Functional Characterization Of Omega-3 Fatty Acid Desaturase (-3 Fad) Gene Involved In The Fatty Acid Biosynthesis Pathway In Chlorella Vulgaris (Strain Umt-M1)

Abstract

A large number of microalgae have been studied in term of lipid class and fatty acids composition as microalgae such as Chlorella vulgaris have great potential sources of polyunsaturated fatty acids (PUFAs). Genetic engineering of the fatty acid biosynthesis pathway has been applied to improve PUFAs production of microalgae. Thus, it is essential to identify the genes coding for the key enzymes that contribute to fatty acid synthesis and accumulation. One of the enzyme involved in desaturation is omega-3 fatty acid desaturase (-3 FAD) enzyme which takes part in conversion of linoleic acid (LA, C18:2) to alpha linolenic acid (ALA, C18:3n3). ALA is the precursor for the synthesis of other essential fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this study, the promoter of -3 FAD gene was successfully isolated from genomic DNA of C. vulgaris (strain UMTM1)

using PCR-Genome Walking method. From five GenomeWalker libraries (DraI, EcoRV, PvuII, SmaI and StuI), StuI managed to produce higher putative fragment with approximately 2.3 kb. Alignment analysis revealed that from the 2.3 kb fragment, only 170 bp sequence contained 100 % of homology regions with the full-length -3 FAD cDNA sequence isolated from the same species and the remaining sequence of the fragment (2186 bp) upstream from the -3 FAD putative transcription start site (+1) was undoubtedly a fragment of -3 FAD gene promoter