三角褐指藻Δ^6脂肪酸脱饱和酶基因克隆及其表达分析

Cloning and Expression of Δ^6-Fatty Acid Desaturase Gene from Phaeodactylum tricornutum in Pichia pastoris

γ-亚麻酸在人类的健康与营养中具有重要的功能,利用基因工程手段提高其产量是满足其需求日益增大的途径之一。Δ6脂肪酸脱饱和酶是γ-亚麻酸合成途径中的关键酶,催化亚油酸(C18∶2Δ9,12)到γ-亚麻酸(C18∶2Δ6,9,12)的反应。通过克隆三角褐指藻的Δ6脂肪酸脱饱和酶基因,并连入酵母表达载体pPIC3.5K中,在毕赤酵母中进行了表达,结果表明:重组酵母经甲醇诱导后表达转入的外源基因,气-质联用分析显示重组毕赤酵母中含有γ-亚麻酸和SDA(18∶4Δ6,9,12,15),其含量分别为2.5%和0.8%。

γ-linolenic acid plays important roles in human nutrition and health. Genetic manipulation is one of preferable alternatives to increase production that meets the needs of increasing demand of 5,-linolenic acid. Δ^6-fatty acid desaturase is the rate-limiting enzyme of the desaturation from the linoleic acid （C18：2 Δ^9,12） to γ-linolenic acid. In this study, the Δ^6-fatty acid desaturase gene in the Phaeodactylum tricornutum was cloned; the recombinant Pichia pastoris expression vector of pPIC3.5KD6 which contains the cloned gene was constructed and expressed in P. pastoris strain. The results showed that the recombinant P. pastoris was induced by methanol for expression. The GC- MS analysis indicated that intracellular fatty acid composed γ-linolenic acid and stearidonic acid （SDA, C 18：4 Δ^6,9,12,15） in the recombinant yeast, the contents were 2.5% and 0.8% , respectively.