深黄被孢霉Δ~6-脂肪酸脱氢酶基因敲除及鉴定

Disruption and Identification of the Fatty Acid Δ~6-Desaturase Gene in the Oil-producing Fungus Mortierella isabellina

以产油丝状真菌深黄被孢霉M6—22（Mortierella isabellina 6-22）为出发菌株，利用亚硝基胍诱变技术筛选到一株对潮霉素药物敏感的菌株Mortierella isabellina 6-22—4，同时构建了含有潮霉素抗性基因表达单元和深黄被孢霉△6-脂肪酸脱氢酶基因5’与3’端部分同源序列的重组质粒PD4M16，用于对深黄被孢霉△6-脂肪酸脱氢酶基因的敲除.通过PEG／CaCl2介导的原生质体转化法，将质粒PD4M16转化入深黄被孢霉M6—22—4菌株．转化子经潮霉素抗性选择平板筛选、分子检测获得了一个△^6-脂肪酸脱氢酶基因缺失的突变株Mut6．气相色谱分析结果显示，该突变株γ-亚麻酸特征峰消失，相应地亚油酸产量显著增加．培养结果表明该突变株的菌落形态、颜色及生长状况与出发菌株M6—22或M6—22—4相比均有显著改变。

Mortierella isabellina 6-22 was an important strain which can produce γ-linolenic acid. The fatty acid △6- desaturase is a key enzyme for producing γ-linolenic acid in filamentous fungi. To clarify the biosynthetic pathways for polyunsaturated fatty acids in this strain, a disruption vector PD4MI6, including 5＇ and 3＇ regions of the fatty acid △6-desaturase open reading frame as homologousre combination elements and the Escherichia coli hygromycin B resistance gene（hpt） as selectable marker, was successfully constructed. Following transformation of PD4MI6 into the hygromycin B-sensitive recipient strain, M. isabellina 6-22-4, one fatty acid △6-desaturase gene-defective mutant strain designated Mut6 was selected that was unable to produce γ-linolenic acid as determined by molecular analysis and gas chromatography. The morphology and physiology of Mut6, such as colonial shape, color and growth rate, were changed dramatically compared with that of strain M. isabellina 6-22-4.