Functional characterization of aΔ6 fatty acid desaturase gene and its 5′-upstream region cloned from the arachidonic acidrich microalga Myrmecia incisa Reisigl(Chlorophyta)

摘要

It is suggested thatΔ6 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.OneΔ6 FAD gene(MiD6fad)from an arachidonic acidrich microalga Myrmecia incisa Reisigl(Chlorophyta)was fi rst heterologously expressed in Saccharomyces cerevisiae for the identifi cation of function.The fatty acid profi le of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6FAD could convert linoleic and?-linolenic acids toγ-linolenic and stearidonic acids,respectively,demonstrating that MiD6fad encoded aΔ6 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 fl uorescent protein(GFP)gene to replace the GAL1 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 verifi ed by fl uorescence intensity quantifi cation and Western blot analysis using anti-GFP antibody.The conversion effi ciency(approximately 2%?3%)of MiD6FAD was much lower than the reported?3 FAD andΔ6 elongase in this microalga,suggesting that MiD6FAD catalysed the possible ratelimiting step for ArA biosynthesis.The presence of several putative cis-acting regulatory elements in this identifi ed promoter sheds new light on the regulation mechanism research ofΔ6 FAD transcription for the ArA production in M.incisa in changing environmental factors.