中长链羟基脂肪酸聚酯(medium-chain.length-PHAs,mcl-PHAs)属于微生物聚酯.羟酰-CoA-ACP-转移酶和Ⅱ型PHA合酶是mcl-PHAs生物合成途径中的两个关键酶.将编码羟酰-CoA-ACP-转移酶的基因phaG与水稻叶绿体psbA基因的启动子和终止子连接构建表达盒RpsbA-pro-phaG-RpsbA-ter,将Ⅱ型PHA合酶的基因phaC与水稻叶绿体16S rRNA基因的强启动子Prm及rbcL基因的终止子连接构建表达盒prrn-phaC-RrbcL-ter,连同壮观霉素抗性基因aadA表达盒prrn-aadA-TpsbA-ter-起克隆进烟草叶绿体基因组同源片段rbcL和accD之间,得到烟草叶绿体表达载体pTGC.用包裹有质粒pTGC的金粉子弹轰击烟草无菌苗叶片,经壮观霉素筛选后获得6株叶绿体型转基因植株.对T_0代和T_1代转基因植株进行PCR检测和Southern blot分析表明,外源基因已整合进烟草叶绿体基因组中,且T_1代转基因植株已同质化.RT-PCR分析结果证实外源基因已在转录水平上表达.转基因植株的自交及正反交结果表明,外源基因在转基因后代中能够稳定遗传,遗传方式遵循母性遗传规律,不存在转基因的花粉漂移现象.%Polyhydroxyalkanoates (PHAs) belong to the group of microbial polyesters, which are a class of polymers produced by various species of bacteria as source of carbon and energy reserve. Due to their properties of biodegradable thermoplastics and elastomers, PHAs have been regarded as ideal alternatives to traditional petroleum-derived plastics in medical areas and many other areas of high technology and high-value. Compared with short-chain-length-PHAs (scl-PHAs), such as polyhydroxybutyrate (PHB), medium-chain-length-PHAs (mcl-PHAs) copolymers are less crystalline, and are more flexible polymers with low melting points, and generally regarded as elastomers. PHAs were produced commercially by bacterial fermentation method, but the proc-ess was not economically competitive with petrochemical-based polymers. At present, novel efforts are focused on using the transgenic plants as bioreactors to produce PHAs. Both 3-hydroxyacyl-CoA-ACP-transferase and type Ⅱ PHA synthase are the key enzymes for mcl-PHAs biosynthesis. The gene phaG encoding 3-hydroxyacyl-CoA-ACP-transferase was placed under the control of psbA-pro and psbA-ter of rice to construct phaG expression cassette, and the gene phaC encoding type Ⅱ PHA synthase was placed under the control of prm and rbcL-ter of rice to construct phaC expression cassette, which were ligated with the screening marker gene aadA expression cassette prm-aadA-TpsbA-ter. These recombined fragments were cloned between the plastid rbcL and accD genes of tobacco for targeting to the large single copy region of chloroplast genome. Chloroplast expression vector of pTGC was constructed and then transformed into tobacco chloroplast genome through particle bombardment. Six trans-plastomic tobacco plants were obtained by spectinomycin screening. PCR and Southern blot analysis confirmed integration of phaG andphaC genes into chloroplast genome of T_0 and T_1 transgenic plants, and T_1 transgenic plants exhibited homogenization. The expression of phaC and phaG at transcription level was detected by reverse transcriptase-polymerase chain reaction (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.
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