深黄被孢霉Δ^(12)-脂肪酸脱氢酶基因RNAi表达载体的构建  

Construction of an RNAi plasmid targeting Mortierella isabellina Δ^(12)-fatty acid desaturase gene

作　　者：丁丽娜[1] 胡彬彬[1] 林连兵[1] 季秀玲[1] 魏云林[1] 张琦[1] 

机构地区：[1]昆明理工大学生命科学与技术学院生物工程技术研究中心,云南昆明650500

出　　处：《云南大学学报（自然科学版）》2012年第2期242-248,F0003,共8页Journal of Yunnan University(Natural Sciences Edition)

基　　金：国家自然科学基金资助项目(31160016);云南省应用基础研究基金资助项目(KKSA201126005)

摘　　要：深黄被孢霉是国内研究生产γ-亚麻酸(γ-linolenic acid,GLA)和花生四烯酸(Arachidonic acid,AA)等多不饱和脂肪酸(Polyunsaturated fatty acid,PUFA)的主要产油丝状真菌.前期的实验结果表明,深黄被孢霉M6-22具有潮霉素抗性,而且目前也没有关于深黄被孢霉营养缺陷型菌株的报道,限制了一些基于深黄被孢霉菌株进行遗传操作的研究.研究以红色荧光蛋白DsRED基因作为报告基因,构建能同时用于丝状真菌外源基因和RNAi表达载体pS-DsRED.通过PEG/CaCl2原生质体转化法将pS-DsRED导入深黄被孢霉M6-22中进行表达,成功获得产粉红色的阳性菌落,并在此基础上构建了深黄被孢霉Δ12-脂肪酸脱氢酶基因RNAi表达质粒pSREDMID12RNAi,为下一步目的基因的敲除和基因功能分析奠定了基础.Mortierella isabellina is an oil-producing fungal species which has been widely studied for producing polyunsaturated fatty acids including γ-Linolenic acid and arachidonic acid in China.Our previous research showed that Mortierella isabellina M6-22 is a hygromycin resistant fungal strain,and no auxotrophic mutant strains from M.isabellina were constructed so far,which limits its applications of genetic manipulation.In this study,DsRed gene which encodes a red-fluorescent protein was used as a report gene to construct a vector for both heterologous filamentous fungal gene and RNAi expression.The resultant vector pS-DsRED was further delivered into M.isabellina M6-22 by PEG/CaCl2-mediated protoplast transformation for expression,and acquired pink positive colonies.Based on the pS-DsRED,an RNAi expression plasmid pSREDMID12RNAi targeting Monierella isabellina delta-12-fatty acid desaturase gene was constructed,which provided a basis for the subsequent target gene knockdown and gene functional analysis.

关 键 词：深黄被孢霉 DsRED筛选标记 RNAi表达载体 Δ12-脂肪酸脱氢酶基因 

分 类 号：Q782[生物学—分子生物学]