番茄质体多顺反子定点整合表达载体的构建及其转烟草的研究

根据烟草叶绿体高频同源重组片段的已知序列(GenBank Z00044.1)设计引物，用PCR的方法克隆到1个3.663 kb的番茄叶绿体DNA片段（psbD/trnG），命名为ctDNA。该片段与GenBank中烟草的相应片段有96.7%同源性, 与本文所用的烟草的相应片段有95.8%同源性。以其为外源多顺反子定点整合介导的同源重组片段，与来自烟草叶绿体的强启动子Prrn和终止子psbA3’，以及甘露聚糖酶基因man、绿荧光蛋白基因gfp、氨基糖苷3’-腺苷酰基转移酶基因aadA构建番茄质体多顺反子定点整合表达载体pLM2(-psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3’-trnG-)。将该载体用基因枪轰击烟草叶片，用添加了壮观霉素的选择分化培养基筛选，获得质体转基因烟草3株。用PCR、激光扫描、Western Blot、RFLP等方法检测都证实man、gfp、aadA基因已整合到烟草质体基因组中，且均得到表达。用番茄质体多顺反子定点整合表达载体成功实现在烟草质体中的表达。

Construction of Tomato Chloroplast Multicistron Site Integration Expression Vector and Its Transgenic Tobacco

According to the published DNA sequence (GenBank Z00044.1) of tobacco chloroplast high-frequency homologous recombination fragment, primers were designed to amplify a 3.663 kb DNA fragment named ctDNA from chloroplast genome of tomato by PCR. The fragment was not only 96.7% homology compared with the tobacco fragment reported in GenBank, but also 95.8% homology compared with the tobacco fragment used in this paper. The tomato chloroplast multicistron expression vector pLM2（Fig.1） (-psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA -psbA3’-trnG-) was constructed with ctDNA, Prrn, man, gfp, aadA and psbA3’. And the tobacco leaves were bombarded with golden particles coated with the vector pLM2. Three transgenic tobacco shoots were obtained on the screening medium. The function of aadA gene was identified by screening medium（Fig.3）, and the function of gfp gene was confirmed by laser scanner（Fig.4）. The expression of man gene was identified by Western blot（Fig.5）. The genes man, gfp and aadA being in the chloroplast of tobacco were confirmed by PCR（Fig.6）. And the tobacco chloroplast multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP（Fig.7）. The results were showed that the three genes( man, gfp, aadA ) which were in the tomato chloroplast multicistron expression vector pLM2 were expressed in tobacco chloroplasts.