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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 clone the gene M-C-methyl-D-erythritol-2, 4-pyrophosphate synthetase (MCS), a key enzyme in MEP pathway from Artemisia annua and study the sequence characterization, prokaryotic expression and tissue expression. Methods According to the EST sequence of A. annua MCS (AaMCS) gene, the MCS cDNA and its genomic sequence were cloned. The prokaryotic expression vector pET-21a (+) - MCS was constructed by recombining the MCS coding region with the expression vector pET-21a (+). The prokaryotic expression vector was transformed into E.coli BL21 (DE3) and induced by IPTG. Semi-quantitative RT-PCR detection of MCS tissue expression. Results AaMCS cDNA was 994 bp in length and contained a 681 bp ORF encoding 226 amino acids. The full-length cDNA of AaMCS was 2 540 bp in length and contained 3 exons and 2 introns. The pET-21a (+) - MCS recombinant plasmid was constructed and a stable prokaryotic expression system of pET-21a (+) - MCS was obtained. AaMCS was expressed in roots, stems, leaves and flowers, with higher expression in flowers and lower expression in roots and stems. Conclusion The AaMCS gene was cloned and a stable prokaryotic expression system of pET-21a (+) - MCS was established, which laid the foundation for the study of the activity and function of AaMCS protein.