《植物生理学报》 2021, 57(1): 59-68

中国樱桃乙烯响应因子PpcERF5基因克隆与功能分析 

高玉迪^1,2，李可^1,2，朱友银^2,3,*，刘向蕾^2,3，尹亚红^1,2，王月^1,2，郭卫东^1,2,*

¹浙江师范大学化学与生命科学学院, 浙江金华321004；²浙江省特色经济植物生物技术研究重点实验室, 浙江金华321004；³金华职业技术学院农学院, 浙江金华321007

通信作者：朱友银；E-mail: zhuyouyin@jhc.edu.cn；郭卫东；E-mail: gwd@zjnu.cn

摘　要：

乙烯响应因子(ethylene responsive factor, ERF)广泛参与植物生长与环境响应。从中国樱桃(Prunus pseudocerasus)休眠花芽转录组中发现一个ERF编码基因, 对其克隆后发现该基因开放阅读框长度为1 059 bp, 编码352个氨基酸, 只含有1个AP2/ERF (APETALA2/ethylene responsive factor)结构域。同源比对分析发现该基因编码蛋白与桃(P. persica) PpERF5以及拟南芥(Arabidopsis thaliana) AtERF5、 AtERF6同源性较高, 推测与ERF5、 ERF6具有相似的功能, 因此将其命名为PpcERF5。 RT-qPCR分析表明, PpcERF5基因受4°C、 PEG2000、脱落酸(abscisic acid, ABA)及H2O2诱导, 说明该基因响应低温、干旱、 ABA以及氧化胁迫。进一步分析发现PpcERF5启动子序列中含有低温响应元件LTR (low-temperature responsive element, CCGAAA)、干旱诱导元件MBS (MYB binding site, CAACTG)以及脱落酸响应元件ABRE (ABA responsive element, ACGTG); 双荧光系统分析表明PpcERF5启动子能在烟草(Nicotiana tabacum)叶片中表达, 且受到低温和ABA诱导。超量表达PpcERF5基因显著提高转基因拟南芥种子萌发率, 抑制转基因植株莲座叶生长, 但促进开花。

关键词：中国樱桃; 乙烯响应因子; PpcERF5; 基因克隆; 功能分析

收稿：2020-10-15   修定：2020-12-09

资助：浙江省自然科学基金(LQ17C150004)和浙江省农业新品种选育重大科技专项(2016C02052-9)

Cloning and functional analysis of ethylene responsive factor gene PpcERF5 from Chinese cherry

GAO Yudi^1,2, LI Ke^1,2, ZHU Youyin^2,3,*, LIU Xianglei^2,3, YIN Yahong^1,2, WANG Yue^1,2, GUO Weidong^1,2,*

¹College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, Zhejiang 321004, China; ²Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants, Jinhua, Zhejiang 321004, China; ³School of Agriculture, Jinhua Polytechnic, Jinhua, Zhejiang 321007, China

Corresponding author: ZHU Youyin; E-mail: zhuyouyin@jhc.edu.cn; GUO Weidong; E-mail: gwd@zjnu.cn

Abstract:

Ethylene responsive factors (ERFs) are widely involved in plant growth and response to environmental factors. Herein, an ERF gene was identifed from the dormant flower bud-derived transcriptome of Chinese cherry (Prunus pseudocerasus). The ERF gene has an open reading frame length of 1 059 bp, encodes 352 amino acids containing a single conserved AP2/ERF (APETALA2/ethylene responsive factor) domain. Homology analysis revealed that the protein encoded by this gene had higher homology with PpERF5 in P. persica and AtERF5, AtERF6 in Arabidopsis thaliana, which was speculated to have similar functions with ERF5 and ERF6, so it was named as PpcERF5. Quantitative real-time PCR results suggested that the expression level of PpcERF5 gene was induced by 4°C, PEG2000, abscisic acid (ABA) and H2O2, indicating that the gene may respond to low temperature, drought, ABA and oxidation stress. By analyzing the promoter sequence, LTR (low-temperature responsive element, CCGAAA), MBS (MYB binding site, CAACTG) and ABRE (ABA responsive element, ACGTG) were identified. The dual fluorescence analysis suggested that PpcERF5 promoter could express in tobacco (Nicotiana tabacum) leaves and be induced by
low temperature and ABA. Overexpression of PpcERF5 signifcantly increased seed germination rate and inhibited rosette leaf growth, but promoted flowering of transgenic A. thaliana.

Key words: Prunus pseudocerasus; ethylene responsive factor; PpcERF5; gene cloning; function analysis