Cloning and partial characterization of putative acy-ACP thiosterase gene in coconut (Cocos nucifera L.)

Coconut (Cocos nucifera L.) is the major export crop of the Philippines due to the high content of lauric acid with endosperm meat. Lauric acid is a medium fatty acid with a high market value in industry and medicine. The high levels of medium chain fatty acids in coconut oil are primarily due to the presence of acyl-ACP thiosterases specific for medium chain fatty acids. To be able to control the fatty acid composition in coconut oil, a good molecular strategy is to control the gene encoding for the acyl-ACP thiosterase specific for medium chain fatty acids. The first step therefore is to clone the gene encoding for the medium chain fatty acid specifically acyl-ACP thiosterase. Three cloning strategies were employed to identify the putative thiosterase gene. The RT-PCR strategy was able to generate a 1.59 kb DNA fragment that contains a highly conserved amino acid region, DRFDW which is also present in the other plant thiosterases. This 1.59 kb PCR fragment, (obtained using TH10 1 and TH10 2 as gene specific primers for PCR) is not the full-length cDNA and the strategy to extend the 5' and 3' end of the partial cDNA was to employ 5' RACE and 3' RACE, respectively. A 460 bp PCR fragment was generated by 3' RACE but no PCR fragment was generated by 3' RACE but no PCR fragment was generated at the 5' end due to technical difficulties. With cDNA library, the full-length thiosterase gene was isolated by a modified colony PCR screening method using gene specific primers. Based on PCR screening of the coconut cDNA library, a similar 460 bp fragment was also produced from plasmids 47 and 48 which suggests that these two plasmids contain the full-length sequence. This study sets the groundwork for the identification of genes playing important roles in the fatty acid synthesis of coconut