乌头DFR基因的克隆与表达特征分析

李媛, 李伟, 王奎玲, 刘庆超, 姜新强, 刘庆华*
青岛农业大学园林与林学院, 山东青岛266109

通信作者:刘庆华;E-mail: lqh6205@163.com

摘 要:

二氢黄酮醇4-还原酶(DFR)是花青素合成途径中的关键酶。本研究以乌头(Aconitum carmichaeli)不同开放时期的花蕾为材料, 测定其花青素含量, 并采用同源克隆和cDNA末端快速扩增(RACE)技术从乌头花中获得了DFR基因cDNA全长, 命名为AcDFR (GenBank登录号KY272864)。结果显示, 乌头花青素含量随着花的开放呈先上升后下降的趋势。AcDFR的cDNA全长为1 233 bp, 开放阅读框为1 014 bp, 预测编码337个氨基酸。氨基酸序列比对显示, AcDFR在NADPH结合区域和底物结合区域都高度保守, 属于NADB_Rossmann超家族。系统进化分析表明, AcDFR与翠雀(Delphinium grandiflorum)DFR亲缘关系最近。AcDFR主要在乌头的花中表达, 在根、茎、叶中几乎不表达, 其表达量与花青素含量的变化呈正相关, 随着不同的开花级数呈现先上升后下降的趋势。

关键词:乌头; DFR; 基因克隆; 表达分析

收稿:2017-06-26   修定:2017-11-10

资助:山东省农业良种工程重大课题(2313215)。

Cloning and expression analysis of DFR gene in Aconitum carmichaeli

LI Yuan, LI Wei, WANG Kui-Ling, LIU Qing-Chao, JIANG Xin-Qiang, LIU Qing-Hua*
College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: LIU Qing-Hua; E-mail: lqh6205@163.com

Abstract:

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis pathway of anthocyanin. The anthocyanin content of seven developmental stages of Aconitum carmichaeli flowers were detected in this study. And a full-length cDNA sequence of DFR gene was obtained from the flower with homology-based cloning and rapid-amplification of cDNA ends (RACE) method, named AcDFR (GenBank accession no. KY272864). The results showed that anthocyanin content was first increased and then decreased along with the opening of flowers. The full-length cDNA sequence of AcDFR was 1 233 bp in length and contained 1 014 bp open reading frame encoding 337 amino acids. Multiple sequence alignment showed that AcDFR protein contained an NADPH binding domain and a substrate binding site domain, which belonged to NADB_Rossmann superfamily. Phylogenetic tree analysis indicated that AcDFR had the closest relationship with DFR of Delphinium grandiflorum. AcDFR was mainly expressed in flowers, whereas was rarely expressed in leaf, stem or root of A. carmichaeli. And the positive correlation between AcDFR expression level and anthocyanin content, showed a trend of increasing and declining later with the different developmental stages of flower.

Key words: Aconitum carmichaeli; DFR; gene cloning; expression analysis

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