文心兰HSP70基因的克隆及表达分析

冯保云 李蓉 赖钟雄 林玉玲

摘  要：为研究文心兰热激蛋白70基因（命名为OnHSP70）的分子特性及表达特点，以文心兰‘柠檬绿（Oncidium hybridum ‘Honey Angel）为材料，采用RT-PCR技术克隆OnHSP70，利用生物信息学方法分析其分子特性，通过qRT-PCR技术分析其在不同组织及不同非生物胁迫处理下的表达特点。生物信息学分析表明：该基因开放阅读框为1944 bp，编码647个氨基酸，翻译的蛋白为稳定蛋白，属于HSP70超家族，其蛋白二级结构由41.11%的α-螺旋、17.77%的延伸链、7.73%的β-转角和33.38%的无规卷曲组成，具有2个高度保守的功能域。聚类分析表明：该蛋白与铁皮石斛和深圳拟兰的HSP70亲缘关系较近。qRT-PCR分析表明：文心兰HSP70在4种不同组织器官中具有表达差异性，在花中的表达量最高;高温处理后基因的表达量明显上升;40 ℃高温胁迫4 h时表达量达峰值;茉莉酸甲酯及水杨酸处理时表达量呈现下调响应。本研究为后期该基因功能研究及提高文心兰对高温的适应性提供理论基础。

关键词：文心兰;HSP70;克隆;非生物胁迫;表达分析

中图分类号：Q786;Q949.718.43      文献标识码：A

Abstract： In order to study the molecular characteristics of the heat shock protein gene （named OnHSP70） and its expression patterns in O. hybridum ‘Honey Angel， OnHSP70 was cloned by RT-PCR， its molecular characteristics was analyzed by bioinformatics， and its expression patterns in different tissues and under different abiotic stress treatment were carried out by qRT-PCR. Bioinformatics analysis showed that the open reading frame of the gene was 1944 bp， encoding 647 amino acids， and the translated protein was a stable protein belonging to the HSP70 superfamily. The secondary structure of the protein consisted of 41.11% α-helix， 17.77% extended chain， 7.73% β-turn and 33.38% random coil， and had two highly conserved functional domains. Clustering analysis showed the protein had closely relative to HSP70 from Dendrobium catenatum and Apostasia shenzhenica. qRT-PCR analysis showed OnHSP70 differentially expressed in four tissues and the highest expression level was found in flowers; the expression of OnHSP70 gene increased significantly after high temperature treatment， and reached the peak under 40 ℃ for 4 h treatment; OnHSP70 showed down-regulated under methyl jasmonate and salicylic acid treatment. The study would provide a theoretical basis for studying the gene function and improving the adaptability to high temperature of Oncidium hybridum.

Keywords： Oncidium hybridum; HSP70; cloning; abiotic stress; expression analysis

DOI： 10.3969/j.issn.1000-2561.2020.04.016

文心蘭（Oncidium hybridum）是兰科（Orchi-daceae）文心兰属（Oncidium），又名吉祥兰、舞女兰、金蝶兰等。起源于美洲热带地区，喜湿润和半阴环境，属于热带气生兰。植株轻盈、花茎自然下垂、花朵奇特、静时形似优雅舞者，动又如翩翩金蝶，是世界重要的切花和盆花种类之一，具有较高的经济价值。商业用文心兰多为花色优美、常年开花的优良杂交种。文心兰生长发育进程受温度影响较大，福建省地处东南沿海，属于温暖湿润的亚热带气候，是文心兰的适宜栽植区，但不同区域气候差异性较大。……