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| 水稻叶绿体表达体系的建立及抗PPT叶绿体转化植株的获得 | |
| 引用本文： | 李轶女,孙丙耀,苏宁,孟祥勋,张志芳,沈桂芳.水稻叶绿体表达体系的建立及抗PPT叶绿体转化植株的获得[J].中国农业科学,2007,40(9):1849-1855. |
| 作者姓名： | 李轶女 孙丙耀 苏宁 孟祥勋 张志芳 沈桂芳 |
| 作者单位： | 1. 中国农业科学院生物技术研究所,北京,100081 2. 苏州大学生命科学学院,苏州,215123 3. 中国农业科学院作物科学研究所,北京,100081 |
| 基金项目： | 引进国际先进农业科技计划(948计划);江苏省自然科学基金 |
| 摘 要： | 【目的】建立外源基因在水稻叶绿体中的表达体系。【方法】通过分析已公布的水稻叶绿体基因组全序列及其基因分布特点，选择ndhF与trnL的基因间序列作为除草剂PPT抗性基因bar定点整合的位点。以水稻叶绿体基因组DNA为模板，采用PCR方法克隆了两个适于水稻叶绿体基因组遗传转化的同源片段，构建了含水稻叶绿体16S高效表达启动子、外源基因bar、psbA基因的3′序列（终止子）以及两个同源片段的水稻叶绿体特异表达载体pRB。【结果】采用基因枪法转化水稻品种中花10号幼穗诱导的愈伤组织，并再生植株，获得转化体后代种子。对转化植株进行的分子检测结果表明，外源基因bar 可能被整合到水稻叶绿体基因组中。后代种子的遗传学分析显示，外源基因bar可正常表达并遗传。【结论】利用所建立的水稻叶绿体外源基因表达体系，外源基因bar既可在水稻叶绿体基因组中正常表达，还可作为转化体筛选时的除草剂抗性选择标记。 |
| 关 键 词： | 水稻 同源片段 bar基因 叶绿体转化 |
| 收稿时间： | 2006-5-15 |
| 修稿时间： | 2006-05-12 |
Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants |
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| LI Yi-nü,SUN Bing-yao,SU Ning,MENG Xiang-xun,ZHANG Zhi-fang,SHEN Gui-fang.Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants[J].Scientia Agricultura Sinica,2007,40(9):1849-1855. | |
| Authors: | LI Yi-nü SUN Bing-yao SU Ning MENG Xiang-xun ZHANG Zhi-fang SHEN Gui-fang |
| Institution: | 1 Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;2 College of Life Sciences,Suzhou University,Suzhou 215123;3 Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081 |
| Abstract: | 【Objective】The aim of this study was to establish an expression system for foreign genes in rice chloroplast genome.【Method】By analyzing the published complete nucleotide sequence of rice chloroplast genome and its gene distribution pattern, the intergenic region of ndhF and trnl on chloroplast genome was selected as target for site-specific integration of PPT-resistant bar gene. Two fragments were cloned from rice chloroplast genome DNA by using PCR technique, which were suitable for homologous recombination in the genetic transformation of rice genome. A gene expression cassette was generated by fusing a modified 16S rRNA gene promoter to bar gene and using 3’ sequence of psbA gene as terminator. Based on this cassette and the cloned homologous fragments, the chloroplast-specific expression vector pRB was constructed. 【Result】Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with plasmid pRB. Subsequently, the regenerated plantlets, and seeds of progeny arising from reciprocal cross to the wild-type lines, were obtained. Molecular analysis on the transformed plants indicated that foreign gene bar has been integrated into rice chloroplast genome in a site-specific way. Genetic analysis with seeds of progeny revealed that bar gene expressed normally in rice chloroplast genome, and could be stably transmitted into next generation. 【Conclusion】Therefore, it was suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique. The bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. |
| Keywords: | Oryza sativa L Homologous fragments bar gene Chloroplast transformation |
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