意大利蜜蜂铁蛋白基因AmFer3HCH的克隆及其在温度胁迫下的应答  

Cloning of ferritin gene AmFer3HCH and its response to temperature stress in the Italian honeybee,Apis mellifera ligustica (Hymenoptera:Apidae)

作　　者：马荣琴 王林玲[1] 衡惠 周泽扬[1] 李治[1] MA Rong-Qin;WANG Lin-Ling;HENG Hui;ZHOU Ze-Yang;LI Zhi(College of Life Sciences,Chongqing Normal University,Chongqing 401331,China)

机构地区：[1]重庆师范大学生命科学学院,重庆401331

出　　处：《昆虫学报》2018年第9期1029-1039,共11页Acta Entomologica Sinica

基　　金：国家高技术研究发展计划("863"计划)项目(2013AA10250-7);重庆师范大学博士科研启动基金项目(13XLB00-9);重庆市教委科学技术研究项目(KJ1703051;KJ1703053);重庆市自然科学基金项目(31302037);重庆市前沿与应用基础研究计划项目(cstc2014jcyjA80039;cstc2015jcyj-A80014)

摘　　要：【目的】本研究旨在克隆鉴定意大利蜜蜂Apis mellifera ligustica铁蛋白基因Am Fer3HCH,分析其在意大利蜜蜂应对温度胁迫时的组织表达变化,探讨铁蛋白Am Fer3HCH在蜜蜂应对环境温度胁迫中的作用。【方法】PCR扩增意大利蜜蜂铁蛋白基因Am Fer3HCH的cDNA序列,并对其编码氨基酸序列进行生物信息学鉴定;实时定量PCR分析该基因在不同温度(40℃和5℃)胁迫下的意大利蜜蜂工蜂成虫胸、中肠和脂肪体中的表达变化。【结果】PCR扩增获得意大利蜜蜂铁蛋白基因Am Fer3HCH,cDNA片段大小为507 bp,其核苷酸序列与GenBank意大利蜜蜂基因组数据库中的铁蛋白基因Fer3HCH(GenBank登录号:GB55416)的完全一致,确认为Am Fer3HCH基因序列。Am Fer3HCH鉴定为重链型铁蛋白,在系统进化上聚类于重链型铁蛋白分支,其氨基酸序列具有重链型铁蛋白特有的Fe2+氧化酶活性中心,含有铁蛋白家族成员所共有的水合氧化铁成核中心和铁离子通道。实时定量PCR分析显示,与对照组相比在短时高温(40℃,0~48 h)胁迫过程中,Am Fer3HCH在意大利蜜蜂工蜂成虫胸、中肠和脂肪体中的表达水平整体呈先上升、后下降再上升的趋势;在低温(5℃,0~6 h)胁迫处理过程中,中肠和脂肪体中Am Fer3HCH基因的表达量呈先上升后下降趋势,而在胸中表达量逐渐下降。【结论】意大利蜜蜂铁蛋白Am Fer3HCH为重链型铁蛋白,在意大利蜜蜂对极端温度胁迫的应答中扮演了重要的角色。【Aim】This study aims to clone and identify the ferritin gene AmFer3HCH from Apis mellifera ligustica,to analyze its tissue expression changes in response to temperature stress,and to explore the role of ferritin in encountering the extreme temperature stress.【Methods】The cDNA sequence of AmFer3HCH gene was amplified from A.m.ligustica by PCR,and its amino acid sequence was identified by bioinformatic tools.The expression levels of this gene in the thorax,midgut,and fat body of A.m.ligustica worker adults under temperature stress(40℃and 5℃)were detected by real-time quantitative PCR.【Results】A ferritin gene named AmFer3HCH was cloned from A.m.ligustica by PCR and its cDNA fragment is 507 bp in length.Its nucleotide sequence has 100%identity to that of the Fer3HCH gene(GenBank accession no.:GB55416)in A.m.ligustica genome in GenBank database.AmFer3HCH was identified as a ferritin heavy-chain subunit and clustered in one phyletic clade with other heavy-chain ferritins.The amino acid sequence of AmFer3HCH has a typical ferroxidase activity center,ferrihydrite nucleation center and iron channel as in most ferritin family members.Real-time quantitative PCR analysis showed that the expression levels of AmFer3HCH in the thorax,midgut and fat body of A.m.ligustica worker adults after brief exposure to high temperature stress(40℃for 0-48 h)presented a tendency of upregulation first,then downregulation and upregulation again as compared to the control group.However,after the adults were exposed to brief low temperature stress(5℃for 0-6 h),the expression level of AmFer3HCH was up-regulated first,and then down-regulated in the midgut and fat body,but showed a falling tendency in the thorax.【Conclusion】AmFer3HCH is a heavy-chain ferritin and plays an important role in response to extreme temperature stress in A.m.ligustica.

关 键 词：意大利蜜蜂 铁蛋白 基因克隆 温度胁迫 表达分析 

分 类 号：Q966[生物学—昆虫学]