《植物生理学报》 2018, 54(2): 282-290

水芹类黄酮3'-羟化酶基因的克隆与表达特性分析

康美玲, 冯凯, 段希, 王柯文, 王枫, 熊爱生^*

南京农业大学园艺学院, 作物遗传与种质创新国家重点实验室, 农业部华东地区园艺作物生物学与种质创新重点实验室, 江苏南京210095

通信作者：熊爱生；E-mail: xiongaisheng@njau.edu.cn

摘　要：

类黄酮3'-羟化酶(flavonoid 3′-hydroxylase, F3'H)属于细胞色素P450家族(cytochromeP450, CYP450), 在植物主要成色物质花青素的合成中发挥重要作用。本实验以‘八卦洲水芹’以及紫色叶柄突变型水芹为实验材料, 利用RT-PCR方法, 从紫色叶柄突变型水芹的cDNA中克隆得到编码类黄酮3'-羟化酶的基因, 命名为OjF3'H1。序列分析显示, OjF3'H1基因全长1 575 bp, 共编码524个氨基酸。OjF3'H1蛋白相对分子质量为58 292.59, 理论等电点为6.77。系统进化分析显示, OjF3'H1蛋白具有高度保守性, 与同属伞形科的胡萝卜F3'H1进化关系最近。OjF3'H1编码的蛋白属于疏水蛋白, 无序化比例为5.53%, 空间结构主要由α-螺旋和β-折叠组成。实时定量PCR分析显示, OjF3'H1在不同品种水芹茎中相对表达量有明显差异, 紫色叶柄突变型水芹中OjF3'H1的表达量明显比‘八卦洲水芹’中高。

关键词：水芹; 类黄酮3'-羟化酶基因; 克隆; 叶柄; 表达分析

收稿：2017-12-26   修定：2018-01-04

资助：国家大学生创新创业训练计划(201710307031)、国家自然科学基金(31272175)、教育部新世纪优秀人才支持计划项目(NCET-11-0670)和江苏高校优势学科建设项目(PAPD)。

Cloning and expression analysis of flavonoid 3'-hydroxylase in Oenanthe javanica

KANG Mei-Ling, FENG Kai, DUAN Xi, WANG Ke-Wen, WANG Feng, XIONG Ai-Sheng^*

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: XIONG Ai-Sheng; E-mail: xiongaisheng@njau.edu.cn

Abstract:

Flavonoid 3'-hydroxylase (F3′H), belongs to the family of cytochrome P450 monooxygenases (CYP450), plays important roles in anthocyanins biosynthesis in plant. In this study, ‘Baguaizhou shuiqin’ and the purple-petiole mutant Oenanthe javanica were used as plant materials. A gene encoding flavonoid 3'-hychoxylase was cloned from the cDNA of purple-petiole O. javanica by RT-PCR and designated as OjF3'H1. The OjF3'H1 gene contained a full-length open reading frame of 1 575 bp, which encodes 524 amino acids. The relative molecular mass and theoretical isoelectric point of OjF3'H1 protein was 58 292.59 and 6.77, respectively. Sequence alignments and phylogenetic analysis indicated that OjF3'H1 was highly conserved and had the closest phylogenetic relationships with the F3'H protein from carrot. Quantitative real-time PCR analysis demonstrated that the relative expression levels of OjF3'H1 gene in the stems of different O. javanica varieties were significantly different. The relative expression levels of OjF3'H1 gene in purple O. javanica was higher than that in ‘Baguaizhou shuiqin’.

Key words: Oenanthe javanica; flavonoid 3'-hydroxylase; clone; petiole; expression analysis