Identification And Function Analysis Of Piwi-interacting RNA In The Brain Of Silkworm Bombyx Mori

Silkworm is the model insect of the Lepidoptera,the nervous system of which is the type of abdominal nerve cord.The development of nervous system of silkworm was regulated by a variety of genes and small RNA,such as microRNAs,siRNAs?small interfering RNAs?and piRNAs?Piwi-interacting RNAs?.Previous studies of piRNAs have been concentrated more on germ cells,but less on nervous tissues.Therefore,these works of exploring function of piRNA in the nervous tissue of silkworm will lay a good foundation on the study of new function of piRNA and also provide new ways on Lepidoptera pest control.Total RNAs of the head of the 3rd day of fifth instar larvae of silkworm were extracted.To obtain a homologous small RNA?sRNA?library of brain of silkworm,rRNAs and mRNAs were removed by Ambion Poly?A?Purist?Kit fishing before deep sequencing identified piRNAs present in the brain tissue.Potential target genes of these piRNA were predicted by bioinformatics.Moreover,we further confirmed that piRNA had a impact on target gene's expression by overexpression of piRNA in celluar level and body,and explored the regulation mechanism of piRNA on the development of the nervous system silkworms.The main results were as following.First of all,the sRNA library of brain of silkworm was constructed and sequenced by Illumina Hiseq2000^TM.With Rfam&Repbase database and genome comparison respectively,a total of 189,415 sequences matched to the Rfam&Repbase database were acquired.Further analysis showed that rRNAs accounted for 54.05%,which indicated that the removing method of rRNAs was not so effective,and we need to modify the pursuiting probes of rRNAs.While tRNAs accounted for 25.57%,other types of RNAs in the library accounted for 12.66%.After statistically analyzing the length of obtained sRNAs,we found that most of the sRNAs had the length between 18-30 nt,indicating that the sRNA library not only had mi RNAs but also included piRNAs.Secondly,we analyzed mi RNAs and piRNAs that obtained in the sRNA library and predicated their target genes.Results showed that there were 684 miRNAs,including 357known miRNAs in the silkworm,26 miRNAs were homologous to other insect species,the other 301 miRNAs had homologs to humans.In addition,we found 74 novel miRNAs of silkworm.Target gene prediction showed that a total of 1015 target sites that guiding by 634known miRNAs and 65 novel miRNAs were predicated.Besides we also found 5805 novel piRNAs in silkworm and homologs to Drosophila one known piRNA.We found that 391piRNAs had 1191 target sites of protein coding genes.GO annotation and KEGG pathway analysis of target genes of piRNA showed that these piRNAs presented in the brain of silkworms might play an important role in the development regulation of nervous system of silkworm.Thirdly,three piRNAs?that is piR-1841,piR-1886,piR-4682?and their predicted target genes were selected as the research object to explore the expression patterns of ten tissues in the 3rd day of fifth instar larvae of silkworm.Results showed that piR-1841 had more expression in the testis and ovarian,but little in the brain and ventral thoracic nerve,while its target gene neuronal PAS domain-containing protein 1/3 accumulated more in the brain and ventral thoracic nerve.The expression pattern of piR-1886 was similar to piR-1841,but the expression of its target gene agrin was extremely high in the epidermis,except for its high expression in the brain and ventral thoracic nerve.piR-4682 had relatively high expression levels in the testis,ovary and hemolymph,however,its target gene neuromedin U receptor with higher expression in the brain,silk glands,epidermis and fat body.These results demonstrated that these three piRNAs existed in different tissues and might play different roles,they also might play converse regulation roles to their target genes related to neural development.Fourthly,further research found that one piRNA and one miRNA can be transcribed from the same genomic locus?such as piR-4682,miR-305?,expression analysis showed that unlike the expression pattern of piR-4682,miR-305 was highly expressed in the hemolymph of silkworm,but low in the testis,ovary and ventral thoracic nerve.Finally,functions of piRNAs were studied.Recombinant vector psiCHECK[small wing]was co-transfected to the mammalian HEK293T cell line with piR-4682 and miR-305 analogues?mimics?respectively.Results showed that the expression of small wing gene decreased when the piR-4682 or miR-305 was overexpressed,and the inhibitory effect of miR-305 on small wing gene was stronger than piR-4682.Furthermore,analogues of piR-4862?agomir?were injected to the head of the 3rd day of fifth instar larva of silkworm.Results showed that piR-4862 was significantly overexpressed after injection of piR-4682 agomir,while the expression of piR-4682 target gene,small wing gene decreased,which indicated that piR-4682 may play key roles in the regulation of small wing gene.