Identification,expression And Functional Study Of Long Non-coding RNA 63 Of Bombyx Mori

Long non-coding RNAs（lnc RNAs）are a class of endogenous RNA molecules with a transcript length of more than 200 nucleotides and do not or rarely participate in proteins coding.At present,there are many studies on the functions of lnc RNAs in mammals,but the research on lnc RNAs in insects is still very limited.Due to the relatively low conservation of lnc RNAs among species,it is an arduous task to study their functions and mechanisms in different organisms.As a kind of special economic animal in China,Bombyx mori is also a typical completely metamorphosis insects,which has important economic value and scientific significance.A lnc RNA（lnc RNA63）in response to 20-hydroxyecdysone（20E）regulation was screened based on the transcriptome sequencing analysis of B.mori larval fat body in the previous study.In this study,the lepidoptera model insect,B.mori,was taken as the research object.The sequence and expression of lnc RNA63 in response to 20E regulation and its function in the development of B.mori were studied at molecular,cellular and individual levels.The main results are as follows:1.Sequence analysis,cloning and sequencing showed that lnc RNA63 was a real non-coding RNA in B.mori.Bioinformatics prediction revealed that one of the co-expressed genes of lnc RNA63 was a hormone receptor 3（Hr3）gene in response to 20E regulation.2.q RT-PCR was used to detect the expression of lnc RNA63 and Hr3 in different tissues of the 5^th instar larvae and silk gland tissues at different developmental stages of the 5^th instar.The results showed that the expression characteristics of lnc RNA63 and Hr3 were basically consistent.The expression of Hr3 was significantly up-regulated after lnc RNA63overexpression and significantly down-regulated after lnc RNA63 interference,indicating that Hr3 is a co-expressed gene of lnc RNA63.3.The subcellular localization of lnc RNA63 showed that lnc RNA63 mainly existed in the nucleus,but 20E could induce the nucleocytoplasmic migration of lnc RNA63.After lnc RNA63 was overexpressed in Bm N cells,the expression of co-expressed gene Hr3 and autophagy marker gene ATG8 were significantly up-regulated,and the red fluorescence of ATG8 was significantly enhanced by immunofluorescence detection,indicating that lnc RNA63 overexpression increased the autophagy level of Bm N cells.4.When lnc RNA63 was interfered in B.mori,the expression of co-expressed gene Hr3and autophagy marker gene ATG8 were significantly down-regulated.The results of phenotypic analysis showed that lnc RNA63 interference could affect the development from larva to pupa and the development of silk gland.The changes of glycogen and triglyceride contents in laval fat body of silkworm showed that lnc RNA63 could also affect the metabolism of larval fat body.5.RNA-seq results of silkworm silk gland after lnc RNA63 interference showed that there were 31 significantly differentially expressed genes after interference.GO enrichment analysis showed that these differentially expressed genes were mainly related to gene expression regulation.KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in metabolism,development and immune-related signaling pathways,suggesting that lnc RNA63 might play a role in silk gland development,fat body metabolism and immune regulation.In conclusion,this study took the lepidoptera model insect,Bombyx mori,as the research object,and carried out sequence identification,expression analysis and functional study on lnc RNA63 in response to 20E regulation at the molecular,cellular and individual levels.It was found that 20E could not only regulate the expression of lnc RNA63,but also induced the nucleocytoplasmic migration of lnc RNA63.Hr3,a hormone receptor gene involved in autophagy regulation of B.mori,is a co-expressed gene of lnc RNA63.LncRNA63 might participate in silk gland development and fat body metabolism during the metamorphosis and development of silkworm larva to pupa through the co-expressed gene Hr3.This study lays a foundation for further study on the function and regulation mechanism of lnc RNA63 in the metamorphosis and development of silkworm,and provides potential targets and strategies for biological control of lepidopteran pests based on the metamorphosis and development regulation of B.mori,which has important application value and practical significance.