The Roles Of Glycerol-3-Phosphate Acyltransferase And Lysophosphatidic Acid Acyltransferase In Lipid Metabolism In Brassica Napus

Oil content and fatty acid composition are the key parameters of varieties in oil crops. It is of importance either theoretically or practically to investigate the genes related to lipid biosynthesis.Phosphatidic acid (PA) is an important precursor for the synthesis of triacylglycerol. In plants, glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT) are essential for the de novo synthesis pathway of PA. PA is an important intermediate in lipid metabolism including triacylglycerol (TAG) and phospholipid synthesis. In addition, PA functions as a signaling molecular in regulating biological processes. However, the molecular regulation mechanisms of GPAT and LPAAT are yet to be identified in crop plants. In this study, the genes encoding GPAT and LPAAT are isolated and cloned from Brassica napus for functional characterization. The main results are as follows:1. The full length cDNA sequences of GPAT and LPAAT in Brassica napus were identified by searching EST database using the sequcence of homologous gene in Arabidopsis. The full length cDNAs of six genes including BnGPAT9, BnLPAAT2, BnLPAAT3, BnATS2, BnATSl and BnLPAAT5 had been cloned from Brassica napus.2. The full length cDNAs of BnGPAT9, BnATSl, BnATS2, BnLPAAT2, BnLPAAT3 and BnLPAAT5 were cloned into pET42a(+) vector for protein expression in E. Coli cells. The proteins will be used for the enzyme activity assay in the future work.3. The expression profiling of BnGPAT9, BnATSl, and BnLPAAT2 was performed by semi-quantitative PCR and real-time PCR. BnGPAT9 is expressed in young leaf, inflorescence, flower, old leaf and stem with the order from higher to lower. The expression level of BnATSl is highest in young leaf, and is higher in inflorescence and old leaf, but is lower in flower and stem. BnLPAAT2 is expressed in old leaf, young leaf, inflorescent, stem and flower with the order from higher to lower.4. The full length cDNAs of BnGPAT9, BnATSl, BnATS2, BnLPAAT2, BnLPAAT3 and BnLPAAT5 were cloned into the binary vector pBI121 under the control of 35S promoter and then were transformed into Brassica napus via Agrobacteria mediation. More than 20 independent transgenic plants containing cDNA of BnGPAT9, BnATSl and BnLPAAT2 were generated, respectively. The expression levels of BnGPAT9, BnATSl and BnLPAAT2 in the transgenic plants are much higher than those of wild type plants by semi-quantitative PCR.5. Overexpressing BnGPAT9, BnATS1 and BnLPAAT2 in Brassica napus enhanced oil accumulation in both leaves and seeds. The oil content of BnGPAT9-OE, BnATS1-OE and BnLPAAT2-OE plants were increased significantly, as compared with wild type plants. The oil compositions of leaves in OE plants were also different from that of wild type plants.6. Overexpressing the specific gene resulted in the decrease in the expression level of genes in the same family, but enhanced the expression of the genes that functions as upstream or downstream in the Kennedy pathway.7. The homozygous mutants of Arabidopsis LPAAT3, LPAAT5, ATS1, and heterozygous mutants of AtLPAAT2 were isolated for BnLPAATs and BnATSl functional complementary study.Taken together, the overexpression of BnGPAT9, BnATSl, and BnLPAAT2 had significant impact on oil content and fatty acid composition of Brassica napus. Different GPAT and LPAAT exhibited heterogeneity role in regulating fatty acid composition. This study provides a theoretical basis for the genetic regulation of the lipid biosynthesis in oil crop plants.