Cloning Of Pepc Gene Of Dunaliella Salina And Construction Of Expression Vector For Microalga Using DGAT Genes Cloned From Brassica Napus

Background:Dunaliella salina is a unicellular eukaryotic green alga, and is capable of growth in salinities ranging from 0.05 to 5.0 M NaCl. The most important advantage of Dunaliella salina is the simple culture condition. Effectively culturing a large number of algae can greatly reduce the cost, and therefore Dunaliella salina is suitable as an algal candidate for the bio-energy microalgae. In a variety of enzymes involved in the synthesis of triacylglyceride (TAG), phosphoenolpyruvate carboxylase (PEPC) and diacylglycerol acyltransferase (DGAT) are the key enzymes in the process of lipid metabolism. Phosphoenolpyruvate carboxylase has the negative regulation in the lipid synthesis, by catalyzing the phosphoenolpyruvate into phosphoric acid and oxaloacetate, so that the phosphoenolpyruvate can be utilized in protein synthesis. Therefore, Phosphoenolpyruvate carboxylase determines the proportion of protein and TAG by controlling the flow of phosphoenolpyruvate. Diacylglycerol acyltransferase has the positive regulation in the lipid synthesis, and is a key enzyme in the final step of catalyzing triacylglyceride biosynthesis in plants and animals. DGAT1 has a broader role in triacylglyceride metabolism, while DGAT2 is focused on the accumulation of specific fatty acids, and both of them can perform the final step in the triacylglyceride synthesis pathway. Obviously, DGAT1 and DGAT2 have a similar effect in lipid synthesis.Aim:To investigate the function of phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina, a full length of the PEPC cDNA sequence was isolated. And in order to investigate whether diacylglycerol acyltransferase will affect the triacylglyceride biosynthesis in microalga, the cloned DGAT sequences were utilized for constructing eukaryotic expression vectors and the vectors were transferred into the cells of Dunaliella salina by microprojectile delivery.Methods:a pair of degenerate primers was designed according to the conserved motifs of the PEPC of Chlamydomonas reinhardtii, Arabidopsis thaliana, Arachis hypogaea, etc. A cDNA fragment was obtained from Dunaliella salina by using RT-PCR, and then a full length of the cDNA was isolated by 3’and 5’RACE. And the DGAT1 and DGAT2 were obtained from Brassica napus by using RT-PCR and then cloned into pMD18-T Simple vectors. The cDNA fragments indentified correctly by sequencing- were subcloned into eukaryotic expression vectors. The vectors were transferred into the cells of Dunaliella salina by microprojectile delivery, and then the transformants were selected by phosphinothricin (PPT).Results:A cDNA fragment was obtained from Dunaliella salina by using RT-PCR, and then a full length of the cDNA was isolated by 3’and 5’RACE. The isolated cDNA sequence was 3523 bp in length with a 2949 bp coding region that encoded 982 amino acid residues with the predicted relative molecular mass of 110560.5 Dolton. And the lengths of the cloned DGAT1 and DGAT2 were 1512 bp (99%) and 1026 bp (98%) respectively, and they were utilized for constructing eukaryotic expression vectors pSV40DGAT1/CaMVBar and pSV40DGAT2/CaMVBar. The vectors were transferred into the cells of Dunaliella salina by microprojectile delivery, and then the transformants were selected by phosphinothricin (PPT). The results showed that all the transformants of Dunaliella salina were dead under the selection pressure.Conclusion:The full length of the PEPC cDNA sequence was isolated from Dunaliella salina. And the cloned DGAT1 and DGAT2 were utilized for constructing eukaryotic expression vectors pSV40DGAT1/CaMVBar and pSV40DGAT2/CaMVBar. All the transformants of Dunaliella salina were dead under the selection pressure, but the study still provided some relevant information for investigating the the triacylglyceride biosynthesis in microalga.