棉花2个新的PPR基因的克隆及表达模式分析

doi:10.3864/j.issn.0578-1752.2014.11.020

摘要/Abstract

摘要： 【目的】从棉花中克隆2个新的PPR基因，分析其序列结构、基因表达和亚细胞定位，为深入研究这2个基因在棉花中的功能提供依据。【方法】利用棉花EST库，通过RT-PCR从陆地棉徐州142中克隆得到2个PPR基因：GhPPRH1和GhPPRH2；应用生物信息学方法对2个基因编码蛋白序列进行预测分析，利用半定量RT-PCR和实时荧光定量PCR的方法分析目的基因在不同组织及纤维不同发育时期的表达模式；利用棉花子叶瞬时表达系统对上述2个基因编码的蛋白进行亚细胞定位。【结果】GhPPRH1和GhPPRH2均属于PPR基因家族的PLS亚家族；GhPPRH1和GhPPRH2的开放读码框的长度分别为1 917和2 556 bp，编码638和851个氨基酸；GhPPRH1蛋白有18个PPR基序，包含1个E+结构域和1个DYW结构；GhPPRH2蛋白有17个PPR基序，包含1个E结构域，1个E+结构域和1个DYW结构；将GhPPRH1和GhPPRH2蛋白的氨基酸序列与GenBank数据库中的9条同源蛋白以及棉花中已经报道的5个PPR蛋白的氨基酸序列进行比对，构建系统进化树，结果显示，GhPPRH1与水稻RF1b蛋白亲缘关系较近，推测其可能与胞质雄性不育的育性恢复相关，而GhPPRH2与玉米PPR5蛋白亲缘关系最近，推测可能会影响细胞器一些转录本的RNA编辑；半定量RT-PCR和实时荧光定量PCR的结果表明，GhPPRH1和GhPPRH2在根、茎、叶、花及纤维发育不同时期均有表达，GhPPRH1在根中表达较高，而GhPPRH2在根、叶及15 d的纤维中表达较高；亚细胞定位结果显示，GFP标记的GhPPRH1蛋白的绿色荧光与线粒体Marker的红色荧光重叠，表明该蛋白可能定位于线粒体，GFP标记的GhPPRH2蛋白的绿色荧光与叶绿体自发的红色荧光重叠，表明GhPPRH2可能定位在叶绿体。【结论】从棉花中获得2个新的PPR基因，均属于PLS亚家族成员，亚细胞定位显示可能在线粒体和叶绿体中，推测其功能可能与细胞器中RNA的加工、修饰等过程有关。

关键词: 棉花 , PPR蛋白 , 基因克隆 , 表达分析 , 亚细胞定位

Abstract: 【Objective】 The aim of this study was to clone two novel PPR genes in cotton. Their sequence characteristics were investigated, and their expression in different tissues was analyzed and subcellular localization was identified. All these results could provide a support for further studying PPR genes function of cotton. 【Method】 Two PPR genes were cloned on the basis of ESTs from cotton using RT-PCR techniques. The bioinformatics method was used to analyze the putative amino acid sequence, semi- quantitative RT-PCR and real-time PCR were used to analyze the expression of target genes in tissues and different development periods of fiber. Transient expression system of cotton cotyledons was used to analyze subcellular localization. 【Result】 GhPPRH1 and GhPPRH2 belonged to PLS subfamily of PPR gene family. The full length ORF of both genes is 1 917 and 2 556 bp, encoding 638 and 851 amino acids, respectively. GhPPRH1 has 18 PPR motifs, including one E+ domain and DYW structure. GhPPRH2 has 17 PPR motifs with one E domain, E+ domain and DYW structure. Phylogenetic analysis of homologue PPR proteins of other plants and five cotton PPR proteins showed that GhPPRH1 has a close relationship with OsRF1b of rice. It implied that GhPPRH1 may involve in fertility restoration. Whereas GhPPRH2 might relate to RNA editing because of clustered together with ZmPPR5 of Zea mays. The expression pattern of GhPPRH1 and GhPPRH2 can be found in root, stem, leaf, petal and different development periods of fibers, and GhPPRH1 has relatively higher expression level in root, however high expression level of GhPPRH2 was observed in root, leaf and 15 DPA fibers. Subcellular localization showed that green fluorescence of GhPPRH1 was merged well with red fluorescence of mitochondria marker; green fluorescence of GhPPRH2 was merged very well with chloroplast autofluorescence, which indicated GhPPRH1 and GhPPRH2 might locate in mitochondria and chloroplast, respectively.【Conclusion】Two novel PPR genes were cloned and characterized from Gossypium hirsutum, which belonged to the typical PLS subclass family of PPR. The results of gene expression pattern and subcellular localization implied that two novel genes might participate in organelles RNA processing and modification.

Key words: cotton , PPR protein , gene cloning , expression analysis , subcellular localization

何鹏, 黄鹏, 黄圣, 钱慧, 俞嘉宁. 棉花2个新的PPR基因的克隆及表达模式分析[J]. 中国农业科学, 2014, 47(11): 2271-.

HE Peng, HUANG Peng, HUANG Sheng, QIAN Hui, YU Jia-Ning. Cloning and Expression Analysis of Two Novel PPR Genes in Gossypium hirsutum L.[J]. Scientia Agricultura Sinica, 2014, 47(11): 2271-.

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