Functional characterization of a Δ6 fatty acid desaturase gene and its 5′-upstream region cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl(Chlorophyta)  

作　　者：ZHANG Li CAO Haisheng NING Pu ZHOU Zhigang 张利;曹海生;宁璞;周志刚

机构地区：[1]Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Conferred by Ministry of Education,Shanghai Ocean University [2]National Demonstration Center for the Experimental Teaching of Fisheries Science,Shanghai Ocean University [3]International Research Center for Marine Biosciences Conferred by Ministry of Science and Technology,Shanghai Ocean University

出　　处：《Journal of Oceanology and Limnology》2018年第6期2308-2321,共14页海洋湖沼学报（英文）

基　　金：Supported by the National Natural Science Foundation of China(No.31172389);the Special Project of Marine Renewable Energy from the State Oceanic Administration(No.SHME2011SW02);the Shanghai Universities Peak Discipline Project of Aquaculture

摘　　要：It is suggested that Δ6 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 Δ6 FAD gene( MiD6 fad) from an arachidonic acidrich microalga M yrmecia incisa Reisigl(Chlorophyta) was first heterologously expressed in S accharomyces cerevisiae for the identification of function. The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6 FAD could convert linoleic and ?-linolenic acids to γ-linolenic and stearidonic acids, respectively, demonstrating that M iD6 fad encoded a Δ6 FAD. A 1 965-bp fragment of the cloned 2 347-bp 5′-upstream region of M iD6 fad was next subcloned and fused upstream with green fluorescent 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 verified by fluorescence intensity quantification and Western blot analysis using antiGFP antibody. The conversion efficiency(approximately 2%-3%) of MiD6 FAD was much lower than the reported ? 3 FAD and Δ6 elongase in this microalga, suggesting that MiD6 FAD catalysed the possible ratelimiting step for ArA biosynthesis. The presence of several putative c is-acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of Δ6 FAD transcription for the ArA production in M. incisa in changing environmental factors.It is suggested that Δ6 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 Δ6 FAD gene( MiD6 fad) from an arachidonic acidrich microalga M yrmecia incisa Reisigl(Chlorophyta) was first heterologously expressed in S accharomyces cerevisiae for the identification of function. The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6 FAD could convert linoleic and ?-linolenic acids to γ-linolenic and stearidonic acids, respectively, demonstrating that M iD6 fad encoded a Δ6 FAD. A 1 965-bp fragment of the cloned 2 347-bp 5′-upstream region of M iD6 fad was next subcloned and fused upstream with green fluorescent 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 verified by fluorescence intensity quantification and Western blot analysis using antiGFP antibody. The conversion efficiency(approximately 2%-3%) of MiD6 FAD was much lower than the reported ? 3 FAD and Δ6 elongase in this microalga, suggesting that MiD6 FAD catalysed the possible ratelimiting step for ArA biosynthesis. The presence of several putative c is-acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of Δ6 FAD transcription for the ArA production in M. incisa in changing environmental factors.

关 键 词：arachidonic acid(ArA) fatty ACID desaturase(FAD) green fl uorescent protein(GFP) green MICROALGA Saccharomyces cerevisiae 5′-upstream region(5′-USR) 

分 类 号：Q943.2[生物学—植物学]