生菜查尔酮异构酶基因克隆及体外酶活测定  

Cloning and catalytic analysis of chalcone isomerase in vitro in lettuce(Lactuca sativa)

作　　者：徐朝阳 张苛苛 王晨晨 张彬 刘雪 唐金富[3] 周军 刘敏 李大勇 XU Zhaoyang;ZHANG Keke;WANG Chenchen;ZHANG Bin;LIU Xue;TANG Jinfu;ZHOU Jun;LIU Min;LI Dayong(College of Life Sciences,Shandong Normal University,Jinan 250014,China;Beijing Key Laboratory of Vegetable Germplasm Improvement,Vegetable Research Institute,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]山东师范大学生命科学学院,济南250014 [2]北京市农林科学院蔬菜研究所,蔬菜种质改良北京市重点实验室,北京100097 [3]中国中医科学院中药资源中心,道地药材国家重点实验室,北京100700

出　　处：《植物生理学报》2023年第11期2135-2143,共9页Plant Physiology Journal

基　　金：中央本级重大减支项目(2060302);北京市财政局现代农业产业技术体系北京市设施蔬菜创新团队项目(BAIC01-2022-5);北京市农业局北京市种质创新和新品种育种联合攻关项目(G20220628003-01)。

摘　　要：查尔酮异构酶(chalcone isomerase,CHI)是植物黄酮类化合物合成的关键限速酶之一。生菜(Lactuca sativa)是全球性的叶用蔬菜,含有丰富的黄酮类物质。本研究在生菜基因组中鉴定到一个编码查尔酮异构酶的基因LsCHI,该基因编码235 aa的蛋白质,具有典型的查尔酮异构酶活性残基位点。系统进化分析显示,LsCHI属于I型查尔酮异构酶。基因表达模式分析表明,LsCHI在生菜的地上部分表达量明显高于地下部分,且在花序中表达量最高。构建原核表达载体pET28a-LsCHI并诱导表达,纯化后获得LsCHI蛋白。体外酶活实验表明,LsCHI蛋白催化柚皮素查尔酮转化为柚皮素。本研究为进一步探讨查尔酮异构酶在生菜中的功能以及在黄酮类化合物生物合成中的作用提供一定基础。Chalcone isomerase(CHI)is a key rate-limiting enzyme in flavonoids biosynthesis in plants.Lettuce(Lactuca sativa)is an important leafy vegetable worldwide,which contains abundant flavonoids.Here,a gene encoding chalcone isomerase was identified from lettuce,which was designated as LsCHI.LsCHI encodes a protein of 235 amino acids with a typical chalcone isomerase active residue site.Phylogenetic analysis shows that LsCHI belongs to the type I chalcone isomerase.Gene expression patterns analysis shows that LsCHI expression was significantly higher in the above-ground part of lettuce than in the underground part,with the highest expression in the inflorescence.The prokaryotic expression vector pET28a-LsCHI was constructed and induced to express LsCHI.LsCHI protein was obtained after purification.In vitro enzymatic assay shows that LsCHI protein catalyzed the conversion of naringenin chalcone to naringenin.This study provides a basis for further study on the function of chalcone isomerase and its roles in the biosynthesis of flavonoids in lettuce.

关 键 词：生菜 查尔酮异构酶 黄酮类化合物 酶活鉴定 

分 类 号：S636.2[农业科学—蔬菜学]