Cloning And Functional Analysis Of Diacylglycerol Acyltransferase Genes In Eustigmatos Cf. Polyphem

Eustigmatos cf. polyphem is a yellow-green unicellular edaphic microalga, which belongs tothe Heterokontophyta, Eustigmatophyceae. Previous research had proved the formation ofhigh-value bioactive subatances in E. cf. polyphem cells, such as polyunsaturated fatty acid,beta-carotene, chrysolaminarin and so on, especially that the cells accumulated large amount ofstorage lipids at the late growth phase under nitrogen-limited condition, therefore, E. cf.polyphem could be a potential microalga with the advantage of exploiting and utilizing biologicsand bioenergy. In our study, we cloned the full-length cDNA of genes that encoded thediacylglycerol acyltransferase (DGATs) involved in TAG synthesis of E. cf. polyphem,performed heterologous expression assay to identify their function, investigated the changes inthe expression level of the DGATs mRNA following nitrogen limited condition associated withTAG accumulation. Our aim was to understand the molecular and regulatory mechanisms ofTAG synthesis in the E. cf. polyphem cells, and provide theoretical basis to the exploitation ofmicroalgae biological energy. The following are the results:(1) We cloned the full-length cDNA of a DGAT gene (EcfpDGATb) from E. cf. polyphem of2347bp by the RACE technology，and gained the3’-end cDNA of the other two genes ofEcfpDGATa (309bp) and EcfpDGATc (480bp), respectively.(2) We found a23bp5’-UTR and a338bp3’-UTR from the EcfpDGATb full-lengthsequence by DNA sequence analysis, as well as a ORF encoding661animo acids. The wholeprotein included a43aa predicted signal peptide and9possible transmemberane regions, and6motifs within the DGAT domain, indicated EcfpDGATb to be a member of the family thatencoded the II type DGAT in E. cf. polyphem.(3) The heterologous expression experiment revealed that the mutant yeast harborEcfpDGATb restored lipid bodies in the cells and appeared to display a preference forincorporating saturated16C and18C fatty acid into TAG, which confirmed the fact thatEcfpDGATb involved in the TAG synthetic process.(4) The relative abundance of mRNA level in two sodium nitrate concentration conditionsboth went up first and dropped after following the growth phase, the maximal mRNA relativeabundance showed at the36h when cultured in higher nitrogen concentrate of18mmol/L NaNO3and96h at the lower one of6mmol/L NaNO3, demonstrated that EcfpDGATbresponsived to the N-limited condition at the level of RNA abundance.