马缨杜鹃查尔酮异构酶（RdCHI1）重组蛋白的制备及功能验证

王聿晗; 孙世宇; 鞠志刚; 孙威; 徐小蓉

doi:10.19303/j.issn.1008-0384.2023.05.009

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

摘要

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。

Abstract:
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.

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