Functional Study On Two Subtypes Of Glycerol Triphosphate Dehydrogenase From Phaeodactylum Tricornutum

Microalgae is an important producer in the ecosystem,which has the advantages of rapid growth and easy cultivation and high lipid content in the field of biomass energy.In recent years,some oil-producing algae strains have been put into large-scale cultivation.Among them,the diatom model organism,Phaeodactylum tricornutum is considered to be an ideal source of biodiesel.However,microalgae also constrains its industrial development due to its own production and extraction costs.With the development of modern molecular biology technology,we can carry out genetic level transformation of the corresponding algae strains.How to improve the total lipid content in Phaeodactylum tricornutum has become a research hotspot.Based on the glycerol triphosphate dehydrogenase found in the previous studies,which increasedd the total lipid and glycerol production of Phaeodactylum tricornutum,the research found two subtypes of GPDH in the Phaeodactylum tricornutum by protein analysis.Asssay by molecular biology,overexpression and artificial microRNA interferencewere processed to study its function through the mature genetic transformation system,successfully obtaining GPDH2 and GPDH3 endogenous gene-transformed algae;artificial microRNA dry algae MiGPDH2 and MiGPDH3;The function of the two subtypes of this gene was studied through physiological and biochemical analysis of these four transformed algal strains,and in wish of obtaining high-yield oil or glycerol engineering algae strain.According to previous studies,GPDH also has multiple subtypes in other species.Its main function is to catalyze the production of G3 P by dihydroxyacetone phosphate and to fix fatty acids or produce glycerol by G3 P.Some special subtypes have special subcellular organelles and contain functions such as salt tolerance and antioxidant reduction.The results showed that the growth rate and chlorophyll a content of the strains overexpressing GPDH2 and GPDH3 were significantly increased in the early logarithmic phase,and the photosynthesis efficiency was slightly changed.The total lipid content of the two GPDH2 overexpressing mutant algae decreased by 25% and 18%,respectively,and the total glycerol yield increased by 27% and 24%,respectively.The lipid content of the twoGPDH3 transformed algae increased by 114% and 83% compared with the wild type.The glycerin content was reduced by 7% and 5%.Compared with the changes of different fatty acid contents,GPDH2 had certain preference for C14:0,C16:1 fixation,and GPDH3 had a greater promotion effect on the increase of C16:0 content.The results of the study indicated that GPDH2 has stronger triglyceride activity and can induce more dephosphorylation of G3 P to produce glycerol.GPDH3 has stronger fatty acid fixation,and G3 P is more biased to bind fatty acids.The formation of phosphatidic acid ultimately synthesizes triglycerides to increase total lipid content.In addition,the experiment of specifically reducing the expression of GPDH2 and GPDH3 by artificial microRNA also accorded with the above conjecture.In addition,co-expression of glycerol and lipid in transformed algae increased;this study significantly increased total lipid while maintaining normal biomass.These results enhance our understanding of the different subtypes of triglyceride dehydrogenase in Phaeodactylum tricornutum and provide a theoretical basis and new ideas for promoting the high value utilization of microalgae resources.