Cloning Of The CDNAs Of DGAT1Genes And The Prokaryotic Expression From Camelia Oleifera

As a specie of woody fuel plants of great importance in the Southern part of China, Camellia oil is mostly composed of unsaturated fatty acids (UFA), such as oleic acid and linoleic acid,So Camellia oil is a high quality edible oil. However, because of the camellia seed oil rate is low, there are many serious impact on the development and economic value of Camellia oleifera. TAG is the main storage camellia lipid, DGAT is the only rate-limiting enzyme that acts in the final and committed step of TAG synthesis, Its main effect is catalytic DAG plus acyl fatty acids form TAG.So the study of DGAT gene in Camellia Oleifera is important for revealing the lipids molecular biosynthesis patterns. The main results in the paper are as follows:(1) Cloning of partial cDNA sequence of DGAT1gene from Camellia oleifera.So "DGAT" was used as the keyword in the search in GeneBank, and six amino acid sequences and full-length cDNAs of DGAT gene in other plants were found. Based on the alignment of the five amino acid sequences and cDNA sequences, a pair of degenerate primers was designed in the conservative region. Total RNA isolated from the riping seed from the state authorized qualities "HUA SHUO"which was also the raw material in the process of the construction of the cDNA library was used to generate cDNA by reverse transcription. A sequence about500bp in length was cloned by degenerate PCR, after sequencing we got partial cDNA of506bp.According to the results of Blast online, confirmed the fragment products as the part of camellia Oleifera DGAT1gene, But to be through the3'end and the5'integrity verification.(2) Molecular clone and bioinformatics analysis of DGAT1gene from C. oleifera. Based on the partial cDNA sequence of DGAT1gene from Camellia oleifera, three gene specific primers were designed for5'RACE, and two for3'RACE. Total RNA isolated from the riping seed of from "HUA SHUO" was used to generate RACE ready cDNA by reverse transcription. Finally, we got Full-length cDNA cloning of DGAT1gene and was named by co-dgatl. The results showed that this gene is1893bp in length, and it has an open reading frame of1548bp and121bp5'UTR and229bp3'UTR,its encoding515aminoacids Application of some bioinformatics software online analysis, the results showed that:Camelia oleifera FBPase protein molecular weight is58.44KDa, theoretical isoelectricpoint is8.70, where the number of negatively charged amino acid residues46, thenumber of positively charged amino acid residues38, and it is a unstable protein, instability coefficient is46.41. The hydrophobic index of DGAT1was from-3.31to 3.744, and has ten obvious transmembrane domain.(3) Camelia oleifera DGAT1gene expression in the original fusion.The fragment encoding DGAT1protein from Camellia was inserted into a prokaryotic expression vector pET-30a and was overexpressed in E coli BL21.And were successfully expressed in E.coli.