不结球白菜BcICE1基因的克隆与功能分析

Cloning and functional analysis of BcICE1 gene in non-heading Chinese cabbage

[目的]本文旨在分析不结球白菜BcICE1基因的表达模式并探索其在冷胁迫中的功能。[方法]以不结球白菜品种‘NHCC001’为材料,采用同源克隆的方法获得BcICE1基因的全长序列,并进行生物信息学分析;对‘NHCC001’进行4℃低温和PEG-6000模拟干旱处理,检测BcICE1基因对胁迫的响应;构建过表达载体pEarlyGate-BcICE1,利用注射法将载体农杆菌菌液导入烟草细胞进行BcICE1基因的瞬时表达并检测;构建酵母诱饵载体pGBKT7-BcICE1,转化AH109验证其转录激活活性;将pEarlyGate-BcICE1农杆菌菌液利用蘸花法进行BcICE1基因的遗传转化,并用RT-qPCR和Westernblot对阳性苗进行鉴定。通过RT-qPCR对BcICE1过表达拟南芥中冷胁迫相关基因的表达水平进行分析。[结果]克隆获得不结球白菜BcICE1基因,其含有1338bp的开放阅读框,编码466个氨基酸,相对分子质量为47.40×10^3,等电点为5.42;氨基酸的多序列比对和系统进化树结果表明,BcICE1蛋白与欧洲油菜、荠菜和拟南芥ICE1蛋白同源关系较近。亚细胞定位表明BcICE1蛋白定位在细胞核中,具有转录激活性且对冷胁迫与干旱胁迫有响应。BcICE1过表达株系能增强植株的抗寒性,同时在低温处理后BcICE1过表达植株中冷胁迫相关基因AtCBF3和COR15A的表达量显著高于野生型植株。[结论]克隆并获得BcICE1过表达拟南芥植株,功能分析表明BcICE1基因能增强植株抗寒性。

[Objectives] The purpose of this study was to analyze the expression pattern of BcICE1 gene in non-heading Chinese cabbage and explore its function in cold stress.[Methods] The full-length sequence of BcICE1 gene was cloned from non-heading Chinese cabbage‘NHCC001’,and basic bioinformatics analysis was performed.The‘NHCC001’plants were treated with 4 ℃ low temperature and PEG-6000 simulated drought to detect the response of BcICE1 gene to stress.To detect the transient expression of BcICE 1 gene,the overexpression vector pEarlyGate- BcICE1 was constructed,and the agrobacterium was introduced into tobacco cells by injection.The yeast bait vector pGBKT7- BcICE1 was constructed and transformed into AH109 to test its transactivation activity.Moreover,the pEarlyGate- BcICE1 agrobacterium was subjected to genetic transformation by the method of sputum,and the positive seedlings were identified by RT-qPCR and Western blot.Finally,the expression level of cold stress-related genes in BcICE1 -overexpressed Arabidopsis thaliana was analyzed by RT-qPCR.[Results] The BcICE 1 gene contained 1 338 bp open reading frame encoding 466 amino acids with a relative molecular mass of 47.40×10^3 and an isoelectric point of 5.42.The multi-sequence alignment and phylogenetic tree analysis of ICE1 amino acids indicated that BcICE1 protein was closely related to ICE1 protein in Brassica napus,Capsella bursa-pastoris and A.thaliana.The BcICE1 protein was localized in the nucleus with strong transactivation activity and was responsive to cold and drought stress.BcICE1 -overexpressed strains could enhance the cold resistance of plants,and the expression profiles of cold stress-related genes AtCBF3 and COR15A in BcICE1 -overexpressed plants were significantly higher than those in wild type plants under low temperature treatment.[Conclusions] Cloning and obtaining BcICE1 -overexpressed A.thaliana plants,functional analysis showed that BcICE1 gene could enhance the cold resistance of plants.