Cloning of important genes involved in the fatty acid metabolism of coconut

Genetic manipulation of coconut to modify its fatty acid composition has been initiated through cloning of important genes involved in fatty acid synthesis. Initially, three different approaches were taken in cloning and isolating the thioesterase gene from coconut endosperm. Strategy 1 involving RT-PCR [Reverse Transcription-Polymerase Chain Reaction] using THIO 1 and THIO 2 as primers identified a fragment encoding the gene. The partial sequence obtained from clone 23 shows a conserved amino acid region, DRFPDW, which has been shown to be similar and identical to other thioesterases. Phylogenetic analysis of the partial putative coconut thioesterase gene revealed a greater relationship between Brassica napus and that of coconut thioesterase compared to other plant thioesterase. Five putative bands were originally obtained which could represent isoforms or closely related genes. Strategy 2 based on RACE protocol, produced a 460 bp product corresponding to the 3'end with some segments related to enolase. Strategy 3 using the plasmids obtained from single clones from the coconut cDNA library as template, and gene specific primers in a modified colony PCR protocol, identified 2 clones bearing the full-length putative thioesterase. PCR products produced had a 460 bp size, similar to that obtained from strategy 2. The other genes namely, KAS3, PAP, ACP, and ACCAse involved in fatty acid synthesis were cloned using strategy 3 involving cDNA library screening. Highly conserved amino acid regions based on publication and multiple sequence alignment were the basis of primer design of the forward primer for each gene, while using the coconut cDNA library as template. Plasmids 87 and 88 were positive for KAS3 with a 425 bp band. Plasmids 91 and 92 were positive for PAP with a 400 bp band. Plasmids 2, 8, 37 and 38 were positive for ACCse with a 300 bp band. Since ACP complexes with other genes, it was difficult to screen-out the uncomplexed form of ACP using the cDNA library. Cloning strategy 2, RACE, was adaptive cloning for the KAS3, PAP and ACP genes. Furthermore, 4, 5 and 6 mo. old coconut endosperm were used as template. For PAP, three isoforms were produced from the 6 mo. old coconut endosperm, namely: 700 bp, 500 bp and 400 bp. For KAS3, two isoforms were produced from both the 5 and 6 mo. old coconut endosperm, namely: 725 bp and 425 bp. For ACP, a single 300 bp RACE product was produced from the 4 mo. old coconut endosperm only. This suggests a possible ontological significance in the expression of the genes involved in fatty acid synthesis