To Improve The Fatty Acid Content Of Chlamydomonas Reinhardtii By Using Lpaat And Gpd1 Gene

With the economic globalization and the rapid amplification of total population, energy demand is increasing. However, non- renewable energy such as fossil oil is facing the danger of depletion so that people are urgent to found renewable energy. Biodiesel is thought the ideal renewable energy, and fatty acid methyl esters(FAME) is the major constituent of biodiesel. Biodiesel is made by transesterification using renewable resource as raw material, hence it has the similar performance with petroleum and diesel. The production of oil from microalgae has many advantages, such as less farmland, easy to cultivation and collection, high oil content and so on. Based on the above advantages, the microalgae yields oil has become a hot research topic in recent years. Genetic engineering is a commonly method of genetic transformation to achieve the purpose of orientation and obvious effect. In the fatty acid synthesis pathway of Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase and lyso-phosphatidic acid acyltransferase are two key enzymes, which play a key role in the regulation of fatty acid content. Here, lyso-phosphatidic acid acyltransferase gene(lpaat) from Brassica napus and glycerol-3-phosphate acyltransferase gene(gpd1) from Saccharomyces cerevisiae were inserted into the genome of Chlamydomonas reinhardtii and showed transcript activity leading to increase the fatty acid content of Chlamydomonas. The main results are as follows:1. The sequence of lpaat and gpd1 genes were obtained from NCBI and optimized according to the codon bias of Chlamydomonas reinhardtii in order to be functioned in Chlamydomonas reinhardtii. The modified sequence were synthesized and connected to p H124 to construct an expression vector, named p H124-c-lpaat and p H124-c-gpd1. The expression vector were introduced into Chlamydomonas reinhardtii by glass-bead method, respectively. The transgenic algae were screened by Zeomycin resistance, PCR and GC-MS. The transgenic algae contained c-lpaat were named Tranc-lpaat-J, K, L, M, N, Q, 1, 2, 3, 4, 5, 14, 15. And the transgenic algae contained c-gpd1 were named Tranc-gpd1-P, T, C, O, N, 10, 15, 16.2. Expression level of c-lpaat and c-gpd1 in the transgenic algae were analysized by RT-PCR and real-time fluorescence quantitative PCR. Results of semi-quantitative RT-PCR showed that the expression level of target gene in Tranc-lpaat-15 and Tranc-gpd1-T were the highest. After heat shock one time, the expression level of c-lpaat and c-gpd1 were increased respectively by 1.93 fold and 2.98 fold. After heat shock three times, the expression level of c-lpaat was increased respectively by 3.01 fold, 4.46 fold and 5.30 fold. The expression of c-gpd1 was increased respectively by 3.6 fold, 5.42 fold and 8.58 fold.3. The total fatty acid content and the components in transgenic algae were analysized by GC-MS. The results were as follows:(1) Transgenic algae contained the highest fatty acid were screened by GC-MS and the transgenic algae were named Tranc-lpaat-15 and Tranc-gpd1-T which respectively containing c-lpaat gene and c-gpd1 gene.(2) After heat shock one time, the fatty acids in Tranc-lpaat-15 transformant increased by 16.8%. Among the fatty acids, the content of C18:1t was the most. It increased by 177.27% when comparing with the control group Chlamydomonas reinhadtii CC-849. The fatty acids in Tranc-gpd1-T transformant rose to 26.7%. Among the fatty acids, the content of C18:1t is the most. It inceased by 270.91% when comparing with the control group.(3) After heat shock three times, the fatty acids in Tranc-lpaat-15 transformant rose to 44.5% comparing with the control group Chlamydomonas reinhadtii CC-849. Among the fatty acids, the content of C18:0 and C18:1t were the most which increased by 355.29% and 220.11% respectively. The fatty acids in Tranc-gpd1-T transformant rosed to 67.5%.Among the fatty acids, the content of C18:0 and C18:1t were the most which increased by 428.24% and 394.18% respectively. In Tranc-gpd1-T and Tranc-lpaat-15, the fatty acid content of C16:0,C18:0,C18:1t, C18:2t were improved the most.The introduction of exogenous genes which insert into the nuclear genome of Chlamydomonas reinhardtii by genetic recombination will improve the lipid synthesis in transgenic algae. Transgenic algae which express exogenous genes can maintain their integrity generation by generation in our lab. The exogenous genes of lpaat and gpd1 can be regulated by Hsp70 A promoter when inducing by heat shock. Finally, the synthesis of triacylglycerol could be regulated and the fatty acid content in Chlamydomonas reinhardtii could be increased. This research could obtain transgenic algae with high oil content, which provided the experimental basis for the production of biodiesel.