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目的克隆独行菜强心苷生物合成途径关键酶甲羟戊酸激酶(mevalonate kinase,MK)基因,进行生物信息学分析,原核表达、纯化和组织特异性分析。方法通过分析独行菜转录组数据,设计基因特异性引物,克隆了LaMK基因的cDNA序列,构建p ET-32a-La MK原核表达载体,诱导表达LaMK重组蛋白。结果 LaMK基因的开放阅读框(opening reading frame,ORF)全长为1 137 bp,编码379个氨基酸。生物信息学分析显示,La MK蛋白可能定位于细胞质中,没有跨膜区不含信号肽,含有GHMP激酶家族保守结构域和ATP结合位点。系统进化分析显示,La MK蛋白与拟南芥等十字花科植物的MK蛋白同源性较高。在大肠杆菌BL21(DE3)菌株中成功表达La MK重组蛋白,利用Ni2+亲和色谱得到了纯化的La MK重组蛋白。荧光定量PCR结果表明,LaMK基因在花中表达量最高,叶和根中次之,茎中较低,在幼苗中表达量最低。结论本实验为下一步制备La MK蛋白抗体,研究La MK基因在独行菜强心苷生物合成途径中的功能奠定了基础。
Objective To clone the mevalonate kinase (MK) gene, a key enzyme of cardiac glycoside biosynthesis pathway in lonicera, for bioinformatics analysis, prokaryotic expression, purification and tissue specificity analysis. Methods The gene sequence of LaMK gene was cloned by analyzing transcriptome data of Lysozyme. The cDNA sequence of LaMK gene was cloned, and the prokaryotic expression vector p ET-32a-La MK was constructed to express LaMK recombinant protein. Results The complete reading frame (ORF) of LaMK gene was 1 137 bp, encoding 379 amino acids. Bioinformatics analysis showed that La MK protein may be located in the cytoplasm, no signal peptide in the transmembrane domain, a conserved domain of GHMP kinase family and an ATP binding site. Phylogenetic analysis showed that the MK protein of La MK protein has high homology with that of cruciferous plants such as Arabidopsis thaliana. La MK recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain, and the purified La MK recombinant protein was obtained by Ni2 + affinity chromatography. Fluorescent quantitative PCR results showed that LaMK gene was the highest expression in flowers, followed by leaves and roots, lower in stems and lowest in seedlings. Conclusion This experiment lays the foundation for the further preparation of La-MK protein antibody and the study of the function of La-MK gene in the pathway of cardiac glycoside biosynthesis.