茄子低温响应基因SmICE1的克隆及其功能分析  

Cloning and functional analysis of cold response gene SmICE1 in eggplant

作　　者：周露 刘杨[2] 崔群香 陈火英[2] ZHOU Lu;LIU Yang;CUI Qunxiang;CHEN Huoying(College of Horticulture,Jinling Institute of Technology,Nanjing 210038,China;School of Agriculture and Biology,Shanghai Jiaotong University,Shanghai 200240,China)

机构地区：[1]金陵科技学院园艺园林学院,江苏南京210038 [2]上海交通大学农业与生物学院,上海200240

出　　处：《南京农业大学学报》2022年第2期244-253,共10页Journal of Nanjing Agricultural University

基　　金：金陵科技学院高层次人才科研启动项目(jit-b-202053)。

摘　　要：[目的]本文旨在探索茄子SmICE1(Inducer of CBF expression 1)基因在低温响应和花青素合成途径中的功能。[方法]以光敏型茄子品种‘蓝山禾线’为材料,通过同源克隆获得SmICE1基因的全长序列,并对其进行生物信息学分析;4℃低温处理4叶期的茄子幼苗,分析SmICE1基因的表达水平;构建植物表达载体PHB-SmICE1-YFP,通过农杆菌介导法在烟草叶片中进行瞬时转化,研究SmICE1蛋白的亚细胞定位;通过农杆菌蘸花法在拟南芥中过表达SmICE1基因,分析转基因植株的耐寒性、生理指标及AtCBF(CRT binding factor)基因表达情况;将PHB-SmICE1-YFP农杆菌和SmCBFpro-LUC农杆菌菌液瞬时共转入烟草细胞中进行双荧光素酶试验;构建诱饵载体SmYABBY-pGBKT7和猎物载体SmICE1-pGADT7转化至酵母感受态AH109中进行酵母双杂交试验;构建植物表达载体SmYABBY-nYFP和SmICE1-cYFP,利用注射法将其农杆菌转化至烟草细胞中进行双分子荧光互补试验。[结果]克隆获得茄子SmICE1基因,其含有1524 bp的开放阅读框,编码507个氨基酸,预测相对分子质量为54.75×10^(3),理论等电点为5.6。多序列比对和进化树结果显示SmICE1与番茄SlICE1同源关系最近。荧光定量PCR表明SmICE1基因的表达受低温诱导。亚细胞定位显示SmICE1蛋白定位在细胞核中。SmICE1蛋白具有转录激活活性,双荧光素酶试验表明SmICE1可以促进SmCBF的表达。在拟南芥中异源过表达SmICE1基因可以增强植株的抗寒性。低温处理后,转基因植物的脯氨酸含量和AtCBF基因的表达量高于野生型,而电解质渗透率低于野生型。酵母双杂交和双分子荧光互补试验表明SmICE1与SmYABBY蛋白存在相互作用。[结论]SmICE1可以诱导SmCBF表达并增加转基因植株的抗寒性,SmICE1与SmYABBY互作可能参与调控茄子花青素生物合成。[Objectives]This research aimed to explore the function of SmICE1(Inducer of CBF expression 1)gene in cold response and anthocyanin biosynthesis in eggplant.[Methods]Using photosensitive eggplant variety‘Lanshanhexian’as material,the full-length sequence of SmICE1 was homology-based cloned and bioinformatics analysis was performed.The seedlings with four leaves treated at 4℃were used to analyze the expression level of SmICE1 gene.To study the subcellular localization of SmICE1,the PHB-SmICE1-YFP vector was constructed and transformed into the tobacco leaves via Agrobacterium-mediated transient transformation.SmICE1 gene was overexpressed in Arabidopsis thaliana using the Agrobacterium-mediated floral dip method.Then,the cold resistance,physiological indexes and the expression levels of AtCBF(CRT binding factor)genes were analyzed in transgenic plants.The Agrobacterium containing the PHB-SmICE1-YFP and SmCBFpro-LUC was transiently co-transformed into the tobacco cells to perform the dual luciferase(Dual-LUC)assay.In order to carry out the yeast two-hybrid(Y2H)assay,the yeast bait vector SmYABBY-pGBKT7 and prey vector SmICE1-pGADT7 were constructed and transformed into yeast strain AH109.The plant expression vector SmYABBY-nYFP and SmICE1-cYFP were constructed,and the Agrobacterium was injected into tobacco cells to carry out bimolecular fluorescence complementation(BiFC)assay.[Results]SmICE1 gene was cloned,and it contained 1524 bp open reading frame encoding 507 amino acids with a predicted molecular weight of 54.75×10^(3) and a theoretical isoelectric point of 5.6.Then,it was found that the SmICE1 had the high similarity to SlICE1 in Solanum lycopersicum by multiple sequence alignment and phylogenetic tree analysis.Quantitative real-time PCR indicated that low temperature could induce the expression of SmICE1 gene.SmICE1 protein was localized in the nucleus according to the subcellular localization analysis.The SmICE1 protein had strong transactivation activity,and Dual-LUC assay indicated that SmICE1 cou

关 键 词：茄子 SmICE1 基因克隆 低温响应 功能分析 花青素 

分 类 号：S641.1[农业科学—蔬菜学]