Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants  

作　　者：LI Yi-nü SUN Bing-yao SU Ning MENG Xiang-xun ZHANG Zhi-fang SHEN Gui-fang 

机构地区：[1]Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China [2]College of Life Sciences, Suzhou University, Suzhou 215123, P.R.China [3]Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

出　　处：《Agricultural Sciences in China》2009年第6期643-651,共9页中国农业科学（英文版）

基　　金：funded by the 948 Program of the Ministry of Agriculture of China (991020);by the Natural Science Foundation of the Science and Technology Department of Jiangsu Province, China (BK2001139)

摘　　要：In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator ofpsbA gene 3 sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator ofpsbA gene 3 sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

关 键 词：Oryza sativa L. chloroplast transformation bar gene homologous fragments 

分 类 号：Q943.2[生物学—植物学] Q786