Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids

Abstract

Abstract. Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acylacyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had~80% and~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.

Identification of Genes Involved in Lipid Biosynthesis through de novo Transcriptome Assembly from Cocos nucifera Developing Endosperm

“…Following the assembly and quality analysis of the de novo coconut endosperm transcriptome, the transcriptome was initially used as a BLAST sequence database for the identification and confirmation of previously discovered and characterized genes from coconut, including the thioesterases (Jing et�al. 2011, Yuan et�al. 2013) and LPAAT (Knutzon et�al.…”

Genes Involved in Lipid Metabolism in Coconut

“…Thus, another key pathway for oil storage-triglycerides (TAG) synthesis is analyzed for coconut palm and 69 genes were identified (Table 2). Key genes in the two pathways were deeply analyzed through in vivo and in vitro assays, including FatB, LPAAT, and orthologs of Arabidopsis WRINKLED 1 (WRI 1) [9,11,14,15].…”

“…Moreover, laurate is found enriched at sn-2 position, which is catalyzed by membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzyme. Davies et al detected an enzyme from coconut endosperm, which is a laurate-CoA-preferring LPAAT and active during endosperm maturation [9]. The LPAAT enzyme prefers acyl-CoAs containing C10:0, C12:0, and C14:0 acyl groups as acyl-donor substrates [9].…”

“…Davies et al detected an enzyme from coconut endosperm, which is a laurate-CoA-preferring LPAAT and active during endosperm maturation [9]. The LPAAT enzyme prefers acyl-CoAs containing C10:0, C12:0, and C14:0 acyl groups as acyl-donor substrates [9]. Knutzon et al [11] performed the LPAAT protein purification and cloned the corresponding cDNA of this gene from coconut.…”