意大利蜜蜂工蜂幼虫nkd基因的生物信息学分析及功能研究  

Bioinformatic analysis and functional study of nkd gene in larvae of Apis mellifera ligustica workers

作　　者：郭意龙 余岢骏 赵萧 钱加珺 赵浩东 张婕 张扬 赵红霞 徐细建 骆群 陈大福 郭睿 GUO Yilong;YU Kejun;ZHAO Xiao;QIAN Jiajun;ZHAO Haodong;ZHANG Jie;ZHANG Yang;ZHAO Hongxia;XU Xijian;LUO Qun;CHEN Dafu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;Apitherapy Institute,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;Institute of Zoology,Guangdong Academy of Sciences,Guangzhou 510260,Guangdong,China;Apicultural Research Institute of Jiangxi Province,Nanchang 330000,Jiangxi,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002 [2]福建省蜂疗研究所,福建福州350002 [3]广东省科学院动物研究所,广东广州510260 [4]江西省养蜂研究所,江西南昌330000

出　　处：《微生物学报》2022年第12期5005-5017,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31702190);国家现代农业产业技术体系(CARS-44-KXJ7);福建农林大学硕士生导师团队项目(郭睿);江西省蜜蜂生物学与饲养重点实验室开放基金(JXKLHBB-2020-04);福建省大学生创新创业训练计划(202210389127,202210389131)。

摘　　要：【目的】意大利蜜蜂(Apis mellifera ligustica,简称意蜂)是西方蜜蜂(Apis mellifera)的亚种之一。蜜蜂球囊菌(Ascosphaera apis)侵染意蜂幼虫导致白垩病。本研究对西方蜜蜂裸表皮蛋白(naked cuticle,Nkd)进行保守基序预测和系统进化分析,并通过RNAi明确nkd基因对意蜂工蜂幼虫体重及宿主响应蜜蜂球囊菌胁迫的免疫应答的影响,以期丰富西方蜜蜂基因nkd的信息,并揭示意蜂幼虫nkd的功能。【方法】通过MEME软件预测西方蜜蜂和其他9个物种Nkd蛋白的保守基序。采用MEGAX软件对西方蜜蜂及其他9个物种的Nkd蛋白进行系统进化分析。通过饲喂dsRNA对意蜂幼虫肠道内的nkd进行RNAi。使用电子天平对幼虫进行称重。利用RT-qPCR检测nkd基因的干扰效率及免疫基因的相对表达量。【结果】西方蜜蜂与东方蜜蜂、柑橘凤蝶、家蚕和金凤蝶的Nkd蛋白均含有3个保守基序(motif 1、motif 2和motif 3),说明上述5个昆虫物种的Nkd具有较高的保守性。西方蜜蜂与东方蜜蜂的Nkd蛋白聚为一支,说明二者的亲缘关系近。与dsRNA-egfp组相比,dsRNA-nkd组5日龄和6日龄幼虫肠道内nkd的表达量均极显著下调(P<0.001),干扰效率分别为49.60%和56.40%。另外,dsRNA-nkd组幼虫体重较dsRNA-egfp组显著下降,说明nkd显著影响幼虫体重。RT-qPCR结果显示,4日龄幼虫肠道内abaecin、apidaecin、birc5、defensin-1和PGRP-S2均被激活表达;5日龄幼虫肠道内abaecin、apidaecin、birc5和defensin-1均被激活表达,PGRP-S2的表达受到抑制;6日龄幼虫肠道内abaecin被激活表达,而apidaecin、birc5、defensin-1和PGRP-S2的表达均受到抑制,说明上述5个免疫基因在宿主响应胁迫的过程中呈不同的表达趋势,均参与宿主的免疫应答,nkd与abaecin和apidaecin的表达存在负向调控关系。【结论】西方蜜蜂的Nkd蛋白含有3个保守基序(motif 1、motif 2和motif 3),西方蜜蜂与东方蜜蜂的Nkd蛋白亲缘关系最近,通过饲�[Objective]Apis mellifera ligustica is one of the subspecies of Apis mellifera.Ascosphaera apis exclusively infects bee larvae and leads to chalkbrood.In this study,we predicted the conserved motifs and analyzed the phylogenetic relationship of naked cuticle(Nkd)protein in A.mellifera.Further,we employed RNA interference(RNAi)to study the effect of nkd gene on the larval body weight of A.m.ligustica and the immune response to A.apis stress,aiming to enrich the information of A.mellifera nkd gene and reveal the role of nkd in A.m.ligustica larvae.[Methods]The conserved motifs of Nkd proteins from A.mellifera and nine other species were predicted by MEME.MEGA X was used for the phylogenetic analysis of these Nkd proteins.RNAi of the nkd in the guts of the worker larvae of A.m.ligustica was carried out by feeding dsRNA-nkd.The larvae were weighed via an electronic balance.RT-qPCR was employed to determine the relative expression of nkd and immune response genes.[Results]The nkd proteins from A.mellifera,Apis cerana,Papilio xuthus,Bombyx mori,and Papilio machaon contained three conserved motifs(motif 1,motif 2,and motif 3),which indicated that the Nkd proteins from the above-mentioned five insect species were highly conserved.The Nkd proteins in A.mellifera and A.cerana shared the same clade,showing the closest genetic relationship.Compared with the dsRNA-egfp group,the dsRNA-nkd group showed down-regulated expression of nkd in the guts of 5-and 6-day-old larvae(P<0.001),with the interference efficiencies of 49.60%and 56.40%,respectively.Additionally,the body weights of all the larvae in the dsRNA-nkd group were significantly lower than those in the dsRNA-egfp group.The expression of abaecin,apidaecin,birc5,defensin-1,and PGRP-S2 was activated in the guts of 4-day-old larvae.In the guts of 5-day-old larvae,the expression of abaecin,apidaecin,birc5,and defensin-1 was activated,while that of PGRP-S2 was inhibited.In the guts of 6-day-old larvae,the expression of abaecin was activated,while that of apidaecin,birc5,defen

关 键 词：西方蜜蜂 意大利蜜蜂 蜜蜂球囊菌 RNA干扰 裸表皮蛋白 功能 免疫应答 

分 类 号：S895.137[农业科学—特种经济动物饲养]