黄花蒿MCS基因的克隆及其序列分析与原核表达  

Cloning and sequence analysis of MCS gene in Artemisia annua and its prokaryotic expression

作　　者：王英[1] 贾伟章[2,3] 谭明俊[2] 张军[2] 王丹[2] 刘顺会[2] 

机构地区：[1]华南农业大学园艺学院,广东广州510640 [2]广东药学院生命科学与生物制药学院,广东广州510006 [3]广东省生物技术候选药物研究重点实验室,广东广州510006

出　　处：《中草药》2013年第16期2288-2293,共6页Chinese Traditional and Herbal Drugs

基　　金：广东药学院博士启动基金项目(43555022)

摘　　要：目的从黄花蒿中克隆MEP途径关键酶2C-甲基-D-赤藓糖醇-2,4-环焦磷酸合成酶(MCS)基因,进行序列特征分析、原核表达以及组织表达的研究。方法根据黄花蒿MCS(AaMCS)基因EST序列,克隆MCS cDNA及基因组序列。将MCS编码区与表达载体pET-21a(+)重组,构建pET-21a(+)-MCS的原核表达载体并转入大肠杆菌BL21(DE3),IPTG诱导获得MCS融合蛋白。半定量RT-PCR检测MCS的组织表达情况。结果 AaMCS cDNA全长994 bp,包含681 bp的开放阅读框,编码226个氨基酸,基因全长2 540 bp,包含3个外显子和2个内含子。构建pET-21a(+)-MCS重组质粒,获得稳定的pET-21a(+)-MCS原核表达体系。AaMCS在根、茎、叶、花中均有表达,其中花中表达量较高,根和茎中表达量低。结论克隆了AaMCS基因,建立pET-21a(+)-MCS稳定的原核表达体系,为研究AaMCS蛋白的活性及其功能奠定了基础。Objective To obtain the indispensable key enzyme involved in the MEP pathway, the 2C-methyl-D-erythritol-2, 4-cyclodi-phosphate synthase gene （MCS） was cloned from Artemisia annua, and bioinformatic analysis, prokaryotic expression, and tissue-specific expression were conducted. Methods According to AaMCS EST sequence, the full length of cDNA and genomic sequences were obtained by the design of specific primers, rapid-amplification of cDNA ends （RACE） and genome amplification. The coding region of MCS gene was cloned into the expression vector pET-21a （＋） by gene recombination technology, the recombinant plasmid pET-21a （＋）-MCS was transformed into E. coli BL21 （DE3）, and the expression of recombinant protein was induced by IPTG. Semi-quantitative RT-PCR was used to detect the expression of AaMCS transcripts. Results The AaMCS cDNA was found to be 994 bp containing an open reading frame （ORF） of 681 bp that translated into a putative peptide of 226 amino acid, The full length of MCS was 2 540 bp consisting of three exons and two introns. A recombinant pET-21a （＋）-MCS was constructed by genetically fusing the MCS to pET-21a （＋） vector system, and was successfully expressed in E. coli BL21. The tissue expression patterns indicated that the expression level of AaMCS transcripts in flowers was higher than that in the roots and stems. Conclusion The MCS gene is cloned from A. annua, and the stable prokaryotic expression system of pET-21a （＋）-MCS is constructed. This work is helpful for investigating the activities and other physiological functions of MCS protein.

关 键 词：黄花蒿 2C-甲基-D-赤藓糖醇-2 4-环焦磷酸合成酶 基因克隆 序列分析 原核表达 

分 类 号：R282.12[医药卫生—中药学]