The effective acyl flux between phospholipids and neutral lipids is critical for a high level of the biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA,20:4-5,8,11,14), eicosapentaenoic acid (EPA, 20:5-5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) which are essential for human health and wellbeing. Three membrane-bound enzymes, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) from VLCPUFA-producing fungi were selected as candidates for my thesis research based on the hypothesis that these enzymes play important roles in acyl trafficking between phosphatidylcholine (PCs) and diacylglycerols (DAGs) during the biosynthesis of VLCPUFAs. Two putative PDCT cDNAs (PiPDCT1 and PiPDCT2) were cloned from Phytophthora infestans which encode polypeptides with two conserved domains and about 15% of amino acid identity to an Arabidopsis PDCT. However, in vitro assays in yeast Saccharomyces cerevisiae showed they did not have any PDCT activity. Four putative CPT and EPT cDNAs (PiCPT1, PiCPT2, PiEPT and TaCPT) were cloned from P. infestans and Thraustochytrium aureum which encode proteins with a conserved CDP-alcohol phosphotransferase motif and 22% to 26% of amino acid identity to the yeast CPT. In vitro assays indicated PiCPT1 and TaCPT had CPT activity, PiEPT had EPT activity and PiCPT2 did not have either activity. Substrate specificity assays showed that all the three functional CPT and EPT preferred VLCPUFA-containing DAGs as substrates with PiCPT1 being the most specific towards ARA-DAG and DHA-DAG. Real-time qPCR analysis revealed that the expression of PiCPT1 was up-regulated in P. infestans fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFAs-specific CPT which may play an important role in shuffling VLCPUFAs from phospholipids to storage neutral lipids, would thus have potential use in metabolic engineering of VLCPUFAs in heterologous systems including oilseed crops.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2012-06-520 |
| Date | 2012 June 1900 |
| Contributors | Qiu, Xiao |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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