Effects On Lipid Synthesis Of ACP1and ACP2Genes Of Chlamydomonas Reinhardtii

Renewable biodiesel is an ideal substitute of traditional fossil fuels. Many microalgae can efficiently synthesise oils as raw materials for biodiesel production. Nowadays microalgae biodiesel is researched vigorously by many countries and remarkable progress have been achieved, such as microalgae selective breeding and modification, molecular mechanisms of lipid metabolism, large-scale cultivation and harvest. A series of demonstration bases of microalgae biodiesel have also been constructed; however, the cost of microalgae biodiesel is still higher than fossil fuels. Several high-yield oil algae strains have been obtained through natural selection and genetic breeding, but they often grow poorly or requre harsh culture conditions which is not conducived to large-scale cultivation.To solve this problem, we attempt a comprehensive understanding of microalgae substances metabolism and its regulation of molecular mechanisms, and provide a theoretical basis for the genetic improvement of algae species.Chlamydomonas reinhardtii is a unicellular eukaryotic green algae. As a eukaryotic model organism, the mature genetic transformation methods of nucleus, chloroplast, mitochondrial genome have been established. In addition, its genome sequencing is completed. Therefore. C. reinhardtii is ideal to explore the general lipid metabolic machenisms of microalgae. In this study, the role of ACP1and ACP2(acyl carrier protein) in the oil synthesis of C. reinhardtii were studied.It was reported that ACPI and ACP2gene were closely related to lipid synthesis of terrestrial plants. But there are not directly evidences in C. reinhardtii. The study is tried to analyse the effects of ACPI and ACP2on oil systhesis in Chlamydomonas reinhardtii through amiRNA interference method. The major works are as followings:(1) The amiRNA expression vectors pmiR1162-ACPl and pmiR1162-ACP2targeted to ACP1and ACP2respectively were constructed based on one-step PCR and MAGIC;(2) C.reinhardtii Cells were transformed with vectors pmiR1162-ACP1and pmiR1162-ACP2, respectively. The ratio of positive transgenic lines containing amiR1162-ACP1or amiR1162-ACP2was about80%;(3) The results of quantitative RT-PCR suggested that there were about28-57%reduction of ACP1or ACP2transcript level in the transgenic lines;(4) The ACP2protein was expressed in Escherichia.coli BL21(DE3), and the ACP2antibody was prepared;(5) ACP2protein levels dropped significantly in the transgenic line which containing pmiR1162-ACP2via the detection of Western blot.(6) The neutral lipid content decreased in these two transgenic lines containing amiR1162-ACP1or amiR1162-ACP2under conditions of nitrogen deficiency, compared with C. reinhardtii.