【摘要】 构建了含有外源目的基因表达盒 ( psbA5′ nifH psbA3′)和选择标记基因表达盒 (Prrn aadA TpsbA)以及两段质体基因组同源片段的烟草叶绿体转化载体 pTRCH2 0 5。基因枪轰击受试外植体后 ,经抗生素筛选获得转化再生植株。为探索提高转基因植株同质化程度的可能途径 ,对再生植株在高浓度的壮观霉素选择压下进行了 3轮次分化再生 ,并结合腋芽繁殖的方式继代 6～ 10次。PCR扩增和以同源片段为探针的Southern印迹分子检测结果表明 ,外源基因已整合进入质体基因组中 ,且已达到同质化状态更多还原

【Abstract】 Scientists pay more and more attention to the resear ch on plastid engineering for its following advantages: foreign genes can be inte grated site specifically into the plastid genome (plastome); there are no posi tion effects as experienced with random insertion of transgenes in nuclear trans formation; pollen mediated dispersion of transgenes can be avoided because chl oroplasts are maternally transmitted; gene silencing does not occur in plastids and therefore transgene expression is stable in progeny of transplastomic plant s; and the high ploidy level of the plastome in leaf cells makes high levels of transgene expression feasible. At the same time, however, the highly polyploid plastome makes it difficult to get transplastomic plants homoplasmic for foreign transgenes. In this work, chloroplast transforming vector pTRCH205, which carri es two psbA5′ nifH psbA3′ and Prrn aadA TpsbA cassette s flanked by plastid DNA sequence to target their insertion between psbA and trnK operons, was constructed. Plastid transformation of Nicotiana tabacu m was carried out by the biolistic delivery of transforming plasmid pTRCH205 D NA into leaf cells. Integration of nifH and aadA by two homologous recom bination events via the flanking ptDNA sequences, and selective amplificatio n of the transplastomes on MS medium with high concentration of spectinomycin, y ielded resistant cell lines. All the independent transplastomic lines were subje cted to three additional rounds of regeneration and were subcultured for 6-10 t imes through stem sections on MS medium containing 500 mg/L spectinomycin, to ob tain homoplasmic tissues. The results of PCR assay and Southern blot hybridizati on, probed with 0.9 kb BglII/SnaBI homologous fragment, indicated that f oreign genes had been integrated into the plastomes of transgenic plants, which finally became homoplasmic for foreign transgenes.更多还原