Differential Expression Of SRNA And Function Of MD17 Encoded By CSBV In Apis Cerana Infected With CSBV

Objective In this study,Illumina high-throughput sequencing and bioinformatics methods were used to sequence the s RNA of CSBV-infected Apis cerana larvae and healthy Apis cerana larvae,and to analyze and predict differential expression of host mi RNAs,vi RNA derived from CSBV and its potential vmi RNA,then screening a vmi RNA(MD17)for preliminary study of its function,laid the foundation for us to sduty the anti-CSBV mechanism of Apis cerana and the function of CSBV vmi RNA.Methods Two s RNA libraries were constructed by artificially infected CSBV larvae(L_I)and healthy Apis cerana larvae(L_H),and sequenced by Illumina high-throughput sequencing technology.After filtering datas,the Bowtie software was used to annotate the length and classification of s RNA of two libraries.Then using mi RBase as a reference,clustering expression levels of the host known mi RNA by DEGseq(2010)software,and predicting the host novel mi RNA by integrating the mi REvo and mirdeep2 prediction software.At the same time,the obtained clean reads were mapped to the CSBV genome to predict the viral vi RNA,the target gene of viral vi RNA was predicted by RNAhybrid,and the potential vmi RNA was predicted by VMIR software.On this basis,GO functional enrichment and KEGG PATHWAY enrichment analysis were performed on the target gene of host mi RNA and virus vi RNA.The predicted vmi RNA precursors were detected and verified using the q RT-PCR method.We aimed to study the function of vmi RNA MD17 which targeting gene VP2.Firstly,we synthesized si RNA targeting MD17 mature sequence,then transfected the si RNA with p EGFPN1-CSBV-VP2 recombinant expression plasmid into 293 T cells to research the effect of MD17 on CSBV VP2 gene expression at the cellular level.On this basis,si RNA(1?g/?L)and 1×107 copies of CSBV were fed to the 3 day old larvae,the copies of CSBV in larvae were detected by q RT-PCR and larval survival was statisticed.The role of vmi RNA-MD17 in the process of CSBV infection was primary discussed.Results Atotal of 14,933,703 and 18,687,476 clean reads were obtain from L_H and L_I by high throughput sequencing,respectively,length analysis showed that small RNAs of 22 nt were enriched,which may represent the class of mi RNA.The obtained s RNAs were classified and a total of 151 known host mi RNA precursors were identified and two new host mi RNAs were predicted.Meanwhile,882,573 s RNAs derived from sense strand of CSBV and 74,214 s RNAs from negative sense strand were detected,indicating that both the sense and antisense strands of the CSBV genome can produce s RNA during infection.On this basis,cluster analysis of host mi RNA expression levels in L_H and L_I libraries revealed that there were significant differences in expression of 44 mi RNA,of which 26 mi RNAs were up-regulated and 18 mi RNAs were down-regulated.After function alanalysis,these mi RNA target genes are mostly regulatory proteins and proteins involved in signal transduction,and can regulate metabolic pathway,endocytosis and m RNA degradation.By comparison with the CSBV genome,146,960 vi RNAs of CSBV were identified,of which 311 were differentially expressed,and functional analysis were performed to the vi RNAs target gene,results show the vi RNA target gene targeting CSBV is mainly related to viral infection,and the gene targeting the host has a function of binding to a signal transduction-related enzyme(such as GTPase,protein kinase,etc.),mainly involved in the metabolic pathway of the host,and secondly a small number of target genes are involved in host endocytosis and RNA degradation.Six vmi RNA precursors derived from CSBV were predicted by Vir-Mir software,then has been detected in the infected CSBV larvae by q RT-PCR,and the sequencing results of the six vmi RNA precursors were matched with the predicted sequences.Further analysis revealed that MR3 and MD17 are located in the structural protein gene of CSBV,and MR10,MD18,MD27 and MD35 are located in the non-coding region of CSBV.The MD17 which tageting structural protein VP2 gene was seleceted toinvestigate the role of MD17 in CSBV infection.The MD17 can effectively inhibit the expression of CSBV VP2 gene in 293 T cells,and the interference effect was close to 40% to flow cytometry.And oral administration of si RNA reduced the mean virus yields and viral RNA level significantly,which was significantly different between the si RNA group and the CSBV group(P<0.01),indicating that it can effect the replication of CSBV in the Apis cerana by interfering the generation of MD17.Conclusions 1.151 host known mi RNAs and 2 host novel mi RNAs were identified,the target genes of host mi RNAs were involved in the regulation of metabolic pathway,endocytosis and m RNA degradation after larvae infected CSBV.2.146,960 viral vi RNAs and 6 potential vmi RNAs were predicted the vi RNA target gene targeting the virus is mainly related to viral infection,and the gene targeting the host has function to bind signal transduction related enzymes(such as GTPase,protein kinase,etc.),mainly involved in the metabolic pathway of the host,and secondly a small number of target genes are involved in host endocytosis and RNA degradation,then 6 potential vmi RNA precursors were detected in the larvae infected with CSBV,and were all confirmed to be derived from CSBV by sequencing results.3.RNAi interference was performed by si RNA targeting MD17,which can affect the replication of CSBV in the larvae of Apis cerana.It is speculated that MD17 may play an important role in the body's defense against CSBV infection.