马缨杜鹃查尔酮异构酶(RdCHI1)重组蛋白的制备及功能验证  

Preparation and Function Verification of Recombinant Protein of Chalcone Isomerase Gene from Rhododendron Delavayi

作　　者：王聿晗 孙世宇 鞠志刚 孙威[1,3] 徐小蓉 WANG Yuhan;SUN Shiyu;JU Zhigang;SUN Wei;XU Xiaorong(Key Laboratory of Plant Physiology and Development Regulation/School of Life Science,Guizhou Normal University,Guiyang,Guizhou 550025,China;Pharmacy College,Guizhou University of Traditional Chinese Medicine,Guiyang,Guizhou 550025,China;Key Laboratory of State Forestry Administration on Biodiversity Conservation in Karst Mountain Area of Southwest of China,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州师范大学生命科学学院/植物生理与发育调控重点实验室,贵州贵阳550025 [2]贵州中医药大学药学院,贵州贵阳550025 [3]西南喀斯特山地生物多样性保护重点实验室,贵州贵阳550025

出　　处：《福建农业学报》2023年第5期574-582,共9页Fujian Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31760076);贵州省教育厅特色领域项目(黔教合KY字〔2021〕059);贵州省自然科学基金项目(ZK〔2023〕270);贵州省科技支撑计划项目(黔科合支撑〔2020〕4Y028号);贵州省高等学校高山杜鹃病虫害绿色防控重点实验室项目(黔教技〔2022〕044号)。

摘　　要：【目的】制备马缨杜鹃(Rhododendron delavayi)查尔酮异构酶(Chalcone isomerase,CHI)基因表达的重组蛋白并验证其活性,为解析查尔酮异构酶功能提供理论依据,为改良植物花色、增加药用成分奠定基础。【方法】根据所获得的马缨杜鹃查尔酮异构酶RdCHI1基因的序列信息设计引物,构建其原核表达载体,优化RdCHI1可溶性重组蛋白最佳诱导表达条件,制备可溶性重组蛋白并检测其活性。【结果】成功构建RdCHI1原核表达载体,RdCHI1重组蛋白可在上清中表达,最佳诱导条件为:15℃、36 h,IPTG浓度0.35 mmol·L^(-1)。经镍柱纯化得到质量较好的RdCHI1重组蛋白,通过体外酶活反应确定,与对照组相比,RdCHI1可以更快地催化柚皮素查尔酮(Naringenin chalcone)反应生成柚皮素(Naringenin)。【结论】RdCHI1为I型CHI,可极大提高柚皮素查尔酮生成柚皮素的速率,增加黄酮类物质积累量。【Objective】Recombinant protein of chalcone isomerase(CHI)gene in Rhododendron delavayi was prepared and its activity was verified.This will provide theoretical basis for analyzing the function of CHI,and also lays a foundation for improving flower color and increasing medicinal ingredientst.【Method】Primers were designed based on the sequence of RdCHI1,and the prokaryotic expression vector constructed.Conditions to induce the expression of soluble recombinant protein were optimized.Activity of the prepared protein was verified by an in vitro enzymatic assay.【Result】The successfully constructed RdCHI1 prokaryotic expression vector was expressed in the supernatant under the optimized induction that applied 0.35 mmol·L^(-1) IPTG at 15℃for 36 h.The nickel column-purified recombinant protein significantly hastened the conversion of naringin chalcone to naringin in an in vitro assay.【Conclusion】As a type I CHI,RdCHI1 significantly accelerated the convertion from naringin chalcone to naringin and increase the accumulation od flavonoids.

关 键 词：马缨杜鹃 查尔酮异构酶 原核表达载体 可溶性重组蛋白 酶活测定 

分 类 号：Q558[生物学—生物化学]