【作者】 王玉华；

【导师】 杨清； 黄丛林；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 羟基脂肪酸聚酯（polyhydroxyalkanoates，PHAs）属于微生物聚酯，在生物体内主要作为细胞内碳源和能源的贮藏性物质存在，它不但具有与化学合成塑料相似的物理、机械特性，而且具有完全的生物可降解性和良好的生物相容性，被认为是石油化工聚酯的理想替代品。中长链羟基脂肪酸聚酯（medium-chain-length-PHAs，mcl-PHAs）是几种中长链的羟基脂肪酸结构单体的共聚物，比短链羟基脂肪酸聚酯（short-chain-length-PHAs，scl-PHAs）具有更好的物理、机械特性。微生物发酵法生产的PHAs价格过高，无法在市场上与石油化工聚酯竞争。随着分子生物学的发展，人们逐渐将视线转向转基因植物，将细菌中的PHAs合成途径引入植物，利用植物作为生物反应器生产PHAs。 高等植物叶绿体基因工程是植物基因工程发展的新领域，是近十年才开始兴起的一种新的转基因途径，具有传统核转化技术所不具备的独特魅力，已经成为植物基因工程领域研究的热点，因而自问世以来迅速在基础研究和实践应用等方面显示出旺盛的生命力，尤其在以叶绿体为生物反应器合成目标产物方面应用广泛。 本研究通过PCR方法从水稻叶绿体基因组中克隆了叶绿体基因psbA的启动子和终止子以及rbcL基因的3’-端调控序列；从烟草叶绿体基因组中克隆了psbA基因的3’-端调控序列和两段叶绿体同源片段。以aadA（编码氨糖苷-3’-磷酸转移酶）基因做筛选标记，分别构建了含phaC2基因的单价叶绿体遗传转化表达载体pTC2以及同时嵌合phaC和phaG基因的双价叶绿体遗传转化表达载体pTGC，基因枪转化法转化烟草，获得具有壮观霉素（Spectinomycin，Spc）抗性的转pTC2的阳性植株5个、转pTGC的阳性植株6个。对T₀代和T₁代转基因烟草的PCR和Southern blot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代转基因植株已完全达同质化。RT-PCR分析结果证实aadA和phaC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烟草后代中遵循母性遗传规律，不存在转基因的花粉漂移现象。气相色谱法检测转基因株系中的mcl-PHAs含量最高达4.8mg/g干重（株系S4-3），合成的mcl-PHAs单体主要成分为3-羟基辛酸（3-hydroxyoctanoate，3-HO）和3-羟基癸酸（3-dydroxydecanoate，3-HD）。只转化phaC2基因的叶绿体型转基因烟草基本检测不到有mel-PHAs合成，共表达phaC和phaG基因的叶绿体型转基因烟草则能合成相对较高水平的mcl-PHAs。透射电镜观察到转基因烟草叶绿体中确有更多还原

【Abstract】 Polyhydroxyalkanoates （PHAs） belong to the group of microbial polyesters, which are a class of polymers produced by various species of bacteria as a source of carbon and an 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 had been produced commercially by bacterial fermentation method, but the process was not economically competitive with petrochemical-based polymers. Novel efforts focused on using transgenic plants as bioreactors to produce PHAs.Chloroplast genetic engineering in higher plants is a new developmental area of plant genetic engineering, and a novel transgenic approach arisen in recent ten years. Chloroplast genetic engineering has several more unique advantages over traditional nuclear transformation. It has become a research hotspot in plant genetic engineering. Thus, in recent years, chloroplast genetic engineering has been applied successfully in several areas including basic and practical researches, especially in plant bioreactor areas.In this study, we cloned promoter and terminator of chloroplast psbA gene and 3’-untranslated regions （3’-UTR） of rbcL gene from rice chloroplast genome by PCR, and also cloned both 3’-UTR of psbA gene and two chloroplast homologous fragments from tobacco chloroplast genome. With aadA （encoding aminoglycoside 3’-adenylyltransferase） gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. Five positive transgenic lines transferred with pTC2 and six positive transgenic lines with pTGC were obtained. PCR and Southern blot analysis confirmed stable integration of the foreign genes into the chloroplast genomes of T₀ and T₁ transgenic plants, and homoplasmy of T₁ transgenic plants. The expressions of both aadA and phaC2 genes at transcription level were detected by RT-PCR.更多还原