菊花翻译起始因子4E基因的克隆、 表达分析及其互作蛋白的筛选

doi:10.3864/j.issn.0578-1752.2013.09.016

摘要/Abstract

摘要： 【目的】为了研究翻译起始因子4E在菊花中的表达特性及功能，克隆菊花翻译起始因子4E（CmeIF4E）基因，分析其表达特性并初步筛选得到菊花CmeIF4E的互作蛋白。【方法】根据已报道植物的eIF4E序列设计引物，采用RT-PCR和RACE技术克隆菊花eIF4E基因，荧光定量PCR及亚细胞定位分析菊花CmeIF4E的表达特性，并用酵母双杂交系统筛选其互作蛋白。【结果】克隆获得菊花eIF4E基因全长914 bp，其开放阅读框（ORF）654 bp，编码1条包含218个氨基酸残基的多肽，将该基因名为CmeIF4E，GenBank登录号为JQ904591。氨基酸序列比对和系统进化分析表明，菊花CmeIF4E与已报道的莴苣的同源基因亲缘关系最近，该结果与植物分类学相符。荧光定量PCR分析结果显示，CmeIF4E基因在切花菊‘神马’各个器官中均有表达，幼嫩的根中表达量最高，其次是叶片，而茎中表达量最低。亚细胞定位分析发现，CmeIF4E在细胞的核、质、膜上都有表达。筛选得到菊花CmeIF4E互作蛋白，其中包括蛋白翻译和翻译后修饰、光合作用、抗逆和防御等相关蛋白。【结论】CmeIF4E在菊花组织中为组成型表达，亚细胞定位显示其在细胞核、细胞质、细胞膜上都有表达。候选蛋白分析验证了CmeIF4E在翻译起始中的作用，还推测其可能与光合系统、植物的抗逆防御相关，结果为进一步研究该蛋白在菊花中的作用提供了重要依据。

关键词: 菊花 , eIF4E , 病毒 , 表达分析 , 亚细胞定位 , 酵母双杂交系统

Abstract: 【Objective】In order to study the expression patterns and function of translation initiation factor 4E in the Chrysanthemum, the eukaryotic translation initiation factor 4E gene was cloned from Chrysanthemum× morifolium ‘Jinba’ and its expression was analyzed, and the proteins interacting with the CmeIF4E protein were screened subsequently.【Method】CmeIF4E gene was cloned from Chrysanthemum by RT-PCR and RACE, the primers were designed according to the conserve motifs of eIF4E from several plant species, whose sequence of eIF4E has been reported. The expression patterns of CmeIF4E was analyzed by quantitative real-time PCR and subcellular localization, then screened the interacting proteins via yeast two-hybrid system.【Result】The eIF4E gene cloned from Chrysanthemum is 914 bp in length, ORF 654 bp, encoding 218 amino acids, named CmeIF4E. The sequences were submitted into GenBank database with accession No. JQ904591. The amino acid sequence analysis showed that the genetic relationships between Chrysanthemum and lettuce were the closest, and this result was consistent with plant taxonomy. Quantitative real-time PCR analysis showed that the CmeIF4E gene expressed in all tissues of Chrysanthemum ‘jinba’, the expression level of CmeIF4E in the young roots was the highest, next by the leaves, and was the lowest in stems. The subcellular localization assay showed that CmeIF4E was expressed in the cell nucleus, cytoplasm and cell membrane. The interacting proteins of CmeIF4E were related to the pathways of translation and post-translational modifications, photosynthesis, stress tolerance and defense.【Conclusion】CmeIF4E was constitutively expressed in Chrysanthemum tissues, while transient expression showed that CmeIF4E protein was located in the nucleus, cytoplasm and cell membrane. The candidate proteins screened by Y2H system showed that CmeIF4E might play an important role in translation initiation, photosynthetic system, plant resistance defense. All these results provided an important basis for the further study of the role of the CmeIF4E protein in Chrysanthemum.

Key words: Chrysanthemum ×, morifolium , eIF4E , virus , expression analysis , subcellular localization , yeast two-hybrid system

楼望淮, 安娟, 宋爱萍, 陈素梅, 蒋甲福, 陈发棣, 房伟民, 管志勇. 菊花翻译起始因子4E基因的克隆、表达分析及其互作蛋白的筛选[J]. 中国农业科学, 2013, 46(9): 1881-1891.

LOU Wang-Huai, AN Juan, SONG Ai-Ping, CHEN Su-Mei, JIANG Jia-Fu, CHEN Fa-Di, FANG Wei-Min, GUAN Zhi-Yong. Cloning and Expression Analysis of Eukaryotic Translation Initiation Factor 4E Gene and Screening of the Interactive Protein from Chrysanthemum×morifolium[J]. Scientia Agricultura Sinica, 2013, 46(9): 1881-1891.

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