《植物生理学报》 2013, 49(8): 803-810

‘嘎拉’苹果多酚氧化酶基因MdPPO的克隆与表达分析

马长青¹, 柏素花², 戴洪义^1,*

青岛农业大学¹园艺学院, ²生命科学学院, 山东青岛266109

通信作者：戴洪义；E-mail: hydai@qau.edu.cn；Tel: 0532-86080752

摘　要：

多酚氧化酶(polyphenol oxidase, PPO)被认为是植物的防卫蛋白。为获得苹果中多酚氧化酶基因序列信息及其表达情况, 运用RACE技术从苹果品种‘嘎拉’中克隆、鉴定了一个PPO基因, 命名为MdPPO (GenBank登录号为KF032055)。MdPPO全长2 022 bp, 包含一个1 851 bp的开放阅读框, 编码包含616个氨基酸残基的蛋白质。利用荧光定量PCR技术检测MdPPO的表达, 发现在‘嘎拉’苹果的幼茎、幼叶、花瓣、枝皮等组织中均有表达, 在幼叶中的表达量最高, 在花瓣中的表达量最低。不同非生物胁迫及激素处理的时序表达分析表明, NaCl、低温均可显著诱导MdPPO表达, 而H₂O₂能诱导其持续上调表达; 水杨酸、脱落酸、茉莉酸、乙烯等可诱导其上调表达, MdPPO对水杨酸和乙烯的响应最早, 这一表达模式暗示MdPPO可能参与多种非生物胁迫过程, 且不同胁迫因素诱导MdPPO表达的信号转换过程可能存在差异。

关键词：‘嘎拉’苹果; 多酚氧化酶; 基因克隆; 表达分析

收稿：2013-04-15   修定：2013-06-08

资助：国家苹果产业技术体系项目(CARS-28-01-07)、山东省良种产业化工程项目(620902)、青岛市科技计划基础研究项目[12-1-4-5-(1)-jch]和山东省教育厅项目(J10LC12)

Cloning and Expression Analysis of Polyphenol Oxidase Gene (PPO) Identified from ‘Gala’ Apple

MA Chang-Qing¹, BAI Su-Hua², DAI Hong-Yi^1,*

¹College of Horticulture, ²College of Life Sciences, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: DAI Hong-Yi; E-mail: hydai@qau.edu.cn; Tel: 0532-86080752

Abstract:

In order to obtain the full-length sequence of polyphenol oxidase gene (PPO) and investigate its ex-pression, we cloned PPO gene (named as MdPPO) from leaves of ‘Gala’ apple (Malus domestica) using RACE technique. The cDNA of MdPPO had a length of 2 022 bp and contained an opening-reading frame (ORF) of 1 851 bp, which was supposed to encode a 616 amino-acid residues polypeptide of 166.5477 kDa. Quantitative real-time PCR experiment revealed that MdPPO expressed in young stem, young leaf, flower and bark. The highest mRNA expression was found in young leaf, and the lowest in flower. It was speculated that MdPPO gene might be involved in the defense of apple leaf against stress. We also investigated the responses of MdPPO expression to abiotic stresses and hormone treatments using quantitative real-time PCR. The results showed that MdPPO expression was obivously induced by NaCl and cold stress, while, MdPPO up-expression was continously enhanced by hydrogen peroxide (H₂O₂). Abscisic acid (ABA), salicylic acid (SA), ethylene (ETH) and jasmonate (JA) also had inductive effects on MdPPO up-expression, but MdPPO expression was increased rapidly by ethylene and salicylic acid. It seems to be possible that MdPPO gene might be involved in various abiotic stresses; the processes of the signal transduction were probably different in diverse stresses.

Key words: ‘Gala’ apple (Malus domestica); polyphenol oxidase; gene clone; expression analysis