丁丽娜, 胡彬彬, 林连兵, 季秀玲, 魏云林, 张琦. 深黄被孢霉Δ12-脂肪酸脱氢酶基因RNAi表达载体的构建[J]. 云南大学学报(自然科学版), 2012, 34(2): 242-248.
引用本文: 丁丽娜, 胡彬彬, 林连兵, 季秀玲, 魏云林, 张琦. 深黄被孢霉Δ12-脂肪酸脱氢酶基因RNAi表达载体的构建[J]. 云南大学学报(自然科学版), 2012, 34(2): 242-248.
DING Li-na, HU Bin-bin, LIN Lian-bing, JI Xiu-ling, WEI Yun-lin, ZHANG Qi. Construction of an RNAi plasmid targeting Mortierella isabellinaΔ12-fatty acid desaturase gene[J]. Journal of Yunnan University: Natural Sciences Edition, 2012, 34(2): 242-248.
Citation: DING Li-na, HU Bin-bin, LIN Lian-bing, JI Xiu-ling, WEI Yun-lin, ZHANG Qi. Construction of an RNAi plasmid targeting Mortierella isabellinaΔ12-fatty acid desaturase gene[J]. Journal of Yunnan University: Natural Sciences Edition, 2012, 34(2): 242-248.

深黄被孢霉Δ12-脂肪酸脱氢酶基因RNAi表达载体的构建

Construction of an RNAi plasmid targeting Mortierella isabellinaΔ12-fatty acid desaturase gene

  • 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.

     

    Abstract: 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.

     

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