花生AhFAD8基因的克隆、表达及活性分析

Cloning, expression and activity assay of AhFAD8 gene from Arachis hypogaea L.

α-亚麻酸对促进人体健康具有重要作用,花生ω-3脂肪酸脱氢酶(FAD3、FAD7和FAD8)是催化亚油酸(18 2,LA)脱氢生成亚麻酸(18 3,ALA)的关键酶。研究利用电子克隆得到花生AhFAD8全长基因,采用RTPCR方法从花生未成熟种子中扩增出AhFAD8的全长cDNA,连接到毕赤酵母表达载体pPIC3.5K中,转化毕赤酵母GS115得到工程菌GS115-AhFAD8并进行诱导表达,采用气相色谱分析GS115-AhFAD8脂肪酸成分,结果表明,工程菌GS115-AhFAD8中亚油酸去饱和率由25.9%上升到32.5%,上升25.5%,说明在毕赤酵母中表达的花生AhFAD8蛋白具有一定脱氢酶活性。

α-linolenic acid has a very important role in promoting human health, peanut omega-3 fatty acid desaturase （FAD3, FAD7 and FAD8） is the key enzyme which can catalytic conversion of linoleic acid （18： 2, LA） and linolenic acid （18： 3, co-3, ALA）. AhFAD8 gene from peanut had been cloned at first time, amplified to AhFAD8 from cDNA of peanut immature seed by RT-PCR method, connected it to the Pichia pastoris expression vector pPIC3.5K, then transformed into Pichia pastoris GSl15 get engineered yeast GS115-AhFAD8, and then analyzed their fatty acid composition using gas chromato- graphy. The results showed that average desaturation rate of LA in the engineered yeast GS115- AhFAD8 increased from 25.9% to 32.5%, while a 25.5% increased in desaturation rate, AhFAD8 expression in Pichia pastoris had activity of desaturase.