Functional characterization of a △6 fatty acid desaturase gene and its 5'-upstream region cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl (Chlorophyta)

摘要

It is suggested that △6 fatty acid desaturase (FAD) plays a critical role in the biosynthesis of polyunsaturated fatty acids in plants and microalgae.But why does it adapt to the changed environments such as nitrogen starvation is seldom understood.One △6 FAD gene (MiD6fad) from an arachidonic acid-rich microalga Myrmecia incisa Reisigl (Chlorophyta) was first heterologously expressed in Saccharomyces cerevisiae for the identification of function.The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6FAD could convert linoleic and α-linolenic acids to γ-linolenic and stearidonic acids,respectively,demonstrating that MiD6fad encoded a △6 FAD.A 1 965-bp fragment of the cloned 2 347-bp 5'-upstream region of MiD6fad was next subcloned and fused upstream with green fluorescent protein (GFP) gene to replace the GAL 1 promoter of the vector pYES2.The generated construct was transformed into S.cerevisiae for function determination.Confocal microscopic images of the transformed line illustrated that this inserted fragment could drive GFP expression,which was further verified by fluorescence intensity quantification and Western blot analysis using anti-GFP antibody.The conversion efficiency (approximately 2％-3％) of MiD6FAD was much lower than the reported ω3 FAD and △6 elongase in this microalga,suggesting that MiD6FAD catalysed the possible rate-limiting step for ArA biosynthesis.The presence of several putative cis-acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of △6 FAD transcription for the ArA production in M.incisa in changing environmental factors.