Studies On The Production Of PHB, A Biodegradable Thermoplastic, In Transgenic Plants

Poly-3-hydroxybutyrate (PHB) and other related polyhydroxyalkanoates (PHAs) are biodegradable thermoplastic polyesters stored in numerous bacteria as carbon and energy reserves and now have been regarded as ideal alternatives to traditional petroleum-derived plastics, which cause severe environmental and waste management problems. Because PHB/PHAs production by bacteria fermentation is prohibitively expensive, great attention has been focused on the synthesis of PHB in plants using CO2 for carbon and sunlight for energy, which is considered as a promising strategy to obtain price-competitive biodegradable plastics. Although great progress has been made in this area, there is still a long way to go for the realization of large-scale production, which requires an environmentally friendly transgenic plant with high-level PHB accumulation and stable inheritance. Here, we try to optimize the production of PHB in plant so that "green plastics" can compete with traditional plastics in the near future.1. In order to avoid promoter-homology-mediated gene silencing, we isolated another seed-specific promoter nap300 from Brassica napus H165 to replace 75 promoter in constructed expression cassettes. Nap300 was fused to the reporter gene 3 -glucuronidase (GUS) to confirm its heterologous expression ability. Fluorometric and histochemical assay of GUS activity showed that this approximate 300bp DNA fragment was sufficient to direct seed-specific gene expression and the two substitution mutations in B-box led to a slight decrease in the promoter's expression efficiency and almost had no effect on its temporal and spatial expression pattern. Then, nap300 and 75 were fused in a similar way to phbA gene encoding 3-ketothiolase for a direct comparison. The result indicated that the two promoter's expression efficiency reached a peak at the same developmental stage of tobacco, which means they have the advantage of being used simultaneously for expressing different foreign genes in plant. Thus, nap300 will help us to overcome co-suppression in plant-mediated PHB production. Through comparison detailed information about the characteristics of the two promoters can also be obtained, which is necessary for precisely regulated gene expression in plant genetic engineering.2. Chloroplast genetic engineering, a new technology that could overcome many problems associated with nuclear genetic engineering, is of growing interest for bioproduction of valuable materials. It has several advantages including theextraordinarily high level of transgene expression and environmental safety, the favorable environment for prokaryote gene expression, the absence of "position effect" and gene silencing. To investigate whether it is suitable for biodegradable plastic production, we constructed the chloroplast integration and expression vector pTRV-PHB, and integrated all the genes necessary for PHB synthesis into tobacco chloroplast genome through gene-gun transformation.The chloroplast transformants showed phenotypically normal growth and were fertile. Gene silencing, a common phenomenon in PHB production through nuclear transformation, hadn't been observed in the chloroplast transgenic plants analyzed by Northern dot blot. According to RT-PCR results, all the three targeted genes in chloroplast transgenics were expressed at a significantly higher level compared with those in nuclear transgenics. GC analysis proved that transplastomic plants had the ability to synthesize PHB in leaves. These data suggest the feasibility of producing PHB through chloroplast genetic engineering. Although PHB-accumulation in obtained transgenic plants was not as high as expected, we believe that chloroplast engineering can be really applied for commercial production in plant after further improvement.3. The mechanism of DNA integration mediated by homologous recombination in chloroplast transformation was tentatively explored. We isolated two chloroplast DNA fragments from Brassica napus and used them as targeting sequence...