Synthesis Of Medium-chain-length-polyhydroxyalkanoates In Transgenic Plants Via Chloroplast Genetic Engineering

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.