Comparative Transcriptome Of Nilaparvata Lugens Continuous Culture Cells In Response To Rice Ragged Stunt Virus Infection

Rice ragged stunt virus(RRSV)is transmitted by brown planthopper(BPH,Nilaparvata lugens)in a persistent propagative manner with nontranovarial transmission.Rice ragged stunt caused by RRSV is a great threat to rice production.To elucidate the interaction between BPH and RRSV,We sequenced the transcriptomes of viruliferous(RRSV-infected)and nonviruliferous BPH culture cell using the Illumina HiSeqTM 2500 platform.1.A total of 64,888 unigenes were obtained,with 26,904(41.46%)unigenes significantly blast to the NCBI-NR protein database.According to the species distribution of the BLASTX results,The top 4 species were Tribolium castaneum,Acyrthosiphon pisum,Pediculus humanus corporis and Branchiostoma floridae accounting for 15.50%,8.31%,7.75%and 6.31%respectively?2.A total of 1,753 genes were detected differentially expressed in RRSV infect cells,among which,1,280 and 473 genes were up-regulated and down-regulated.1,239 genes were differentially expressed in 1 d-infecting by RRSV,among which,919 genes and 320 were up-regulated and down-regulated;1,374 genes were differentially expressed in 4 d-infecting by RRSV,among which,1,044 genes and 330 were up-regulated and down-regulated.3.The GO annotation analyses indicated that,genes differentially expressed in 1 d-infected cells and in 4 d-infected cells were in a similar distribution in GO annotation.The KEGG pathway analyses to differential expression gene of three samples showed that the MAPK signal pathway,PPAR signal pathway,cAMP signaling pathways and the Ras signaling pathways were active in the early infecting of RRSV,indicated that a series of signal pathways were actived in response to the infecting of RRSV;Genes relative to antibiotic biosynthesis,phagosome,spliceosome,lysosome,gelling connection were differentially expressed only in the late infecting of RRSV which indicated that the infection of RRSV may cause disease to BPH.4.13 continuous up-regulated expression unigenes have been verified by RT-q PCR,and 10 Unigenes(76.92%)are at the same expression trend with the results of sequencing.The BLASTX results of these 13 Unigenes against the NCBI-NR protein database indicated that s-3.comp41631_c0_seql corresponding to DNAJC14 protien(Apis mellifera)and s-3.comp41630_c0_seql o rresponding to IscW ISCW005027(Ixodes scapularis).In order to address whether the replication and proliferation of RRSV are regulated by these two gene,exogenous double-stranded RNA of these two genes are produced to knockdown their expression to see whether they can influence the replication or proliferation of RRSV.The RT-qPCR results of Cells transfected dsRNAs demonstrated that the transfection of dsDNAJC14 significantly induced the accumulation of RRSV P8 gene,while the transfection of dsISCW005027 had no significant effect on the accumulation of RRSV P8 gene.The RT-qPCR results shows that,injecting with dsDNAJC14 and RRSV,the RRSV-infected rate and the accumulation of RRSV P8 gene are induced,while injecting with dsISCW005027 and RRSV result in the increasing of the accumulation of RRSV P8 gene only.5.We compared the splicing sequence results with virus gene pool,three virus were first found in BPH.They are Lishi Spider Virus(LSV),Sanxia Water Strider Virus(SWSV),Equine encephalosis Virus(EEV).By cloning in vitro,we got the part of the sequence information about these three virus:7122 bp of LSV,3153 bp of SWSV and 1075 bp of EEV.In the same time,we detected these three virus in BPH bodies and the newly culture cells,but fail to find EEV in,speculates that EEV is not belong to endogenous viruses of BPH.