利用反义RNA技术抑制酿酒酵母羊毛甾醇合酶基因的表达

发布时间：2018-08-08 17:18

【摘要】：羊毛甾醇合酶催化的2,3-氧化鲨烯环化是麦角甾醇和三萜类化合物生物合成分支形成的关键位点,下调2,3-氧化鲨烯流向麦角甾醇的代谢途径可促进其流向三萜类化合物的代谢途径。有鉴于此,本研究根据酿酒酵母羊毛甾醇合酶基因(lanosterol synthase gene,erg7)序列设计引物,扩增5'长片段(包括erg7基因5'非编码区启动子序列和部分编码区序列)、5'短片段(包括erg7基因5'非编码区部分启动子序列和部分编码区序列)和erg7基因编码区片段,分别将其反向克隆到表达载体p ESC-URA上,构建反义表达质粒。用反义表达质粒转化酿酒酵母INVSc1,在营养缺陷型培养基SD-URA上筛选重组菌株。通过半定量PCR进行检测,结果表明:与INVSc1相比,转入erg7基因编码区反义片段的重组菌株内羊毛甾醇合酶基因表达量没有显著变化,而转入5'长反义片段和5'短反义片段的重组菌株内羊毛甾醇合酶基因表达量明显降低。通过TLC和HPLC方法比较麦角甾醇含量,结果表明:与INVSc1相比,转入5'短反义片段和erg7基因编码区反义片段的重组菌株内麦角甾醇含量没有显著变化,而转入5'长反义片段的重组菌株内麦角甾醇含量明显降低。本研究利用反义RNA技术抑制酿酒酵母中羊毛甾醇合酶基因的表达,为应用合成生物学技术在酿酒酵母中组建三萜类化合物代谢途径奠定了基础。
[Abstract]:The cyclization of squalene, catalyzed by wool sterol synthase, is the key site for the formation of branches of ergosterol and triterpenoid compounds. Down-regulating the metabolic pathway of squalene to ergosterol can promote its metabolic pathway to triterpenoid compounds. In this study, primers were designed based on the sequence of the wool sterol synthase gene (lanosterol synthase geneerg7) of Saccharomyces cerevisiae. Amplification of 5'long fragment (including 5'non-coding region promoter sequence and partial coding region sequence of erg7 gene) and erg7 gene coding region (including partial promoter sequence and partial coding region sequence of erg7 gene), The antisense expression plasmid was constructed by reverse cloning into the expression vector p ESC-URA. The recombinant strain of Saccharomyces cerevisiae INVSc1 was transformed by antisense expression plasmid, and the recombinant strain was screened on the nutrition-deficient medium SD-URA. The results of semi-quantitative PCR analysis showed that there was no significant change in the expression of wool sterol synthase gene in the recombinant strain transformed into antisense fragment of erg7 gene coding region compared with INVSc1. However, the expression of wool sterol synthase gene in 5 'long antisense and 5' short antisense fragments was significantly decreased. The ergosterol content was compared by TLC and HPLC. The results showed that the ergosterol content in the recombinant strain transformed into 5 'short antisense fragment and erg7 gene coding region antisense fragment had no significant change compared with INVSc1. The ergosterol content of the recombinant strain transformed into 5 'long antisense fragment was significantly decreased. In this study, antisense RNA technique was used to inhibit the expression of wool sterol synthase gene in Saccharomyces cerevisiae, which laid a foundation for the metabolic pathway of triterpenoids in Saccharomyces cerevisiae.
【作者单位】：中国医学科学院、北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室卫生部天然药物生物合成重点实验室;
【基金】：北京市自然科学基金资助项目(7122115) 天然药物活性物质与功能国家重点实验室自主课题
【分类号】：R915

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