Expression Analysis Of Genes Involved In Accumulation Of Ricinoleic Acid In Castor Bean

Ricinoleic acid(12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid (HFA) is synthesised on the sn-2 position of phosphatidylcholine (PC) by fatty acid Δ12-hydroxylase, which is encoded by the RcFAH12 gene. Recent studies indicated that the inefficient removal of HFA from HFA-PC and incorporation into TAG are a major bottleneck for the accumulation of HFAs in triacylglycerol (TAG) of transgenic Arabidopsis seeds.Various phospholipases (PLs),long-chain acyl-CoA synthetases (LACSs), acyl-CoA:lysophosphatidylcholine acyltransferases(LPCATs), acyl-CoA binding proteins (ACBPs), and phosphatid-ylcholine:diacyglycerol cholinephosphotransferase (PDCT) may play an important role in turning over HFA-PC into HFA-TAG in castor bean. All these enzymes have multiple isoforms encoded by different genes. To determine which isoforms of these enzymes are important for engineering tri-ricinolein synthesis, we inspected the Castor Bean Genome Database to identify all genes related to these isoforms in castor.Using known A. thaliana genes as queries, a series of Basic Local Alignment Search Tool (BLAST) searches and the second BLASTp round against the Castor Bean Genome Database detected 87 proteins. The further analysis of protein structure and motifs identified 66 genes encoding different enzymes including 51RcPLs,4 RcACBPs,7 RcLACSs,3 RcLPCATs, and 1RcPDCT. Subsequently, a systematic study of the expression pattern of the corresponding genes were characterised in whole seeds at three developmental stages (19,33,47 days after pollination), leaves, and male flowers of castor. In reference to the expression pattern of RcFAH12, quantitative expression pattern analyses revealed nine seed-specific genes including 8 RcPLs and 1RcLACS, which may be involved in HFA removal from HFA-PC. Then CXSN-napin under the control of napin promoters was constructed, which can facilitate research specifically expressed genes involved in accumulation of ricinoleic acid in castor bean seed. The GFP which was connected to the CXSN-napin validates the effectiveness of the vector, and RcACBP was over-expressed in developing seeds of Arabidopsis thaliana transgenic lines CL7 for identifying the gene’s role in removing HFA to TAG. The results presented here may provide information on key target genes that can be used to manipulate castor oil production in transgenic plants.