微孢子虫感染下意大利蜜蜂谷胱甘肽降解酶基因AmCHAC2的表达分析  

Expression Analysis of the Glutathione-Degrading Enzyme Gene AmCHAC2 of Apis mellifera ligustica under Nosema ceranae Infection

作　　者：陈兰春 秦启倩 曾慧妮 张毅 王菲 王悦弟 李治[1] CHEN Lanchun;QIN Qiqian;ZENG Huini;ZHANG Yi;WANG Fei;WANG Yuedi;LI Zhi(Chongqing Key Laboratory of Animal Biology,Chongqing Key Laboratory of Vector Insects,College of Life Sciences,Chongqing Normal University,Chongqing 40133l,China)

机构地区：[1]重庆师范大学生命科学学院媒介昆虫重庆市高校重点实验室,重庆市动物生物学重点实验室,重庆401331

出　　处：《重庆师范大学学报（自然科学版）》2023年第2期43-53,共11页Journal of Chongqing Normal University:Natural Science

基　　金：国家自然科学基金面上项目(No.31101770;No.31302037);重庆市教育委员会科学技术研究重点项目(No.KJZD-K202100502);重庆市自然科学基金面上项目(No.cstc2021jcyj-msxmX0422);重庆高校创新研究群体项目(No.CXQT21013);重庆市大学生创新创业训练计划项目(国家级)(No.202110637013)。

摘　　要：[目的]通过生物信息学鉴定意大利蜜蜂(Apis mellifera ligustica)谷胱甘肽特异性γ-谷氨酰环转移酶2(AmCHAC2),并通过定量PCR分析该蛋白的编码基因AmCHAC2在意大利蜜蜂组织中的时空表达特征,探讨该基因在蜜蜂抵抗微孢子虫感染中的作用。[方法]采用多重序列比对分析AmCHAC2的氨基酸序列特征和结构域组成;在线预测该蛋白的结构、互作蛋白网络、细胞定位特征和功能;构建系统进化树分析该蛋白的的系统分类和进化关系;实时定量PCR分析AmCHAC2在供试蜜蜂组织中的时空表达特征;采用试剂盒检测分析供试蜜蜂组织谷胱甘肽含量的表达特征;采用ELISA法检测分析供试蜜蜂中肠活性氧含量。[结果]AmCHAC2由245个氨基酸残基组成,相对分子质量为28.3 kDa, pI为6.71。AmCHAC2为疏水蛋白,不具备信号肽,定位于细胞质。供试蜜蜂在感染东方蜜蜂微孢子虫(Noseama ceranae)14 d后大量死亡,存活率仅为23%;与对照组相比,感染微孢子虫的供试蜜蜂胸部和中肠组织中AmCHAC2表达明显上调,GSH含量明显下降,且中肠内活性氧含量明显上升而触发氧化应激。[结论]AmCHAC2在受到东方蜜蜂微孢子虫感染后表达上调,这可能在意大利蜜蜂抵抗微孢子虫感染过程中扮演着重要角色。[Purposes]Bioinformatics was used to identify glutathione specific-glutamyl cyclotransferase 2(AmCHAC2)in Apis mellifera ligustica,and quantitative PCR was used to analyze the temporal and spatial expression characteristics of AmCHAC2 in A.mellifera ligustica.To explore the role of this gene in bee resistance to Nosema infection.[Methods]The amino acid sequence characteristics and domain composition of AmCHAC2 were analyzed by multiple sequence alignment.Online prediction of the protein structure,interacting protein network,cell localization characteristics and function.The phylogenetic tree was constructed to analyze the phylogenetic classification and evolutionary relationship of the protein.Temporal and spatial expression characteristics of AmCHAC2 in honeybee tissues were analyzed by real-time quantitative PCR.The expression characteristics of glutathione content in honeybee tissues were analyzed by kit.The content of reactive oxygen species in honeybee midgut was determined by ELISA.[Findings]AmCHAC2 was composed of 245 amino acid residues with a relative molecular weight of 28.3 kDa and a pI of 6.71.AmCHAC2 is a hydrophobic protein with no signal peptide and is localized in the cytoplasm.A large number of honeybees died after 14 days of infection with Noseama ceranae,and the survival rate was only 23%.Compared with the control group,the expression of AmCHAC2 was significantly up-regulated in the breast and midgut tissues of the bees infected with nosema,the content of GSH was significantly decreased,and the content of reactive oxygen species was significantly increased in the midgut,which triggered oxidative stress.[Conclusions]The upregulation of AmCHAC2 after infection with Nosema may play an important role in the resistance of A.mellifera ligustica to Nosema infection.

关 键 词：谷胱甘肽特异性γ-谷氨酰环转移酶 东方蜜蜂微孢子虫 意大利蜜蜂 

分 类 号：Q966[生物学—昆虫学]