大花君子兰查尔酮合酶基因CmCHS的克隆及其功能验证  

Cloning and Functional Characterization of Chalcone Synthase Genes(CmCHS)from Clivia miniata

作　　者：刘玥 李月庆 孟祥宇 黄靖 汤昊[1] 李源恒 高翔[2] 赵春莉[1] LIU Yue;LI Yueqing;MENG Xiangyu;HUANG Jing;TANG Hao;LI Yuanheng;GAO Xiang;ZHAO Chunli(College of Horticulture,Jilin Agricultural University,Changchun 130118,China;Key Laboratory of Molecular Epigenetics of Ministry of Education,Northeast Normal University,Changchun 130024,China;College of Life Science and Bioengineering,Shenyang University,Shenyang 110044,China)

机构地区：[1]吉林农业大学园艺学院,长春130118 [2]东北师范大学分子表观遗传学教育部重点实验室,长春130024 [3]沈阳大学生命科学与工程学院,沈阳110044

出　　处：《园艺学报》2021年第10期1847-1858,共12页Acta Horticulturae Sinica

基　　金：吉林省发展和改革委员会资助项目(2020C024-5)。

摘　　要：为研究大花君子兰(Clivia miniata)查尔酮合酶的结构及功能,基于前期构建的大花君子兰转录组数据库,对参与花青素合成的CHS基因家族进行鉴定和筛选。在大花君子兰中克隆得到4个CmCHS(CmCHS1~CmCHS4),其开放阅读框全长分别为1173、1170、1173和1173 bp,编码390、389、390、390个氨基酸。氨基酸序列比对分析证实,CmCHS具备该基因家族典型的保守结构域。进化树分析结果显示,4条CmCHS属于真实的CHS,编码蛋白属于Ⅲ型聚酮合成酶(PKS),亚细胞定位于细胞质和细胞核中。基因表达特异性分析结果表明,CmCHS1和CmCHS2在花朵初开和盛开期表达量较高;CmCHS1、CmCHS2、CmCHS3在红色叶片和果皮中的表达量大大高于绿色叶片和果皮,CmCHS4则相对较低。构建原核表达载体pET-32-CmCHS,体外酶活检测表明,4个CmCHS重组蛋白皆可催化香豆酰辅酶A和丙二酰辅酶A生成柚皮素查尔酮。In order to investigate the structure and function of Chalcone Synthase from Clivia miniata,members of CHS gene family were screened and identified based on the previously constructed transcriptome database.The results showed that four CmCHSs(CmCHS1,CmCHS2,CmCHS3 and CmCHS4)genes were cloned in C.miniata.The open reading frames of the four CmCHSs were 1173,1170,1173 and 1173 bp and encoding 390,389,390,390 respectively.Amino acid sequence that CmCHSs had typical the conserved domains.Phylogenetic tree analysis showed that the four CmCHS genes clustered with the bona-fide CHSs from other plants and belonged to typeⅢpolyketide synthase(PKS)super family.The results of subcellular localization showed that they were localized in the cytoplasm and nucleus.Gene expressionanalysis showed CmCHS1 and CmCHS2 were highly expressed at the late stage of flower development process,and the transcripts of CmCHS1,CmCHS2 and CmCHS3 were confirmed to the extensively detected in pigmented leaves and red fruit peels.In contrast,the expression level of CmCHS4 gene was relatively low.In order to firmly verified the roles of CmCHSs in the biosynthesis of anthocyanins,prokaryotic expression vectors,pET-32-CmCHS,were constructed and recombinant proteins were prepared and purified,which were subjected to in vitro enzyme activity analysis.Results showed that the four CmCHS recombinant proteins could catalyze coumarinyl-CoA and malonyl-Coenzyme A into naringenin chalcone.This study explored the bioinformatics analysis,gene expression analysis,and functional analysis of CmCHS genes,and laid a theoretical foundation for the genetic transformation and flower color improvement of C.miniata.

关 键 词：君子兰 类黄酮 花青素 查尔酮合酶 酶活 

分 类 号：S682.13[农业科学—观赏园艺]