东方蜜蜂微孢子虫侵染过程中nce-miR-26675及其靶基因NcSec24的研究  

Nce-miR-26675 and its target gene NcSec24 during the infection process of Nosema ceranae

作　　者：赵萧 荆欣 冯佩林 张艺琼 任亚萍 姚雨彤 黄枳腱 郭睿[1,3] ZHAO Xiao;JING Xin;FENG Peilin;ZHANG Yiqiong;REN Yaping;YAO Yutong;HUANG Zhijian;GUO Rui(College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Animal Husbandry Terminus of Sichuan Provincial Department of Agriculture and Rural Affairs,Chengdu,Sichuan 610000,China;Apitherapy Research Institute of Fujian Province,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学蜂学与生物医药学院,福建福州350002 [2]四川省农业农村厅畜牧总站,四川成都610000 [3]福建省蜂疗研究所,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2024年第5期662-670,共9页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金项目(32172792);国家现代农业产业技术体系专项资金项目(CARS-44-KXJ7);福建省自然科学基金项目(2022J01131334);福建农林大学硕士生导师团队项目(郭睿);福建农林大学科技创新专项(KFb22060XA);福建农林大学动物科学学院(蜂学学院)科研扶持项目;福建省大学生创新创业训练计划项目(202310389028､X202310389084)。

摘　　要：【目的】探明nce-miR-26675通过靶向调控蛋白转运体NcSec24基因的表达参与东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂的过程,为其侵染意大利蜜蜂工蜂的机制提供依据。【方法】联用RNAhybrid、miRanda和TargetScan软件预测nce-miR-26675的靶基因,取交集作为靶基因集合。使用Blast工具将靶基因比对到基因本体论(GO)和京都基因与基因组百科全书(KEGG)数据库以获得相应的功能和通路注释。采用RT-qPCR检测东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-26675和NcSec24的表达谱。利用Expasy网站上的相关软件预测和分析NcSec24的理化性质和分子特性。分别使用MEME和TBtools软件鉴定东方蜜蜂微孢子虫和其他物种NcSec24蛋白的保守基序和结构域。通过Mega 11.0软件对东方蜜蜂微孢子虫和其他物种的NcSec24蛋白进行氨基酸序列多重比对,并采用邻接法构建了基于NcSec24蛋白的系统进化树。【结果】nce-miR-26675靶向NcSec24等13个基因,其中,10个靶基因可注释到GO中的37个功能条目,6个基因注释KEGG中的23条KEGG通路。与接种后1 d相比,nce-miR-26675的表达量在接种后2 d显著上调(P<0.05),在接种后3、4 d显著下调(P<0.05),呈现先上升再下降的趋势;NcSec24的表达量在接种后2、3、4 d均显著下调(P<0.05),表现出持续下降的趋势。NcSec24的分子质量约为80.419 ku,等电点为5.60,分子式为C3671H5653N897O1067S31,平均亲水系数为-0.035;NcSec24不含典型信号肽,可同时定位于细胞核和细胞质;NcSec24含4个结构域(Sec23_trunk、Sec23_helical、zf-Sec23_Sec24和Sec23_BS)与5个保守基序(motif1、motif2、motif3、motif4和motif5);东方蜜蜂微孢子虫、家蚕微孢子虫、颗粒病微孢子虫、泰氏康罗汉孢虫的NcSec24聚为一支。【结论】nce-miR-26675通过靶向调控NcSec24的表达参与东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂的过程。NcSec24是潜在的亲水性蛋白和胞内蛋白,东方蜜蜂微�【Objective】The study aimed to provide a scientific basis for unveiling the modulation of nce-miR-26675 by protein transporter gene NcSec24 during Nosema ceranae's infection in Apis mellifera ligustica worker.【Method】RNAhybrid,miRanda and TargetScan software were combinedly used to predict the target genes of nce-miR-26675,with the intersection used as the target gene set,which were subsequently mapped onto gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases for function and pathway annotations.After depicting the expression profiles of nce-miR-26675 and NcSec24 during N.ceranae's infection in A.m.ligustica by RT-qPCR,the physicochemical property and molecular characteristics of NcSec24 were predicted and interpreted by relevant software on Expasy website,and the conservative motifs and structural domains in NcSec24 proteins from N.ceranae and other species were further identified by MEME and TBtools software,respectively.Lastly,multiple alignment of amino acid sequences of NcSec24 from N.ceranae and other species was performed by Mega 11.0 software,and NcSec24-based phylogenetic tree was constructed via neighbor-joining method.【Result】The nce-miR-26675 can target 13 target genes including NcSec24,of which 10 targets could be annotated to 37 functional terms in GO database,and of which 6 targets could be annotated to 23 pathways in KEGG database.In comparison to its expression level at 1 d post inoculation(1 dpi),the expression level of nce-miR-26675 was significantly up-regulated(P<0.05)at 2 dpi but significantly down-regulated(P<0.05)at 3 dpi and 4 dpi,showing an overall up-then-down trend;on the other hand,the expression level of NcSec24 was significantly down-regulated(P<0.05)from 2 dpi to 4 dpi compared to that at 1 dpi,demonstrating a continuously declining expression trend.NcSec24 had a molecular weight of 80.419 ku,isoelectric point of 5.60,molecular formular of C3671H5653N897O1067S31,and average hydrophilic coefficient of-0.035;NcSec24 was simultaneously located in the

关 键 词：蜜蜂 西方蜜蜂 东方蜜蜂微孢子虫 微小RNA 靶基因 调控网络 

分 类 号：S895[农业科学—特种经济动物饲养]