苹果MdJAZ1基因表达及蛋白互作分析

doi:10.3864/j.issn.0578-1752.2016.13.020

摘要/Abstract

摘要： 【目的】克隆苹果茉莉酸信号途径阻遏因子基因MdJAZ1，并对其表达及蛋白互作进行分析，为进一步研究MdJAZ1的功能奠定基础。【方法】以TIFY及Jas功能域为探针，在苹果基因组中进行比对，获得多个同源基因，对其中的一个基因设计引物进行克隆；利用DNAMan软件对该基因编码蛋白的分子量、等电点进行预测；利用SMART在线分析软件对该蛋白的功能域进行分析；利用MEGA5.0对该蛋白与拟南芥JAZ家族蛋白构建系统发育树；利用荧光定量PCR分析该基因在苹果不同组织器官、茉莉酸甲酯及创伤处理下的表达情况；利用PlantCARE在线软件分析该基因启动子区域的顺式作用元件；利用酵母双杂交系统检测MdJAZ1蛋白的二聚化及其与拟南芥AtCOI1蛋白的互作关系。【结果】MdJAZ1开放阅读框为1 149 bp，编码382个氨基酸残基，其蛋白的分子量为40.536 kDa，等电点9。氨基酸序列分析显示，该蛋白包含保守的TIFY及Jas结构域；系统发育树分析显示，MdJAZ1蛋白与拟南芥JAZ家族AtJAZ3、AtJAZ4亲缘关系最近；qPCR结果显示，MdJAZ1在苹果的根、茎、叶、花及果实等组织中都有表达，但表达水平存在明显差异，其中根中的表达量最高，果实中的表达量最低；该基因还能被茉莉酸甲酯及创伤处理诱导表达，均在1 h内达到最高表达水平；启动子分析显示，MdJAZ1启动子中包含多个ABA、乙烯、抗病及逆境胁迫等响应元件，此外，还包含多个MYB结合位点；酵母双杂交结果显示，MdJAZ1蛋白能与其自身相互作用形成同源二聚体，还可与拟南芥中同源性较近的AtJAZ3、AtJAZ4蛋白相互作用形成异源二聚体，并且在冠菌素存在时，MdJAZ1蛋白可与拟南芥F-box蛋白AtCOI1相互作用。【结论】MdJAZ1受茉莉酸甲酯及创伤诱导表达，其蛋白能形成同源及异源二聚体，且在冠菌素存在时可与AtCOI1互作。

关键词: 苹果, 茉莉酸, MdJAZ1, 表达, 蛋白互作

Abstract: 【Objective】MdJAZ1 was isolated from apple which encoded a repressor protein in jasmonate signaling pathway. The expression of MdJAZ1 and the interactions of MdJAZ1 with other JAZ proteins or F-box protein were determined in order to lay a foundation for further functional analysis of this gene.【Method】The MdJAZ1 was selected among multiple genes which were obtained through blasting in the apple genome database using the TIFY and Jas domains. The molecular weight and isoelectric point of MdJAZ1 were analyzed by the DNAMan software, and the functional domain was analyzed using the SMART soft. The phylogenetic tree of the proteins, including MdJAZ1 and the JAZ family proteins from Arabidopsis, was constructed using the neighbor-joining (NJ) method of MEGA5.0 soft. In addition, the expression of MdJAZ1 was detected in different tissues of apple and with MeJA or wounding treatment using qPCR. The cis-acting regulatory elements of the promoter of MdJAZ1 were analyzed through PlantCARE database. Finally, the interactions of MdJAZ1 with other JAZ proteins and AtCOI1 were detected through the yeast two-hybrid assays. 【Result】The open reading frame (ORF) of MdJAZ1 was 1 149 bp in length and encoded 382 amino acids. The calculated molecular mass of MdJAZ1 was 40.536 kDa, and the isoelectric point was 9. Sequence analysis showed that MdJAZ1 protein contained the typical TIFY domain and C-terminal Jas domain. The phylogenetic relationship of MdJAZ1 was clustered with the Arabidopsis JAZ family members AtJAZ3 and AtJAZ4. qPCR analysis revealed that MdJAZ1 was expressed in all apple tissues, including roots, stems, leaves, flowers and fruits, however, the transcriptional level was significantly different in the detected tissues, with the highest expression in roots and the lowest expression in fruits. In addition, MdJAZ1 was induced by MeJA and wounding treatments, with the highest expression at 1 h. Promoter analysis showed that there were multiple putative cis-acting elements which are involved in abscisic acid response, ethylene response, and defense and stress responses; MYB binding sites were also found in the promoter of MdJAZ1 gene. The yeast two-hybrid assays showed that MdJAZ1 formed homo- and heterodimers and also interacted with AtCOI1 which encoded a F-box protein in Arabidopsis.【Conclusion】MdJAZ1 was induced by MeJA and wounding treatments. MdJAZ1 protein formed homo- and heterodimers and also interacted with AtCOI1 in the present of coronatine.

Key words: apple, jasmonate, MdJAZ1, expression, protein interaction

安秀红，厉恩茂，李敏，李壮，张修德，程存刚. 苹果MdJAZ1基因表达及蛋白互作分析[J]. 中国农业科学, 2016, 49(13): 2642-2650.

AN Xiu-hong, LI En-mao, LI Min, LI Zhuang, ZHANG Xiu-de, CHENG Cun-gang. The Gene Expression Assays and Protein Interactions of MdJAZ1 in Apple[J]. Scientia Agricultura Sinica, 2016, 49(13): 2642-2650.

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