苹果MdMYB9、MdMYB11表达及其蛋白互作分析

doi:10.3864/j.issn.0578-1752.2015.11.012

摘要/Abstract

摘要： 【目的】克隆苹果(Malus domestica)中的TT2同源基因MdMYB9和MdMYB11,分析它们的序列特征，并对这两个基因在不同组织器官及光诱导条件下的表达特性及蛋白互作进行分析，为进一步解析这两个基因的功能奠定基础。【方法】采用同源克隆的方法分离得到MdMYB9和MdMYB11；利用DNAMAN软件对这两个蛋白的分子量、等电点等进行预测及对氨基酸序列进行分析，同时利用MEGA4.0软件构建这两个蛋白与拟南芥R2R3家族MYB蛋白的系统进化树；利用定量PCR检测这两个基因在不同组织器官、不同发育时期苹果果皮及种子和光照处理条件下的表达特性；利用酵母双杂交检测这两个MYB蛋白与花青苷合成相关蛋白MdbHLH33之间的互作关系。【结果】序列分析显示，MdMYB9开放阅读框长度为873 bp，编码290个氨基酸残基，与拟南芥TT2序列同源性为38.13%；MdMYB11开放阅读框为861 bp，编码286个氨基酸残基，与TT2同源性为32.44%；蛋白结构分析显示，MdMYB9和MdMYB11蛋白的N端都含有保守的R2R3功能域；进化树分析显示，这两个MYB蛋白都与拟南芥原花青苷合成相关蛋白TT2聚在同一个分支；荧光定量结果表明，MdMYB9和MdMYB11在苹果根、茎、叶和花中均有表达，但各器官中表达水平存在差异；同时，这两个基因在不同发育时期的种子及果皮中都有表达，其中均在发育中期表达水平最高；此外，光照诱导条件下MdMYB9和MdMYB11的表达变化无明显差异。酵母双杂交结果显示，MdMYB9和MdMYB11均能够与花青苷合成相关蛋白MdbHLH33相互作用。【结论】苹果MdMYB9和MdMYB11属于R2R3类MYB蛋白，在不同组织器官及不同发育时期的果皮和种子中都有表达，并与MdbHLH33蛋白相互作用。

关键词: 苹果, 原花青苷, MdMYB9, MdMYB11, 表达分析, 酵母双杂交

Abstract: 【Objective】 This study is aiming at isolating MdMYB9 and MdMYB11 genes that are homologous to TT2 from Arabidopsis, detecting their expression in different tissues and under light treatment, identifying the interaction of MdMYB9 and MdMYB11 with MdbHLH33, and laying the foundation for further function analysis of this two genes. 【Method】 MdMYB9 the protein molecular weight, isoelectric point and the full length amino acid sequences were analyzed using DNAMAN software. In addition, the phylogenetic tree of the MYB proteins, including MdMYB9, MdMYB11 and the R2R2-MYB family proteins from Arabidopsis, was constructed using the neighbor-joining (NJ) method of MEGA 4.0 software. The transcriptional levels of MdMYB9 and MdMYB11 genes were detected in different tissues, fruit skins and seeds at different developmental stages and under light treatment using qPCR. Finally, the interaction of MdMYB9 and MdMYB11with MdbHLH33 protein was detected by Y2H assay.【Result】Sequence analysis showed that the open reading frame (ORF) of MdMYB9 was 873 bp in length, encoding 290 amino acids and sequence alignment revealed 38.13% identity with TT2 in Arabidopsis. The ORF of MdMYB11 gene was 861 bp in length, encoding 286 amino acids and sequence alignment revealed 32.44% identity with TT2. Both the proteins contained the typical R2R3 domain in the N-terminal. qPCR analysis indicated that MdMYB9 and MdMYB11 were expressed in all organs, including roots, stems, leaves and flowers, but their expressions exhibited different levels in the detected tissues. Both genes expressed at different developmental stages of seeds and skins, and the highest expression appeared at the middle stages; however, the expression of MdMYB9 and MdMYB11 were not obviously different under light treatment. Yeast two-hybrid assay showed that both MdMYB9 and MdMYB11 interacted with MdbHLH33.【Conclusion】MdMYB9 and MdMYB11 were expressed in different tissues, developmental fruit skins and seeds, and interacted with MdbHLH33 protein. and MdMYB11 genes associated with proanthocyanin biosynthesis were isolated using homology-based cloning method, and the primers were designed to amplify these two genes. Subsequently,

Key words: apple, proanthocyanin, MdMYB9, MdMYB11, expression analysis, yeast two-hybrid

安秀红，张修德，陈可钦，刘肖娟，郝玉金，程存刚. 苹果MdMYB9、MdMYB11表达及其蛋白互作分析[J]. 中国农业科学, 2015, 48(11): 2208-2216.

AN Xiu-hong, ZHANG Xiu-de, CHEN Ke-qin, LIU Xiao-juan, HAO Yu-jin, CHENG Cun-gang. Expression and Protein Interaction Analysis of MdMYB9 and MdMYB11 in Apple[J]. Scientia Agricultura Sinica, 2015, 48(11): 2208-2216.

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