关键词: 水稻, 多顺反子, 水稻载体构建, 基因枪转化, 烟草质体转化

Abstract: 【Objective】The rice (Oryza sativa) chloroplast multicistron site integration expression vector will be transformed for expression into the tobacco chloroplast.【Method】According to the published correlative DNA sequence, a series of elements for construction of the rice chloroplast multicistron site integration expression vectors have been cloned using a PCR technique, including Prrn (a modified plastid ribosomal RNA operon promoter) , psbA3′ (the 3′ region of the plastid psbA gene), aadA gene (encoding aminoglycoside 3′-adenylytransferase), man gene (encoding mannase), gfp gene (encoding green fluorescence protein) and a rice chloroplast high-frequency homologous recombination crDNA fragment (psbC/trnG，3362bp). A rice chloroplast multicistron expression vector pLM21 (-psbC-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3′-trnG-) was constructed with these elements. Then the tobacco leaves were bombarded 5 times with gold particles coated with the vector pLM21. 【Result】 Four tobacco chloroplast transgenic shoots were obtained after the tobacco leaves had been grown in the screening medium. All the genes man, gfp and aadA , having been transformed to the tobacco chloroplast, were confirmed by PCR. The expression of these genes was identified by the screening medium, the laser scanner and the Western blot. The multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP. 【Conclusion】 All these showed that the genes man, gfp and aadA in the rice vector pLM21 were integrated in the tobacco chloroplast genome DNA and expressed in the tobacco chloroplast.

Key words: Oryza sativa, Multicistron, Construction of rice vector, Bombardment microprojectiles, Tobacoo plastid genetic transformation