| 摘要: |
| 强心苷作为药用植物独行菜(Lepidium apetalum)的活性成分,其化学和药理学研究已有良好的基础,但其生物合成途径目前仍不清楚。该研究以独行菜幼苗为材料,通过分析独行菜转录组数据,设计特异性引物,PCR扩增得到了强心苷生物合成MEP途径的关键酶2-C-甲基赤藓醇-4-磷酸胞苷酰转移酶(MCT)基因的开放阅读框(ORF),命名为LaMCT(Genbank注册号KT832554),并进行序列分析和原核表达。序列分析结果表明:LaMCT基因ORF全长为912 bp,编码304个氨基酸。亚细胞定位和保守结构域分析结果表明:LaMCT蛋白位于叶绿体中,不含信号肽,没有跨膜区,含有类异戊二烯合成酶保守结构域(isoprenoid synthase domain)。系统进化树结果表明:LaMCT蛋白与拟南芥的MCT蛋白具有94%的序列相似性,亲缘关系较近。通过构建pET-32a-LaMCT原核表达载体,成功在大肠杆菌BL21(DE3)菌株中诱导表达LaMCT重组蛋白,并得到了纯化的LaMCT重组蛋白。该研究首次从独行菜中克隆了LaMCT基因,建立其稳定的原核表达体系,为LaMCT蛋白抗体的制备以及研究LaMCT基因在独行菜强心苷类化合物生物合成途径中的功能奠定了基础。 |
| 关键词: 独行菜, MCT, 基因克隆, 序列分析, 原核表达 |
| DOI：10.11931/guihaia.gxzw201510007 |
| 分类号:Q943.2 |
| 文章编号:1000-3142(2016)10-1225-08 |
| Fund project:国家重点基础研究发展计划“973”项目(2013CB531802); 河南中医学院博士科研基金(BSJJ2011-07)[Supported by National Program on Key Basic Research Project(2013CB531802); Doctoral Research Fund of Henan Chinese Medicine(BSJJ2011-07)]。 |
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| Cloning, sequence analysis and prokaryotic expression of LaMCT gene from Lepidium apetalum |
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ZHAO Le1,2, MA Li-Gang1,2, Li Xiao-Yang1, FENG Wei-Sheng1,2, ZHENG Xiao-Ke1,2*
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1. School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China;2. Collaborative
Innovation Center for Respiratory Disease Diagnosis and Treatment &3.Chinese Medicine Development
of Henan Province, Zhengzhou 450046, China
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| Abstract: |
| The active components of Lepidium apetalum, cardiac glycosides have been well studied both chemically and pharmacologically, but little is known about the cardiac glycosides biosynthesis pathway. Using cultivated seedlings as material, the transcriptome data of Lepidium apetalum, designing specific primers and using PCR method, an open reading frame(ORF)of key enzyme 2-C-methylerythritol-4-phosphate cytidylyltransferase(MCT)gene which involved in MEP pathway was isolated and named as LaMCT (GenBank accession no. KT832554). The sequence analysis and prokaryotic expression were also performed. Sequence analysis showed that LaMCT has an ORF of 912 bp, which encoded a protein of 304 amino acid residues. Subcelluar localization and conserved domain analysis indicated that LaMCT protein located in chloroplast had no signal peptide and transmembrane domain, but had a isoprenoid synthase conserved domain. Phylogenetic analysis revealed that LaMCT protein showed the highest homology, 94% similarity, with MCT protein from Arabidopsis thaliana. Through the construction of pET-32a-LaMCT vector, the recombinant LaMCT protein was successfully expressed in Escherichia coli BL21(DE3)cells. Finally, the recombinant LaMCT protein was purified through Ni2+ affinity chromatography. The LaMCT gene was cloned from L. apetalum, and the stable prokaryotic expression system of pET-32a-LaMCT was constructed. This study will provide some basic information for the further antibody preparation of LaMCT protein and would be helpful in functional research of LaMCT gene involved in cardiac glycosides biosynthesis pathway. |
| Key words: Lepidium apetalum, MCT, gene cloning, sequence analysis, prokaryotic expression |
