烟草质体多顺反子定点整合表达载体的构建和转化

构建了烟草质体多顺反子定点整合表达载体pLM4(-psaA-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3'-psbC-).用基因枪将该载体轰击烟草叶片5次,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6株.用PCR、激光扫描、Western blot和RFLP等方法检测都证实多顺反子表达盒中的3个基因甘露聚糖酶基因(man)、绿荧光蛋白基因(gfp)、氨基糖苷3'-腺苷酰基转移酶基因(aadA)已整合到烟草质体基因组中,且均得到表达.

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

According to the published DNA sequence, a serial of elements for constructing the tobacco chloroplast multicistron site integrat- ing expression vectors have been cloned by PCR technique, which include Prrn (a modified plas- tid ribosomal RNA operon promoter), psbA3' (the 3' region of the plastid psbA gene), aadA gene (encoding aminoglycoside 3'-adenylyt- ransferase), man gene (encoding mannase), gfp gene (encoding green flurescence protein) and tobacco chloroplast high-frequency homologous recombination ctDNA fragment (psaA/psbC, 3 463 bp) (Fig.2). A tobacco chloroplast multicist-ron expression vector pLM4 (Fig.1) (-psaA-Prrn-SD-man-SD-gfp-SD-aadA-psbA3'- psbC-) was constructed with these elements. Then the tobacco leaves were bombarded 5 times with gold particles coated with the vector pLM4. After growing on the screening medium, the function of aadA gene was identified (Fig.3), and the function of gfp gene was confirmed by laserscanner (Fig.4), the expression of man was iden- tified by Western blot (Fig.5). All these genes man, gfp and aadA being integrated in the to- bacco chloroplast genome DNA were confirmed by PCR (Fig.6). And the multicistron expression cassette integrating in tobacco chloroplast ge- nome DNA was confirmed by RFLP (Fig.7). All these showed that the three genes in the tomato vector pLM4 were expressed in tobacco chloro- plast genome DNA.