彩斑突变体菊花CmF3′Ha基因及启动子的克隆与分析  

Cloning and Analysis of CmF3′Ha Gene and Its Promoter from Chrysanthemum morifolium

作　　者：王森 陈新娜 李彦慧[1] 陈东亮[2,3] 潘晓飞 黄丛林 WANG Sen;CHEN Xinna;LI Yanhui;CHEN Dongliang;PAN Xiaofei;HUANG Conglin(College of Landscape and Tourism, Hebei Agriculture University, Baoding, Hebei 071000, China;2Beijing Agricultural Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China;3Beijing Functional Flower Engineering Technology Research Center, Beijing 100097, China;4Natural Resources and Planning Bureau of Daming County, Handan, Hebei 056900, China)

机构地区：[1]河北农业大学园林与旅游学院,河北保定071000 [2]北京市农林科学院北京农业生物技术研究中心/农业基因资源与生物技术北京市重点实验室,北京100097 [3]北京市功能花卉工程技术研究中心,北京100097 [4]大名县自然资源和规划局,河北邯郸056900

出　　处：《西北植物学报》2021年第10期1627-1634,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：北京市农林科学院青年基金(QNJJ202010);北京市农林科学院科研创新能力专项(KJCX20200106)。

摘　　要：该研究以菊花彩瓣突变体CQ17-mu为材料,利用RT-PCR克隆获得了类黄酮-3′-羟化酶基因(F3′H),该基因开放阅读框全长1527 bp,编码508个氨基酸,与已知的菊花CmF3′H相似性达到99%,故将其命名为CmF3′Ha。氨基酸序列分析表明,CmF3′Ha编码的蛋白具有保守的F3′H结构域,属于P450超家族。多重序列比对和系统发育分析表明,CmF3′Ha与其他菊花品种的F3′H亲缘关系最近。实时定量PCR分析显示,CQ17-mu花瓣中CmF3′Ha基因的表达水平显著高于CQ17的粉色花瓣,表明CmF3′Ha参与了CQ17-mu花瓣紫色条斑部位的花色素代谢。进一步利用染色体步移法克隆得到了CQ17-mu的F3′H基因上游1086 bp的启动子区序列,经分析显示,该序列中除包含TATA-box等核心启动子元件外,还包括多个MYB、MYC结合位点及多个光响应元件和激素应答元件。研究结果证明了菊花CmF3′Ha基因与植物花青素积累呈正相关,参与彩色条斑部分的花青素合成过程,为菊花的分子育种提供了理论依据。In this study,the flavonoid-3′-hydroxylase(F3′H)gene was cloned from Chrysanthemum morifolium color petal mutant CQ17-mu by RT-PCR.The full-length open reading frame of the gene was 1527 bp,encoding 508 amino acids,which was 99%similarity with the known C.morifolium CmF3′H,so it was named CmF3′Ha.Amino acid sequence analysis showed that the protein encoded by CmF3′Ha had a conserved F3′H domain and belonged to P450 super family.Multiple sequence alignment and phylogenetic analysis showed that CmF3′Ha was closely related to F3′H of other Chrysanthemum.Real time PCR analysis showed that the expression level of CmF3′Ha gene in CQ17-mu petals was significantly higher than that in CQ17 pink petals,indicating that CmF3′Ha was involved in anthocyanin metabolism in CQ17-mu petals.Furthermore,a 1086 bp promoter sequence of F3′H gene upstream of CQ17-mu was obtained by chromosome walking method.Analysis showed that the sequence contained not only core promoter elements such as TATA-box,but also multiple MYB and MYC binding sites,multiple light response elements and hormone response elements.This study proved that the CmF3′Ha gene of C.morifolium was positively correlated with plant anthocyanin accumulation and participated in the anthocyanin synthesis process of color stripe,which provided a theoretical reference for the molecular breeding of C.morifolium.

关 键 词：菊花 彩斑 类黄酮-3′-羟化酶 花青素 启动子 

分 类 号：Q785[生物学—分子生物学] Q786