Acta Entomologica Sinica ›› 2019, Vol. 62 ›› Issue (1): 49-60.doi: 10.16380/j.kcxb.2019.01.006

• RESEARCH PAPERS • Previous Articles     Next Articles

Comprehensive analysis of differentially expressed microRNAs and their target genes in the larval gut of Apis mellifera ligustica during the late stage of Ascosphaera apis stress

GUO Rui#, DU Yu#, ZHOU Ni-Hong, LIU Si-Ya, XIONG Cui-Ling, ZHENG Yan-Zhen, FU Zhong-Min, XU Guo-Jun, WANG Hai-Peng, GENG Si-Hai, ZHOU Ding-Ding, CHEN Da-Fu*   

  1.   (College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2019-01-20 Published:2019-01-24

Abstract: 【Aim】 Ascosphaera apis is a deadly fungal pathogen that specially infects the larval gut of honeybees. MicroRNA (miRNA) plays a key negative role in regulation of mRNA at the post-transcriptional level. This study aims to comprehensively analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the larval gut of Apis mellifera ligustica during the late stage of A. apis stress, so as to provide important information for revealing the roles of DEmiRNAs in the stress response process. 【Methods】Normal and A. apis challenged 6-day-old larval gut of A. m. ligustica (represented as AmCK and AmT, respectively) were sequenced using small RNA-seq (sRNA-seq) technology, followed by prediction and analysis of DEmiRNAs. Target genes of DEmiRNAs were predicted with TargetFinder, and annotations of target genes in GO and KEGG databases were performed using Blast. The regulation networks between DEmiRNAs and the target mRNAs were constructed using Cytoscape. The reliability of the sequencing data was verified by stem-loop RT-qPCR. 【Results】 Deep sequencing of AmCK and AmT samples respectively generated 9 230 496 and 10 823 667 clean tags. There were 15 up-regulated and 6 down-regulated miRNAs in AmCK vs AmT comparison group, binding 3 503 and 3 252 target genes, which could be annotated to 40 and 39 GO terms as well as 104 and 99 metabolic pathways, respectively. Further investigation indicated that 17 DEmiRNAs within the regulation networks could bind to 116 serine proteaserelated mRNAs, and 14 DEmiRNAs were capable of binding to 54 ubiquitin-mediated proteolysis associated mRNAs. The result of stem-loop RT-qPCR showed that the variation trends in the expression levels of randomly selected four DEmiRNAs (miR-251-x, miR-9277-y, miR-1672-x and miR-4968-y) were consistent with those in sRNA-seq data, indicating the credibility and reliability of our sequencing data. 【Conclusion】Our study takes the lead in the prediction and investigation of DEmiRNAs and corresponding target genes in the larval gut of A. m. ligustica during the late stage of A. apis stress, and provides expression patterns and differential expression information of miRNAs. The DEmiRNAs including ame-miR-927b, miR-429-y and miR-8440-y may be involved in the regulation of serine protease and ubiquitin-mediated proteolysis. Complex regulations exist between DEmiRNAs and their target genes. DEmiRNAs are believed to participate in the interactions between the larval gut of A. m. ligustica and A. apis.

Key words:  Apis mellifera ligustica, Ascosphaera apis, stress response, microRNA, immune defense, target gene