卵叶牡丹PqDFR和PqANS及启动子克隆与功能分析  

Cloning and Functional Analysis of PqDFR and PqANS Genes and Its Promoters from Paeonia qiui

作　　者：李琴琴 董山榕 罗建让 张延龙 LI Qinqin;DONG Shanrong;LUO Jianrang;ZHANG Yanlong(College of Landscape Architecture and Arts,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学风景园林艺术学院,陕西杨凌712100

出　　处：《园艺学报》2024年第6期1256-1272,共17页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(32271950,31971709);陕西省重点研发计划项目(2022NY-146)。

摘　　要：为探究卵叶牡丹(Paeoniaqiui)春色叶呈现紫红色的成因,从其叶片转录组数据中筛选出两个花青素合成关键基因PqDFR和PqANS进行克隆、表达模式分析、功能鉴定以及启动子活性分析。PqDFR开放阅读框为1 095 bp,编码364个氨基酸,含有NADPH保守结构域和1个substract-binding结合位点;PqANS开放阅读框为1 065 bp,编码354个氨基酸,含有2OG-FeⅡ_Oxy和结合亚铁离子的保守氨基酸。实时荧光定量PCR结果表明,PqDFR和PqANS在叶片发育过程中表达量先上升再下降,与花青素的积累趋势基本一致。在卵叶牡丹叶盘中分别沉默PqDFR和PqANS基因后,叶盘的花青素含量均明显下降。稳定过表达试验表明转基因拟南芥幼苗相比野生型花青素积累更明显。PqDFR和PqANS启动子序列含有MYB转录因子结合序列,也含有光响应元件(G-box)、脱落酸响应元件(ABRE)、生长素响应元件(TGA-element)等顺式作用元件,GUS染色和定量结果显示启动子均具有良好的活性。研究结果表明PqDFR和PqANS可以促进卵叶牡丹叶片花青素合成。To explore the causes of the spring leaves of Paeonia qiui with purple-red color,PqDFR and PqANS genes were selected as key genes for anthocyanin synthesis based on the leaf transcriptional data.The two genes were cloned,analyzed for expression patterns,functional characterization and analyzed for promoter activity.The open reading frame of PqDFR was 1095 bp,encoding 364 amino acids,containing the NADPH conserved domain and a substract-binding binding site.The open reading frame of PqANS was 1065 bp,encoding 354 amino acids,including 2OG-FeⅡ_Oxy and conserved amino acids binding ferrous ion.The results of real-time quantitative PCR showed that the expression of PqDFR and PqANS genes showed a trend of increasing first,then decreasing during leaf development,which was consistent with the accumulation of anthocyanin.The anthocyanin content of leaf disks was decreased after silencing of PqDFR and PqANS genes in Paeonia qiui.Stable overexpression assays showed that transgenic Arabidopsis seedlings all showed significant anthocyanin accumulation compared to wild-type.Both PqDFR and PqANS gene promoters contain MYB binding sequence,light response elements(G-box),abscisic acid response element(ABRE)and growth factor response element(TGA-element).GUS staining and quantitative results showed that both PqDFR and PqANS gene promoters had good activity.This research suggests PqDFR and PqANS can promote anthocyanin synthesis in Paeonia qiui leaves.

关 键 词：卵叶牡丹 叶片 花青素 结构基因 启动子 

分 类 号：S685.11[农业科学—观赏园艺]