Cloning And Characterization Of A Novel Acly-CoA: Diacylglycerol Acyltransferase2(DGAT2) Gene From Phaeodactylum Tricornutum And Construction Of A General Transformation Vector

Microalge has become a hot spot of biodiesel research for its traits, such as high photosyntheticefficiency, strong environment adaptiveness, short growth cycle and biomass. Among them, about20pecent of the primary productivity of word coming from marine diatoms, they are extremely importantprimary producers in the ocean. As the mode alage of diatom research, the pennate diatom Phaeodactylumtricornutum is the high quality raw material of microalgae biodiesel for its high oil content about20%-30%of total dry weight.In this study, using P. tricornutum as research materials, the gene diacylglycerol acyltransferase2(DGAT2) was cloned, which was the key enzyme in neutral lipid synthesis of microalgae by the method ofextension primer PCR. After then, the bioinformatics analysis based on obtained gene sequences wascarried out. Using many control sequences of the endogenous fcp (fucoxanthin, chlorophyll a/c-bindingprotein) gene clusters and selectable marker bar gene, we constructed general transformation vector for thediatom P. tricornutum. Our research provided the basic materials and available method for functionalcharacterization of the key genes involved in the lipid metabolic pathway as well as construction ofoleaginous recombination algae strains.1. Based on a function unknown gene fragment, using extension primer PCR method, a noveldiacylglycerol acyltransferase2(DGAT2) gene was cloned from P. tricornutum cDNA. We named it asPT-cDGAT2, which was1080bp in length, encoding a protein of359amino acids. Subsequently,diacylglycerol acyltransferase2was isolated from P. tricornutum genome DNA，which was1459bp inlength. We nanmed it PT-gDGAT2, it was probable that this gene conteined two introns (77bp and88bp).The analysis results of the deduced amino sequence indicated that it contained a typical conserved DAGATdomain, including CRAL/TRIO lipid-binding domain; CoA binding motif; PlsC domain and so on. Aftercomprehensive analysis, PT-cDGAT2may possess the function of acyltransferase. It catalyzes the final stepin triacylglycerol (TAG) biosynthesis by by converting diacylglycerol（DAG）and fatty acyl-coenzyme A（CoA）into triacylglycerol. These bioinformatics analysis results we mentioned above will providebeneficial evidences for functional characterization of amino acid sequences encoded by PT-cDGAT2aswell as the related key enzymes research involved in lipid metabolic pathway of P. tricornutum.2. Refering to nuclear transformation general methods and selection measures of eukaryoticmicroalgae, according to P. tricornutum own characteristics, we chose appropriate elements and selectablemarker gene, constructed a general transformation vector for P. tricornutum. Several endogenous elementsof fcp gene clusters were cloned from P. tricornutum genome DNA, these fragments and herbicide bastaresistant bar gene were inserted into the cloning vector pSP73. Subsequently, we checked the vector usingPCR, enzyme digestion and sequence analysis. The results showed that the general transformation vectorpfcpA-MCS/fcpB-Bar was successfully constructed. 3. Recombinant plasmid pfcpA-MCS/fcpB-Bar was transferred into P. tricornutum usingmicro-particle bombardment. Positive monoclonal algaes were selected using the f/2-PPT solid plateaccording to the established selection tag system. The experimental results showed that the bar gene wassuccesssfully expressed in the P. tricornutum cells, revealed the effectiveness of general transformationvector, which provided the available method for functional characterization of PT-cDGAT2as well asconstruction of oleaginous recombination algae strains.