【摘要】 中长链羟基脂肪酸聚酯(medium-chain-length-PHAs,mcl-PHAs)属于微生物聚酯。Ⅱ型PHA合酶是mcl-PHAs生物合成途径中的关键酶。将编码Ⅱ型PHA合酶的基因phaC2与水稻(Oryzasativa)叶绿体psbA基因的启动子和终止子构建表达盒(RpsbA-pro-phaC2-RpsbA-ter),连同壮观霉素抗性基因aadA表达盒(prrn-aadA-TpsbA-ter)一起克隆进烟草叶绿体基因组同源片段rbcL和accD中,构建了烟草叶绿体转化表达载体pTC2。基因枪法转化烟草(NicotianatobacumL.)叶片,获得具有壮观霉!"#$%&’()。+T0代和T1代转基因植株的PCR和Southernblot鉴定结果表明,外源基因确已整合进烟草叶绿体基因组中,T1代转基因植株已达同质化。RT-PCR分析结果证实phaC2基因已在转录水平上表达。转基因植株的正反交试验证明外源基因在转基因后代中遵循母性遗传规律,不存在转基因的花粉漂移现象。通过叶绿体遗传转化,实现中长链羟基脂肪酸聚,-./0123phaC2在烟草叶绿体基因组中的稳定转化。更多还原

【Abstract】 Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHAs biosynthesis is type Ⅱ PHA synthase. The gene phaC2 encoding type Ⅱ PHA synthase was placed under the control of psbA-pro and psbA-ter of rice (Oryza sativa ) to construct phaC2 cassette, which was ligated with the screening marker gene aadA cassette (prrn-aadA-TpsbA-ter) . These recombined fragments were cloned between the plastid rbcL and accD genes for targeting to the large single copy region of chloroplast genome. Chloroplast transformation vector of pTC2 was constructed and introduced into tobacco(Nicotiana tobacum ) chloroplast genome through particle bombardment. PCR and Southern blotting analysis confirmed stable integration of phaC2 into the chloroplast genomes of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homoplasmy. The expression of phaC2 at transcription level was detected by RT-PCR. Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation of phaC2 encoding type Ⅱ PHA synthase in tobacco via chloroplast genetic engineering.更多还原