Molecular Cloning And Functional Characterization Of Long-Chain Acyl-CoA Synthetase Gene From Brassica Napus

In oil crops, the oil (i.e. triacylglycerols) is the most valuable product of the seeds. Enhancing the quantity of oil per weight basis in oil crops would increase the value of the crop. As part of our effort to identify enzymes that is critical for producing large amounts of plant oil in the seed of oil crops.Long-chain acyl-coenzyme A synthetases (LACSs) activate free fatty acid to acyl-CoA thioesters and play an important role in fatty acid anabolism and catabolism. These classes of important enzymes are involved in several fatty acid-derived metabolic pathways, such as the activation and elongation of fatty acids, biosynthesis of phospholipids, triacylglycerol and jasmonate. In this study, four cDNAs encoded long chain fatty acyl-CoA synthetase have been isolated from rapeseed (Brassica napus) flower or seeds, designated as BnLACS1, BnLACS2, BnLACS3 and BnLACS4, respectively. Sequence analysis indicated that the four LACSs possessed typical molecular characteristics of LACS. The phylogenetic analysis showed that AtLACS1, AtLACS2, AtLACS9 and 4 BnLACSs were in group I. AtLACS1 has overlapping functions with AtLACS2 in plant wax and cutin synthesis.The function of genes BnLACSs was confirmed to encode LACS activity by yeast complementation tests. LACS substrate specificity assays indicated that the BnLACSs preferentially activate long chain fatty acids and saturated fatty acids. Yeast strain cells pep4 containing pYES2-BnIACS1 or pYES2-BnLACS2 can improve fatty acids and lipid content than the control yeast. These studies suggest that this enzymes increase lipid stored in yeast and have potential use in plant seed oil and bio-energy produced algea.Compared with low oil content varieties seed, the four genes is strongly expressed in high oil content varieties seeds at 35 days after pollination (DAP). The expression pattern suggested that the four BnLACSs might be involved in the biosynthesis of lipids and oil accumulation in rapeseed. Real-time PCR analysis reveals that BnLACS1 and BnLACS2 were strongly expressed in lipid developmental stages of seeds. The trend is similar to the 'S' trend of lipid synthesis in seed. In high oil content varieties, the two genes expressed more stronger.To study the biological functions of BnLACSs, the plant expression vectors were constructed, and the positive transformants of Agrobacterium Tumefaciens have infected Brassica napus, respectively. At present, we obtained over-expression of BnLACS1 in transgenic Arabidopsis thaliana and Brassica napus plants. Preliminary results showed that 5% increased in transgenic Brassica napus plants seed oil content.