【摘要】 长链多不饱和脂肪酸花生四烯酸(ARA,C20:4n-6)是合成生物活性物质类二十碳烷酸的初级底物神经组织的重要组成成分.Δ6-脂肪酸延长酶是花生四烯酸Δ6-合成途径的关键酶之一.根据已经报道的Δ6-脂酸延长酶基因序列设计引物,分别从产花生四烯酸的丝状真菌高山被孢霉ATCC16266菌株基因组和cDNA扩增得到该序列.经序列分析后,将来源于cDNA的序列连接到毕赤酵母表达载体pPIC3.5K上,得到重组质PIC3.5K-ELO.用电击转化法将重组载体pPIC3.5K-ELO转化到巴斯德毕赤酵母GS115中,在缺省培养基筛选得到毕赤酵母转化菌株pPIC3.5K-ELO.在适宜的培养条件下,添加外源底物γ-亚麻酸(GLA)和诱导物醇诱导基因表达.气相色谱分析表明该基因在毕赤酵母中得到了表达,底物GLA转化为二高γ-亚麻酸GLA),转化效率为28.91%.对Δ6-脂肪酸延长酶进行跨膜分析,表明该蛋白为具有7个跨膜域的膜结合蛋白.更多还原

【Abstract】 The very-long-chain polyunsaturated fatty acid(VLC-PUFA), arachidonic acid(ARA), is a primary substrate for the biosynthesis of biologically active eicosanoids and a component of neuron tissues.Δ6-fatty acid elongase is one of the key enzymes involving in the biosynthesis of arachidonic acid inΔ6-pathway. Two specific sequences were cloned from genome DNA and cDNA of theMortierella alpineATCC16266 using primers designed according to the reported sequences. The sequence from cDNA was ligated into pPIC3. 5K, yielding the recombinant vector pPIC3. 5K-ELO.Pichia pastorisGS115was transformed with pPIC3. 5K-ELO by electroporationmethod. Under the optimum culture conditions, by adding exogenous substrateγ-linolenic acid(GLA)and inducermethanol, GC analysis confirmed the genewas expressed inPichia pastoris. Exogenous substrateGLA was converted into dihomo-γlinolenic acid(DGLA), and the substrate conversion efficiency reached 28. 91%. Transmembrane analysis of theΔ6-fatty acid elongase predicted that the protein is amembrane binding proteinwith seven transmembrane domains.更多还原