【Objective】MicroRNA (miRNA) is a kind of key regulator for negative regulation of mRNA at post-transcriptional level. The objective of this study is to provide miRNA expression patterns and differential expression information, illuminate the function of differentially expressed miRNA (DEmiRNA) in the development of larval gut by comprehensively investigating the DEmiRNAs and their regulation networks during the developmental process of Apis mellifera ligustica larval gut. 【Method】Deep sequencing of the 4-, 5- and 6-day-old larval guts of A. m. ligustica was conducted using small RNA-seq (sRNA-seq) technology, followed by mapping of the data after quality-control with the reference genome of Apis mellifera, and the mapped tags were then compared to miRBase database. The miRNA expression level was normalized by TPM algorithm, and the expression clustering, prediction of secondary structure of precursor and differential expression analysis were performed using related bioinformatic softwares. TargetFinder software was used to predict target gene of DEmiRNA, which was annotated to GO and KEGG databases using Blast, furthermore, miRNA-mRNA regulation networks were constructed using Cytoscape software. Stem-loop RT-qPCR was used to verify the sequencing data in this study.【Result】High-throughput sequencing of larval gut samples produced 10 841 644, 12 037 678 and 9 230 496 clean tags, respectively. In Am4 vs Am5 comparison group, there were16 up-regulated and 10 down-regulated miRNAs, while Am5 vs Am6 comparison group included 5 up-regulated and 7 down-regulated miRNAs, respectively. Among them, Novel-m0031-3p was shared by both Am4 vs Am5 and Am5 vs Am6, binding 5 target genes associated with ecdysone inducible protein, 25 and 11 DEmiRNAs were specific for the above-mentioned two comparison groups. DEmiRNA in Am4 vs Am5 could bind 5 742 target genes, among them 2 725 targets could be annotated to 46 GO terms in GO database, and the largest ones were binding, cellular process, metabolic process and single-organism process. Similarly, 12 DEmiRNAs in Am5 vs Am6 could link 3 733 target genes, among them 2 725 targets could be annotated to 41 GO terms, and mostly enriched terms were binding, cellular process, single-organism process and metabolic process. In addition, 1 046 and 676 target genes of two comparison groups were related to 116 and 92 KEGG pathways, and the number of DEmiRNA target genes in Am4 vs Am5 was more than that in Am5 vs Am6, which annotated to Wnt signaling pathway, Hippo signaling pathway, purine metabolism and endocytosis. Further analysis demonstrated that up-regulated and down-regulated miRNAs in Am4 vs Am5 could bind 611 and 85 target genes, and ame-miR-6052 linked the most target genes and participated in regulating cytochrome P450 via 5 target genes. miR-281-x could bind 49 target genes and indirectly regulate histidine metabolism, TGF-β signaling pathway and Hippo signaling pathway. In Am5 vs Am6 comparison group, up-regulated and down-regulated miRNAs could bind 43 and 431 target genes, respectively, among them miR-iab-4-x linked the most target genes, and it could participate in regulating growth and development related pathways, such as dorso-ventral axis formation, Hippo signaling pathway, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway and mTOR signaling pathway. Regulation network analysis indicated that complex networks formed between DEmiRNAs and target genes, and DEmiRNAs lied in the center while target genes lied in the periphery. Finally, Stem-loop RT-qPCR was carried out to validate the randomly selected three DEmiRNAs, and the result confirmed the reliability of sequencing data. 【Conclusion】The DEmiRNA and corresponding target genes in the A. m. ligustica larval gut were predicted and analyzed at genome-wide level, it was found that A. m. ligustica are capable of regulating the expression of many miRNAs such as ame-miR-6052, miR-iab-4-x, miR-281-x and novel-m0031-3p. The results not only offer the expression pattern and differential expression information of miRNA during the developmental process of A. m. ligustica larval gut, but also lay a foundation for clarifying the molecular mechanisms underlying the larval gut’s development.
