The Functional Studies Of Lipid Metabolic Pathways Related Gene In Chlamydomonas

The world is facing a severe energy crisis and environmental issues. Traditional fossil energy can not longer meet people’s needs. What’s worse, the large-scale use of fossil fuels has caused great damage to the ecological environment. the research of new energy is evoking heat discussion worldwide. Microalgae with the advantages of short growth cycle, easy cultivation, high oil content and occupying arable land and so on, is expected to become a good raw material for the production of environmentally friendly renewable biodiesel. Comparing to the much achievements in the fat anabolic areas of animal and vegetable, research of oil mechanism in microalgae is still in its infancy.Functions of CrPK1, CrPK2, CrPEPCK, CrZnFAN1, CrBbox and CrRING in the oil metabolic pathways of Chlamydomonas reinhardtii was studied. Full-length sequence of CrPKl, CrPEPCK, CrZnFAN1, CrBbox and CrRING gene were cloned and inserted into overexpression vector. Then, the vector was transformed into Chlamydomonas reinhardtii strain CC425by using glass beads method. The results showed that the expression of target gene increased substantially comparing to the control. The oil content in CrPKl, CrPEPCK and CrZnFAN1transformants were increased but decreased in CrBbox and CrRING transformants.pGEX-CrPK1, pGEX-CrPEPCK, pGEX-CrZnFANl, pGEX-CrBbox and pGEX-CrRING were transformed into E. coli BL21. The molecular weight of products were in line with expectations. CrPKl and CrPEPCK protein purified with GST affinity chromatography. The results of enzymatic activity measurement of CrPKl and CrPEPCK indicated that it has the biological activity.Interference fragments of the genes above were cloned to construct RNAi expression vector. After that, they were transformed into the Chlamydomonas reinhardtii CC425. The mRNA level in transformants were significantly reduced respectively comparing to the control. RNAi-CrBbox and RNAi-CrRING transformants accumulated more oil compared with the control group, while KNAi-CrPK1, KNAi-CrPK2, RNAi-CrPEPCK and KNAi-CrZnFANl accumulated less.Protein that may interact with the CrZnFAN1, CrBbox and CrRING was initially selected by yeast two-hybrid. The preliminary functional study of CrPKl, CrPK1, CrPEPCK, CrZnFAN1, CrBbox and CrRING in oil metabolic pathways in Chlamydomonas reinhardtii was carried out. The fact that CrPK1, CrPK2, CrPEPCK and CrZnFANl gene could promote lipid accumulation of Chlamydomonas reinhardtii while CrBbox and CrRING was not conducive to the grease accumulation was initially confirmed. What’s more, the proteins may interact with the CrZnFANl, CrBbox, and CrRING were screened out.