Identification And Molecular Mechanism Of Long Non-Coding RNAs In Response To Nosema Bombycis Infection In Bombyx Mori

Lnc RNAs(long non-coding RNAs)have a variety of complex functions and play an irreplaceable and important role as they can influence not only the epigenetics of an organism,but also the transcription of genes and even the various stages of the life process.Current research on lnc RNAs in pathogen and host interactions has focused on mammals such as humans and mice,as well as some model organisms.As lnc RNA research continues,the functions of lnc RNAs are being revealed,and their role in insect pathogen-host interactions is beginning to receive attention.Along with the development of sequencing technology,more and more insect lnc RNAs are being identified and therefore more molecular biology studies are urgently needed to identify these lcn RNAs.microsporidia are a class of organisms that can infect vertebrates and invertebrates as specialized intracellular parasites and are widely distributed and diverse.The silkworm microsporidia(Nosema bombycis,Nb)cause microparticle disease(P?brine disease)by infecting the silkworm(Bombyx mori)and are a major threat to sericulture production.The control of microparticle disease in silkworm is still mainly preventive in production.Microsporidial infection of the host leads to changes in the expression of a large number of response genes in the host,including lnc RNAs,but the mechanism of the role of host lnc RNAs in silkworm-microsporidial interactions has not been reported.Therefore,the identification of lnc RNAs associated with Nb infection and their functional mechanisms can provide a new theoretical basis for revealing the pathogenesis of Nb infection in silkworm and the mechanism of silkworm-microsporidia interactions,which is of great scientific and theoretical significance and practical application for the development and prevention of silkworm disease.In this study,whole transcriptome sequencing was used to screen the lnc RNAs associated with Nb infection and to investigate the functions of some key lnc RNAs.By analysing the expression of target lnc RNAs at different times of Nb infection,the expression in different tissues,the effects of in vitro overexpression and knockdown of target lnc RNAs on Nb replication,the mechanisms and functions of silkworm lnc RNAs in the host response to Nb infestation were investigated.The relevant findings are as follows:1.Screening of Nb infection-associated lnc RNAs and m RNAsNormal silkworm midgut tissues were used as the control group(CK group),and those infected with Nb for 72 h were used as the experimental group(Nb group),with three replicates in each group.The normal midgut tissues and the midgut tissues infected with Nb for 72 h were collected separately,and total RNA was extracted from the midgut tissues for whole transcriptome analysis.The differentially expressed lnc RNAs and differentially expressed m RNAs were screened by DEseq software with log2FC>1.5 and P value<0.05.The differentially expressed lnc RNAs were predicted using bioinformatics software.acting genes versus trans-acting genes,and GO enrichment and KEGG enrichment analyses were performed.A total of 1444 lnc RNAs were finally obtained from the lnc RNAs samples of the CK and Nb groups.42 differentially expressed lnc RNAs were obtained,of which 27 were upregulated and 15 were down-regulated;305 differentially expressed m RNAs were also obtained,of which 159 were up-regulated and 146 were down-regulated.the results of GO and KEGG enrichment analysis indicated that DElnc RNA target genes The results of GO and KEGG enrichment analysis indicated that DElnc RNA target genes were mainly involved in energy metabolism,RNA binding,immune regulation,apoptosis,immune process and energy material transport.2.Cloning of MSTRG857.1 and its identificationThe MSTRG857.1 gene was cloned by PCR with a sequence length of 420 bp.The highest expression was found in the fat body and midgut.The spatio-temporal expression profile of MSTRG857.1 in silkworm midgut tissues at various time points revealed that the expression level of MSTRG857.1 in midgut tissues increased significantly after 48 h of Nb infection in silkworm compared to the control group.3.Role of MSTRG857.1 in Nb replicationThe insect expression vector p IZ/V5-Mcherry-His and Gene knockdown technology were used to overexpress and knock down MSTRG857.1 in cells,respectively.It was shown that MSTRG857.1 had a promoting effect on Nb replication.In addition,FADD was found to be a trans target gene of MSTRG857.1 by lnc RNA-m RNA association analysis,and overexpression of MSTRG857.1 was found to increase the expression of FADD and knockdown of MSTRG857.1 resulted in a decrease in the expression of FADD.In addition,overexpression of MSTRG857.1 inhibited apoptosis in Bm N cells.