马缨杜鹃RdDFR1基因的克隆分析与可溶性重组蛋白的制备

Cloning and analysis of RdDFR 1 from Rhododendron Delavayiand preparation of soluble recombinant protein

二氢黄酮醇4-还原酶位于类黄酮代谢途径下游,可催化二氢黄酮醇转化为对应的花色素苷,因具有底物特异性,直接影响植物颜色的呈现。通过PCR法克隆得到马缨杜鹃DFR1基因(RdDFR1),序列分析显示,RdDFR1不具有信号肽,定位在细胞质膜的可能性最大,与越桔DFR的亲缘关系最近。随后,将RdDFR1与原核表达载体pET-32a(+)连接,构建重组质粒pET-32a(+)-RdDFR1,并转入大肠杆菌BL21感受态细胞中。诱导表达分析显示,重组蛋白最佳诱导表达条件为:IPTG浓度为0.25 mmol/L,15℃下诱导48 h。按照上述条件制备并纯化得到了符合实验要求的重组蛋白,为研究RdDFR1的生物学功能及性质奠定了基础。

Dihydroflavonol 4-reductase locates in the downstream of flavonoid metabolic pathway and catalyzes the conversion of dihydroflavonol into corresponding anthocyanin.Due to the substrate specificity,it directly affects the presentation of plant color.In this study,the Rhododendron delavayi DFR 1 gene(RdDFR 1)was cloned by PCR.Sequence analysis showed that RdDFR1 had no signal peptide and was most likely to locate in the plasma membrane of the cell,which was closely related to the DFR of Vaccinium vitis-idaea L..Subsequently,RdDFR 1 was subcloned into the prokaryotic expression vector pET-32a(+)to construct the recombinant plasmid pET-32a(+)-RdDFR 1.Then the recombinant plasmid was transferred into Escherichia coli BL21 competent cells.Induced expression analysis revealed that the optimal expression conditions of recombinant strain were as follows:IPTG concentration was 0.25 mmol/L and the recombinant strain was induced at 15℃for 48 h.According to the above conditions,the recombinant protein which meets the experimental requirements was prepared and purified.These results laid a foundation for the biological function and properties study of RdDFR1.