感染二分浓核病毒家蚕相关MicroRNA的筛选与鉴定  

Screening and Identification of MicroRNAs in Silkworm Induced by Bombyx mori Bidensovious Infection

作　　者：杨娟 汪羚 李斌 陈润浈 侯成香[1] 郭锡杰[1] 高坤[1] Yang Juan;Wang Ling;Li Bin;Chen Runzhen;Hou Chengxiang;Guo Xijie;Gao Kun(College of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang jiangsu 212018,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212018

出　　处：《蚕业科学》2020年第2期161-170,共10页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(31402141);江苏省自然科学基金项目(BK20140508)。

摘　　要：二分浓核病毒(bidensovirus,BDV)是家蚕病毒病的主要病原之一。为了筛选和鉴定家蚕体内与BDV感染相关的差异表达microRNA(miRNA),对家蚕品种JS的3龄起蚕添食BDV,36 h后收集家蚕并提取总RNA,采用高通量测序技术分析家蚕感染BDV后应答的miRNA,从中筛选出24个显著差异表达的miRNA,其中14个为已知miRNA,10个为新发现的miRNA。进一步利用反转录PCR(RT-PCR)技术对上述24个miRNA进行验证,发现这些miRNA均在家蚕幼虫体内表达;利用实时荧光定量PCR(qPCR)技术检测家蚕感染BDV后这些miRNA的表达情况,发现其中9个miRNA表达上调,15个miRNA表达下调,其中表达上调倍数最高的为bmo-miR-1923,相对表达上调了8.87倍,表达下调倍数最高的为bmo-miR-13b-3p,相对表达下调了6.97倍。此外,根据测序结果进行生物信息学预测和分析差异表达miRNA的靶基因,并利用GO分析对靶基因进行功能分类,发现这些靶基因主要参与了细胞过程、催化、结合和代谢过程。研究结果将为阐明家蚕抗BDV的分子机制提供数据参考和技术支持。Bidensovirus(BDV)is one of the main pathogens of silkworm virus disease.In order to screen and identify differentially expressed microRNAs(miRNAs)in silkworm related to BDV infection,the 3rd instar larva of silkworm JS strain was fed with BDV virus.Its total RNA was extracted 36 h post feeding.Then,miRNAs in response to BDV infec-tion were analyzed by high-throughput sequencing.Totally 24 significantly differentially expressed miRNAs were ob-tained,including 14 known miRNAs and 10 new miRNAs in silkworm.Additionally,all of 24 miRNAs were further veri-fied by reverse transcription PCR(RT-PCR)to be expressed in silkworm larva.Meanwhile,expression of all miRNAs in BDV infected silkworm were testified by real-time quantitative PCR(qPCR).Results indicated that expression levels of 9 miRNAs were up-regulated,while expression levels of the other 15 miRNAs were down-regulated.Among them,the highest up-regulation was found in bmo-miR-1923 whose relative expression level was 8.87 times higher than that of control,and the lowest down-regulation was found in bmo-miR-13b-3p whose relative expression level was 6.97 times lower than that of control.Besides,target genes of differentially expressed miRNAs were analyzed through bioinformat-ics prediction according to sequencing results and were functionally classified by GO analysis. It was found that these target genes were mainly involved in cellular process, catalysis, binding and metabolic process. These results would provide reference and technical sup- port for the research on molecular mechanism of silkworm in resistance to BDV.

关 键 词：家蚕 MICRORNA 二分浓核病毒 

分 类 号：S884.5[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]