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油菜叶绿体基因组同源重组片段的克隆及phb基因定点整合载体的构建

张中林, 张景昱, 李轶女, 苏宁, 宋艳茹, 沈桂芳

中国农业科学院生物技术研究所.北京 100081；中科院植物研究所.北京 100093

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AF267640和AF267641),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。

关键词： phb基因; 叶绿体转化; 同源片段; 定点整合; 油菜

Cloning of homologous targeting sequences containing chloroplast genes, rbcL and atpB, of Brassica napus and the construction of plastid transformation vectors harboring key enzyme genes responsible for PHB biosynthesis

Zhang Zhonglin, Zhang Jingyu, Li Zhinu, Su Ning, Song Yanru, Shen Guifang

Biotechnology Research Institute,Chinese Academy of Agricultural Sciences.Beijing 100081；China

Abstract

The results of restriction analysis and Southern blot hybridization were in accord with the desired. Transformation and consquent works are in progress.

Key words: Phb genes;Chloroplast transformation;Homologous targeting sequence;Site-specific integration;Brassica napus

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引用本文：

张中林, 张景昱, 李轶女, 苏宁, 宋艳茹, 沈桂芳. 油菜叶绿体基因组同源重组片段的克隆及phb基因定点整合载体的构建[J]. 生理学报 2001; 53 (3): .

Zhang Zhonglin, Zhang Jingyu, Li Zhinu, Su Ning, Song Yanru, Shen Guifang. Cloning of homologous targeting sequences containing chloroplast genes, rbcL and atpB, of Brassica napus and the construction of plastid transformation vectors harboring key enzyme genes responsible for PHB biosynthesis. Acta Physiol Sin 2001; 53 (3): (in Chinese with English abstract).