Cloning And Functionalanalysis Of Glycerol-3-phosphate Dehydrogenase Of Phaeodactylum Tricornutum

Microalgae is an ideal raw material for biodiesel. To further increase its lipidaccumulation by means of genetic modification is the important issue of the microalgaebiodiesel. Glyceraldehyde3-phosphate (Gly3P) is formed from the glycolyticintermediate dihydroxyacetone phosphate (DHAP) via glyceraldehyde3-phosphatedehydrogenase (GPDH). The purpose of the current research was to clone GPDH fromPhaeodactylum tricornutum, and test its function in lipid accumulation. GPDHH genewas first cloned from P. tricornutum, ligated with expression vector pHY18, andtransformed into Phaeodactylum tricornutum for recombinant expression. The GPDHgene transformed microalgae were selected in media supplemented with chloramphenicol,and gene integration was confirmed by genomic DNA PCR. The expression of GPDHHwas analyzed by by Real-time quantitative PCR and western blot, showing thetransformed GPDHH expression in the transcript and protein levels. The lipid contentdetected by Nile red staining showed a signicant increase as expected. Meanwhile, fattyacid composition of16carbons and18carbons significantly increased. Glycerolconcentration per cell in transformed microalgae strains showed6.7times higher than thecontrol, indicating that the expression of the transformed GPDH promoted the conversionof glycolytic intermediates dihydroxyacetone phosphate to glycerol3-phosphate. Theresults of this study show that the cloned GPDH gene has a significant impact on thesynthesis of glycerol and fatty acid composition of microalgae, but has no significanteffect on the lipid content. The results also suggest that a comparative study is required todetermine the other isoenzymes of P. tricornutun GPDH for its role in lipid accumulation.