Molecular Cloning And Charaterization Of Brassica Napus LACS1 And Brassica Napus LACS4

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. This classes of important enzymes are involved in several fatty acid-derived metabolic pathways,such as biosynthesis of phospholipids,triacylglycerol and jasmonate and fatty acidβ-oxidation.Therefore,cloning,analysis and characterization LACS from oilseeds can provide us important information about the process of lipids anabolism and catabolism.In this study,a 2334 bp-long cDNA,designated BnLACS1,was cloned from the flowers of Brassica napus via RACE with Arabidopsis thaliana LACS1 as probe.The results of analysis via bioinformatics methods showed that BnLACS1 contains an open reading frame of 1983 bp which encodes a putative protein with 660 amino acids. Sequence analysis indicated that BnLACS1 belonged to AMP-binding protein (AMPBP)superfamily,and its deduced amino acid sequence shared the highest identity with AtLACS1.This gene has been registered in GenBank under the accession number EU375561.Yeast complementation test confirmed that BnLACS1 encodes LACS activity, and long chain fatty acids and saturated fatty acids are the preferable substrates for BnLACS1.RT-PCR analysis revealed that BnLACS1 was strongly expressed in mature flowers and stems,but was not expressed in germinating seedlings.BnLACS1 was also expressed differently in seeds and pod coats among different oil content varieties at 35 days after flowering,it hypothesize that BnLACS1 may be related to the oil accumulation in rapeseed.A plant overexpression vector for BnLACS1 was also constructed and the related transformation experiment was performed,which build foundations for the further studies.In this study,another BnLACS,named BnLACS4,has been found through in silicon cloning with AtLACS4 as probe.The gene is 2,270 bp long with 2,004 bp of ORE encoding a putative LACS with 667 amino acid residues.Sequence analysis indicated that BnLACS4 possessed typical molecular characteristics of LACS. Expression analysis revealed BnLACS4 expressed in mature roots,stems,leaves and flowers.The gene expressed differently in seeds and pod coats among different oil content varieties at 35 days after pollination(DAP),The expression pattern suggested that BnLACS4 might be involved in the biosynthesis of lipids and oil accumulation in rapeseed.