苹果光响应转录因子MdHY5表达及蛋白互作分析

doi:10.3864/j.issn.0578-1752.2014.21.017

摘要/Abstract

摘要： 【目的】克隆苹果光响应过程中关键的bZIP（basic-leucine zipper）转录因子MdHY5，并进行表达分析及蛋白互作检测，为阐述苹果中MdHY5在光信号过程中的功能及作用机制奠定基础。【方法】对苹果中具有bZIP保守结构的基因设计半定量引物进行表达分析，筛选具有光响应的bZIP转录因子。对光响应的bZIP家族基因进行BLAST比对，根据同源比对分析将目的蛋白命名为MdHY5，同时对cDNA序列设计引物进行克隆；利用MEGA5软件将MdHY5蛋白与拟南芥基因组中所有的bZIP转录因子家族成员进行聚类分析，同时对蛋白进行保守结构域分析；取苹果各组织材料进行MdHY5时空表达分析；通过对MdHY5启动子序列进行预测分析，并结合拟南芥同源基因芯片数据，进一步利用RT-PCR与半定量PCR检测MdHY5对不同光质的表达响应。将MdHY5连接到原核表达载体PGEX-4T-1上，利用IPTG对转化的大肠杆菌BL21融合蛋白进行诱导表达、蛋白杂交检测，并进行蛋白纯化；利用pull down试验验证MdHY5与MdCOP1蛋白的互作。【结果】通过光响应分析，在苹果中筛选到一个bZip转录因子家族成员，聚类分析显示MdHY5基因与拟南芥AtHY5同源性最高，该基因位于苹果基因组12号染色体上，基因编号为MDP0000586302，与拟南芥AtHY5结构相似，MdHY5也含有4个外显子，3个内含子。该基因cDNA序列长度为1 112 bp，其中5′非编码区长度为214 bp，3′非编码区长度为403 bp，开放阅读框长度为495 bp，编码一个含有164个氨基酸残基的蛋白。蛋白保守域分析显示，MdHY5蛋白C端含有一个典型的亮氨酸拉链结构（bZIP domain），其中在bZIP结构域的N端含有一个核定位信号区域（NLS domain）。时空表达分析显示MdHY5在各组织中均表达，其中叶片中表达水平最高，花和种子中表达水平较低。利用PLACE对启动子进行了顺式作用元件分析发现，MdHY5启动子上含有G-box、GT-1-box、I-box等多个光响应的作用元件，而定量表达分析显示MdHY5能够被白光、蓝光、紫外光诱导，而红光对MdHY5表达没有明显影响。将构建好的融合蛋白表达载体转化BL21并进行蛋白诱导，菌体经超声波破碎后显示，融合蛋白主要在上清中存在。将诱导的MdHY5-GST蛋白分别与MdCOP1-HIS及pET-HIS蛋白孵育进行pull down试验，结果显示，MdHY5在体外能够与MdCOP1蛋白互作。【结论】MdHY5为光诱导的bZIP转录因子，是拟南芥光形态建成关键转录因子AtHY5的同源基因，与AtHY5具有类似的基因与蛋白结构，在叶中表达量最高，同时对多种光质具有表达响应，能够与MdCOP1互作。

关键词: 苹果, bZIP转录因子, MdHY5, 光响应, 蛋白互作

Abstract: 【Objective】Cloning of the key light responsive bZIP (Basic-leucine zipper) transcription factors from Malus domestica Borkh, gene expression and protein interaction analysis, which were determined to identify the function and molecular mechanism of MdHY5.【Method】First, semi-quantitative primers used for detecting expression levels were designed from the apple in order to identify the light-responsive bZIP genes. Differentially screened and identified a light responsive gene named as MdHY5 based on comparative analysis of homologous. Subsequently, the primers were designed to clone the MdHY5 gene. Phylogenetic relationship including the MdHY5 protein with Arabidopsis bZIP proteins was analyzed using MEGA5 and the multiple alignment of bZIP domain was constructed, while the conserved protein domain was predicted, spatio expression analysis was made to detect the MdHY5 in different organs. The expression of MdHY5 in response to various light qualities was detected by semi-quantitative RT-PCR based on promoter prediction analysis, combined with the Arabidopsis【Result】A bZIP transcription factor was isolated in apple through the light response analysis. Phylogenetic relationship showed that MdHY5 is a homolog of Arabidopsis AtHY5. This gene is located on chromosome 12 of apple genome, with the gene number MDP0000586302, MdHY5 gene contains four exons and three introns in genome structure, which is similar to its homolog. The length of the cDNA is 1 112 bp, with 214 bp in the 5′ untranslated region, 403 bp in 3′ untranslated region, and 495 bp of open reading frame, which encode a 164 amino acid residues. MdHY5 contains a leucine zipper structure typical (bZIP domain) in the C-terminal of the protein, with a nuclear localization signal domain (NLS domain) included. Expression analysis showed that MdHY5 is expressed in various tissues, with the highest level in leaves. Promoter analysis showed that multiple light responsive cis-acting element exists in the promoters of MdHY5, such as G-box, GT-1-box, I-box and as so on. Quantitative expression analysis showed that MdHY5 gene could be induced by white, blue and UV light. The prokaryotic expression vector was transformed into BL21 and the fusion protein presented mainly in the supernatant after sonication. Pull down assay showed that MdHY5 could interact with MdCOP1 protein in vitro. 【Conclusion】 MdHY5 is a light responsive bZIP transcription factor and homologous to AtHY5, which is the key transcription factor in photomorphogenesis of Arabidopsis, MdHY5 has similar genome structure and conserved protein domain to AtHY5, and can be interacted with MdCOP1. microarray database. Finally, MdHY5 gene was inserted into the prokaryotic vector pGEX-4T-1, and IPTG was used to induce the fusion protein expression in E.coli BL21. Then the fusion protein was purified and detected by Western blot. The interaction between MdHY5 and MdCOP1 was detected by pull down assay.

Key words: apple, bZIP transcription factor, MdHY5, light responsive, protein interaction

李慧峰，王小非，冉昆，何平，王海波，李林光. 苹果光响应转录因子MdHY5表达及蛋白互作分析[J]. 中国农业科学, 2014, 47(21): 4318-4327.

LI Hui-feng, WANG Xiao-fei, RAN Kun, HE Ping, WANG Hai-bo, LI Lin-guang. Expression and Protein Interaction Analysis of Light Responsive bZIP Transcription Factor MdHY5[J]. Scientia Agricultura Sinica, 2014, 47(21): 4318-4327.

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