水稻质体多顺反子表达载体的构建及其对烟草质体的转化

【目的】尝试水稻质体多顺反子定点整合表达载体转化到烟草质体中表达。【方法】根据已发表的相关序列，分别设计引物，通过PCR技术克隆了一系列构建水稻质体多顺反子定点整合表达载体所需的元件：质体核糖体(16S)RNA操纵元启动子（Prrn）、质体psbA基因3′端终止子（psbA3′）、氨基糖苷3′-腺苷酰基转移酶基因（aadA）、水稻质体基因组高频同源重组片段(psbC/trnG，大小3362 bp)，命名为crDNA、甘露聚糖酶基因（man）、绿荧光蛋白基因（gfp）。构建了水稻质体多顺反子定点整合表达载体pLM21(-psbC-Prrn-RBS -man-RBS-gfp-RBS-aadA-psbA3′- trnG-)。将该载体用基因枪轰击烟草叶片5枪，用添加了壮观霉素的选择分化培养基筛选。【结果】获得质体转基因烟草4株。用PCR、激光扫描和Western blot等方法检测都证实man、gfp、aadA三个基因且均得到表达，用RFLP证实表达盒整合到烟草质体基因组中。【结论】水稻质体多顺反子定点整合表达载体已整合到烟草质体基因组中，并得到表达。

Construction of Rice Chloroplast Multicistron Site Integration Expression Vector and Its Transgene to Tobacco Chloroplast

【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.