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蛋白质复合物参与植物多种次生代谢物的合成,其分离对阐明植物次生代谢及提高体外催化效率至关重要。该研究构建了过表达丹参酮合成途径关键酶CYP76AH1的转基因毛状根株系,通过Western杂交筛选得到高表达CYP76AH1蛋白的丹参转基因毛状根,并以此作为后续提取丹参酮合成途径中蛋白质复合物的组培材料。通过优化蛋白提取缓冲液中的去污剂种类和浓度,筛选出含0.5%Triton X-100的缓冲液为最佳提取缓冲液,并分离得到相对大量的可溶性CYP76AH1蛋白质。该研究为后续进一步分离纯化与CYP76AH1相互作用的蛋白质复合物奠定了基础,也为深度解析丹参酮合成代谢通路提供思路。
Protein complexes are involved in the synthesis of many secondary metabolites of plants. Their separation is crucial for elucidating plant secondary metabolism and increasing the efficiency of in vitro catalysis. In this study, transgenic hairy root lines overexpressing CYP76AH1, a key enzyme in the pathway of tanshinone synthesis, were constructed. The hairy roots of Salvia miltiorrhiza were overexpressed by western blotting and were used as the base for the subsequent extraction of protein complexes from the tanshinone synthesis pathway Training materials. By optimizing the type and concentration of detergent in the protein extraction buffer, a buffer containing 0.5% Triton X-100 was screened as optimal extraction buffer and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes that interact with CYP76AH1, and provides an insight into the in-depth analysis of tanshinone anabolic pathways.