the chloroplast genome by homologous recombination. Three genes, phbA, phbB, and phbC, were placed under the control of strong chloroplast promoter PpsbA and Prrn. They were ligated in the order phbC-phbA-phbB, which is the natural order in their host strain. Together with the selectable aadA cassette, the phbC-phbA-phbB sequence was finally cloned into the unique SalⅠsite between two targeting sequences which gave rise to two resulting vectors pRCABZ and pRCABF (Fig.3) for chloroplast transformation. The results of restriction analysis (Fig.4) and Southern blot hybridization (Fig.5) were in accord with the desired. Transformation and consequent works are in progress.更多还原