The CircRNA Expression Profile In The Caste Development Of Apis Mellifera Ligustica Female Larvae

The honeybee is a typical social insect,which usually form a colony that consists of queen,workers and drones.Queen and worker bee are come from fertilized eggs.Although inherited same genetic material,they have huge differences in terms of tissue morphology,physiological function,social division of labor and life span.This phenomenon is called caste differentiation.The mechanisms behind this have been explored in several aspects,including environment,nutrition,DNA methylation level,proteome and transcriptome.To conclude,it is a complex physiological process regulated by multiple factors.While,with the development of biotechnology and the progress of research methods,more factors are found to be involved in this process.In recent years,with the development of RNA-seq,the biological functions of circRNAs have been gradually discovered.An emerging studies have showed that circRNAs can be used as sponges of miRNA to regulate the expression of target genes.But there is limited researches about circRNAs and the expression and function of circRNA have remained to be elucidated until now in bee.Therefore,we start with this study from identifying and analyzing the circRNAs in four samples of Italian bee larvae of different ages and different grades using high-throughput whole transcriptome sequencing technology and circRNA-miRNA-mRNA analysis network,with a view to discovering circRNAs related to bee grade differentiation.The main results of this study are as follows:(1)Construction of sequencing library as well as preliminary analysis of sequencing quality and results.We colleted 10 queen larvae of 3 days old,10 worker larvae of 3 days old,2 queen larvae of 5 days old and 2 worker larvae of 5 days old,and each sample has 4 biological duplicates.Then,we choose 3 of 4 biological duplicates in each sample to establishe the circRNA libraries.The results of sequencing showed the clean reads of QL3d,WL3d,QL5d and WL5d that were analyzed by mass analysis respectively,the matching rate with the reference genome ranged from 85.83%to 97.70%.(2)Expression profile and differential expression of circRNAs.There were 339,181,337 and 266 new circRNAs of QL3d,WL3d,QL5d and WL5d identified in this study.The four kinds of samples were divided into four groups for comparison according to their properties.There were 24 significantly different expression circRNAs between WL3d and QL3d(P<0.05);49 significantly different expression circRNAs between QL5d and QL3d;39 significantly different expression circRNAs between WL5d and QL5d;48 significantly different expression circRNAs between WL5d and WL3d.(3)GO and KEGG analysis of circRNAs.The results showed that the difference gene in 4 kinds of samples was enriched in 734 biological process functional groups(P<0.05);113 cellular components functional group;345 molecular functional functional groups.KEGG analysis revealed that the differential genes between QL3d and QL5d,QL3d and WL3d,QL5d and WL5d,WL3d and WL5d were enriched in 16,6,14 and 20 signaling pathways respectively,including some key signaling pathways in the development and caste differentiation of honeybee larvae,such as the Dorso-ventral axis formation pathways and Hippo signaling pathway.(4)Combined analysis of high-throughput sequencing results and mRNA-miRNA-circRNAs.The differentially expressed circRNAs were randomly selected and the primers were designed in accordance with junction,then quantified by real-time quantitative PCR.The results showed that the quantitative results were consistent with the predicted results,but the specific difference is different.In conclusion,this is the first time to resport the expression profiles of circRNAs in QL3d,WL3d,QL5d and WL5d of honeybee larvae.And,several potential circRNAs that may be involved in the regulation of honeybee differentiation were identified,may provide some reference for the further study in honeybee differentiation.