枇杷叶片发育基因EjGRF5与启动子克隆及其在不同倍性枇杷中的表达  

Molecular Cloning of Leaf Developmental Gene EjGRF5, Its Promoter and Expression Analysis in Different Ploidy Loquat(Eriobotrya japonica(Thunb.) Lindl.)

作　　者：刘超[1] 王玲利 吴頔[1] 党江波[1] 尚维 郭启高[1] 梁国鲁[1] LIU Chao;WANG LingLi;WU Di;DANG JiangBo;SHANG Wei;GUO QiGao;LIANG GuoLu(l College of Horticulture and Landscape Architecture, southwest university, Chongqing 400715;Technical Advice Station of Economic Crop, Chongqing 401120)

机构地区：[1]西南大学园艺园林学院,重庆400715 [2]重庆市渝北区农业委员会,重庆401120

出　　处：《中国农业科学》2018年第8期1598-1606,共9页Scientia Agricultura Sinica

基　　金：‘十二五’国家科技支撑计划(2013BAD02B02-1);国家星火计划(2015GA811003);国家林业局项目(渝林科推2016-03);重庆市林业局项目(渝林科研2016-10);国家青年科学基金(31701876);重庆市科委基础科学与前沿技术研究项目(cstc2017jcyj A1154)

摘　　要：【目的】克隆并解析枇杷(Eriobotrya japonica(Thunb.)Lindl.)中参与调控叶片生长发育的EjGRF5及其启动子序列的结构特征,研究其在不同倍性枇杷中的表达特性,为进一步研究该基因调控不同倍性枇杷叶片生长势差异的机理奠定基础。【方法】从转录组测序数据中挖掘枇杷EjGRF5的参考序列,以此序列设计引物,并以‘龙泉1号’四倍体枇杷基因组DNA为模板扩增EjGRF5全长,参照EjGRF5参考序列获得其CDS序列。利用Bioedit7.2及Signal P4.1对EjGRF5的CDS序列及其蛋白的理化性质进行分析;采用Mega7.0软件构建EjGRF5和其他物种GRF5的系统进化树;采用Loc Tree3及Soft Berry Prot Comp9.0在线软件对EjGRF5蛋白进行亚细胞定位预测;采用染色体步移的方法克隆EjGRF5的启动子序列,并利用Plant CARE在线软件对克隆得到的EjGRF5启动子序列进行生物信息学分析;采用RT-PCR技术对EjGRF5在三倍体枇杷及其亲本(4x、2x)中的表达差异进行初步分析。【结果】将测序结果与转录组测序的参考序列进行比对发现,EjGRF5全长为1 386 bp,含有3个外显子,2个内含子,内含子全长399 bp,CDS全长987 bp。进化树分析表明,枇杷EjGRF5与蔷薇科的其他植物高度同源,且与白梨的亲缘关系最近。亚细胞定位预测结果显示,枇杷EjGRF5蛋白定位于细胞核中。启动子分析显示,EjGRF5启动子区域含有多个顺势作用元件,包括脱落酸、乙烯、高温、厌氧诱导、赤霉素和光响应元件,并且光响应元件多达11个。qRT-PCR结果显示,除F1代A-6和B-3外,其余三倍体子代EjGRF5表达量相对于中间亲本值(MPV)都发生了不同程度的上调,其中A-3的表达量是其MPV的20倍,A-5表达量是其MPV的18倍左右。【结论】获得了与枇杷叶片生长发育相关的EjGRF5、CDS序列及其启动子序列,EjGRF5在三倍体枇杷叶片中的表达呈现出上调趋势。【Objective】 In order to provide more details for further studying the mechanisms of EjGRF5 gene in regulating the growth vigor of different ploidy loquat leaf, the aims of this study are to isolate the code region of EjGRF5 gene which is involved in the regulation of leaf development and its promoter sequence, and illustrate the expression pattern of the EjGRF5 in different ploidy loquat. 【Method】 The primers were designed by using the EjGRF5 reference sequence obtained from theRNA-Seq, and the full length of EjGRF5 was cloned by using the genome DNA of Longquan-1 tetraploid, and then the full length and reference sequences were compared to obtain the targeted sequence. The Bioedit7.2 and Signal P4.1 were used to analyze the structure of the EjGRF5 CDS and the physical and chemical properties of EjGRF5; Mega7.0 was used to construct the EjGRF5 phylogenetic tree. The online software of Loc Tree3 and Soft Berry Prot Comp9.0 was adopted to predict the subcellular location of EjGRF5. The genome walking technique was employed to amplify the promoter sequence, and the online software Plant CARE was adopted to analyze the structure of the promoter. The expression patterns of EjGRF5 in triploid loquat and their parents（4 x, 2 x） were analyzed preliminarily. 【Result】 When Comparing the sequenced data with the reference sequence of EjGRF5, the results showed that the full length of EjGRF5 is 1368 bp and it contains three extron and two intron sequences. The CDS length of EjGRF5 is 987 bp. The results of phylogenetic analysis revealed that the EjGRF5 protein is highly homologous with some other species in Rosaceae and is closest to Pyrus bretschneideri. The result of subcellular localization prediction showed that EjGRF5 protein is located in the nucleus. The promoter analysis indicated that there were multiple putative cis-acting elements involved in the responsive elements, including abscisic acid（ABA）, ethylene, heat, anaerobic inductive, gibberellin（GA） and light. Moreover, the number of light re

关 键 词：枇杷 EjGRF5 基因克隆 启动子克隆 表达分析 

分 类 号：Q943.2[生物学—植物学] S667.3[农业科学—果树学]