番茄胞嘧啶甲基转移酶同源基因SlMET1功能研究  

Cloning and functional analysis of SlMET1 gene in tomato

作　　者：曹徐绿 唐晓凤[1] 苗敏[1] 刘永胜[1] 

机构地区：[1]合肥工业大学食品科学与工程学院,安徽合肥230009

出　　处：《合肥工业大学学报（自然科学版）》2017年第6期829-834,共6页Journal of Hefei University of Technology：Natural Science

基　　金：国家自然科学基金资助项目(31471157)

摘　　要：真核生物中,DNA甲基化作为表观遗传学修饰的重要手段之一,影响了植物的大量生长发育过程。文章通过生物信息学分析确定番茄中的胞嘧啶甲基转移酶同源基因(SlMET1)并对其进行进化关系的分析;利用聚合酶链式反应(polymerase chain reaction,PCR)技术从番茄cDNA中扩增出胞嘧啶甲基转移酶同源基因(SlMET1)全长,构建SlMET1与绿色荧光蛋白融合表达载体,通过在烟草瞬时表达系统,对其进行亚细胞定位;实时定量PCR分析SlMET1基因在番茄不同组织中的表达模式;同时,构建SlMET1与HA标签蛋白融合表达载体,通过免疫共沉淀分析其与DDB1的相互关系。研究结果表明:SlMET1基因在番茄各个时期和组织中都有表达,其中,叶片和花中表达量较高,在果实的变色期和红果时期表达量较低;通过绿色荧光融合蛋白载体的表达,确定SlMET1基因只在番茄细胞核中表达;通过免疫共沉淀分析确定SlMET1和DDB1之间存在相互作用。该研究为研究DDB1以表观遗传学的方式调控植物生长发育过程奠定了理论基础。Abstract.DNA methylation is one of the vital epigenetic modifications in eukaryote, which affects the developmental process of the plant. This paper identified the sequence of the tomato SIMET1 through the bioinformatic analysis. The full-length cDNA sequence of SIMET1 gene was cloned by polymerase chain reaction（PCR）. The GFP fusion expression vector of SlMET1 gene was constructed and transi- ently expressed on the Nicotiana benthamiana leaves to find out its subcellular localization. The level of expression of the SIMET1 among the different tissue of tomato was analyzed by realtime qRT- PCR. In addition, SIMET1 was cloned into pBTEX with HA tag, which was used to test the interac- tion between SIMET1 and DDB1 by the co-immunoprecipitation. The results indicated that expression of SIMET1 was higher in leaves and flowers and lower in fruit that was in mature green（MG） and ripe red（RR） stage. Meanwhile, SIMET1 proteins were located on the nucleus in the plant cell. And the co-immunoprecipitation showed that the SlMET1 interacted with the DDB1. The study can lay the theoretical foundation for the research on regulating plant growth and development with DDB1 by epigenetics modification.

关 键 词：SlMET1基因 过量表达 转基因番茄 聚合酶链式反应(PCR) 亚细胞定位 免疫共沉淀 

分 类 号：S641.203[农业科学—蔬菜学]