Cloning And Expression Of Genes Involved In DHA Biosynthesis In Schizochytrium

Two genes were isolated from the cDNA library of Schizochytrium sp.FJU-512 using bioinformatics methods. The Acyl carrier gene (ACP) and Malonyl transferase gene (MAT) were cloned and then the Acyl carrier gene (ACP) was expressed in E.coli. The results make a foundation for further investigating the mechanism of Docosahexaenoic acid (DHA) synthesis pathway in Schizochytrium sp.FJU-512. The main results of this study were as follows:(1) Sequences are aligned using the Blastn program of the NCBI site, a 429bp ORF was obtained. The deduced amino acid sequence contained 142 residues with isoelectric point of 5.04. The predicted structure of ACP was obtained using SWISS-MODEL, The 2D structure of ACP was constructed by the PHD program. The structure contains fourα-helices ,one-loop and is absence ofβ-sheet. It has the conserved domain: 4'-PP binding site. (4'-phosphopantetheine prosthetic) . Phylogenetic analysis showed ACP was the closest to Isochrysis galbana.(2) To identify the function of cloned ACP-cDNA, the recombinant expression vector was constructed, and the BL21(DE3) was used as a host for expressing ACP gene. The expression of protein was induced by 1%IPTG SDS-PAGE showed that a 26 kD recombinant protein was effectively expressed as enclusion bodies.(3) The genomic DNA and total RNA of Schizochytrium sp.FJU-512 were extracted respectively. The MAT-DNA and MAT-cDNA was amplified using the techniques of LA-PCR and RT-PCR. The total size of MAT-cDNA was made of 1121bp. Amino acid sequence alignments indicates that it has the two conseved domains which are the active sites of MAT as the ACP-binding region.