摘要
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 multiple secondary metabolites in plants, and their separation is essen- tial to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0. 5% Triton X-100 was selected as the best 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 interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.
作者
齐梦蝶
王健
马晓晶
张权
王方方
康莹
刘勇
林慧馨
QI Meng-die WANG Jian MA Xiao-jing ZHANG Quan WANG Fang-fang KANG Ying LIU Yong LIN Hui-xin(State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijiag 100700, China Beijiag University of Chinese Medicine, Beijing 100102, China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2017年第20期3901-3905,共5页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(ZZ2016026)
中央本级重大增减支项目(2060302)
