| Duck Tembusu virus(TMUV)disease is a new infectious disease caused by TMUV.The disease has resulted in sustaining harm to Chinese waterfowl farm since 2010,causing substantial economic losses.LncRNA,more than 200 nucleotides in length,was recognized as pseudo-transcription due to the lack of protein-coding capacity.Emerging reports uncovered that lncRNA,implicated in complex biological processes,played an important role in gene regulation during viral infection.However,little is known for the effect of lncRNA on TMUV infection.In order to explore the possible mechanism of action between lncRNA and TMUV infection,the study aimed to use duck embryo fibroblast cells(DEFs)as experimental material,using high-throughput RNA-sequencing(RNA-Seq)technology to reveal 1ncRNA expression profile induced by TMUV infection,predicting the biological functions of differentially expressed 1ncRNA by bioinformatics analysis,performing the expression validation and preliminary function study of filtered lncRNA.The main research contents are as follows:1?To explore the replication of TMUV and the changes of biological characteristics,DEFs were infected with TMUV by a MOI of 3,and then the viral replication in cells,cell cycle,and cell apoptosis were detected at 12 hpi,24 hpi,and 48 hpi.The result showed that the replication of TMUV in DEFs was gradually increased,and the uptrend was greater in 24~48 hpi than 12~24 hpi.Besides,TMUV infection induced S and G2 M phases arrest of DEFs at 12 hpi and 24 hpi.The significant apoptosis of DEFs was appeared at each time point,and the percentage of apoptotic cells increased gradually with the extension of time.2?Based on the detection of cell biology,the study selected TMUV-infected cells at 12 hpi and 24 hpi for RNA-Seq to carry out the lncRNA expression profile.The RNA-Seq data showed that 34 and 339 differently expressed lncRNA were identified at 12 hpi and 24 hpi in TMUVinfected DEFs compared with mock-infected DEFs.To initially explore the biological role of 1ncRNA during the process of virus infection,cis target genes of differently expressed lncRNA predicted by bioinformatics were used for GO and KEGG analysis.The GO analysis indicated that target genes were closely related to biological regulation,cellular processes,singleorganism processes,cell,cell part,and membrane.The KEGG analysis showed that target genes were mainly referred to the signal pathway categories of endocrine system,signal transduction,signaling molecules and interaction,and immune system.3?In order to validate the RNA-Seq data,4 and 11 differently expressed lncRNAs at 12 hpi and 24 hpi were screen out for RT-qPCR.The results showed that expression changes confirmed by RT-qPCR were consistent with the RNA-Seq data.Furthermore,the correlation analysis revealed that changes in lncRNA expression level were comparable between RNA-Seq and RT-qPCR,with the correlation coefficients of 0.8769(P<0.0001).The above results confirmed that the RNA-Seq data were reliable and accurate.4?To further explore the biological role of 1ncRNA in TMUV infection,lncRNA8505.2 was selected for subsequent study.The overexpression vector was constructed with gene recombination technology.After the DEFs transfected with overexpression vector and infected with TMUV of 3 MOI,the TMUV replication was detected at 12 hpi,24 hpi,and 48 hpi.The result showed that TMUV replication was significantly inhibited at 36 hpi,whereas the inhibiting effect was not significant at 12 hpi and 24 hpi.To explore the role of lncRNA8505.2 in inhibiting TMUV replication,the study detected the transcriptional levels of I-IFN,MX1,and CXCL10 in DEFs.The result showed that transcriptional levels of these innate immunity related genes did not rise significantly.The above results indicated that lncRNA8505.2 could inhibit TMUV replicated,and the inhibiting effect was not achieved through enhancing the transcriptional levels of innate immune factors. |
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