| Porcine Reproductive and Respiratory Syndrome(PRRS)is an immunosuppressive disease that causes reproductive disorders in sows and respiratory diseases in pigslets.In 1987,the Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)was first discovered in the United States.Just a few years,the PRRS epidemic swept the world,Caused huge economic losses.The PRRSV genome is variability and has immune escaping ability,although various prevention and control measures have been taken,the problem still can’t solved.Disease resistance breeding of PRRS has become the only way to solve the PRRS epidemic.Our previous study found that Tongcheng pigs have resistance to PRRSV.The transcriptome,proteome and microRNA(miRNA)levels have been studied in Tongcheng pigs,and we found a large number of differentially expressed mRNA and miRNA.In recent years,long non-coding RNA(LncRNA)has been shown to play an important regulatory role in antiviral immunity.LncRNA can affect the proliferation of virus by regulating mRNA or miRNA.we prepared to study the differential expression of LncRNAs in spleen and inguinal lymph node before and after PRRSV artificial infection in Tongcheng pigs and Large White pigs,in order to reveal the role of LncRNA in host immune response to PRRSV and for the disease resistance breeding of PRRS.The main results obtained are as follows:1.Differential expression analysis of LncRNAs in spleen and inguinal lymph node before and after PRRSV infection in Tongcheng pigs and Large White pigsThe RNA-seq data of spleen and inguinal lymph node before and after PRRSV artificial infection in Tongcheng pigs and Large White pigs were used as materials.Using FastQC,Tophat2,Cufflinks software and LncRNA transcript screening and differential expression analysis procedures,the identification and differential expression analysis of LncRNA in spleen and inguinal lymph node before and after PRRSV infection in Tongcheng pigs and Large white pigs were performed.In the spleen,441 differentially expressed LncRNAs were identified after infection in Tongcheng pigs,and 695 differentially expressed LncRNAs were identified in the Large white pigs.The genetic differences were found in the spleen of the two cultivars,there were 1744 LncRNAs Differential expressions.In the inguinal lymph node,1602 LncRNAs was differentially expressed after PRRSV infection in Tongcheng pigs,and 777 LncRNAs was differentially expressed after PRRSV infection in Large White pigs.There were 282 LncRNAs expressions in the inguinal lymph node showing genetic differences between varieties.2.Screening of LncRNA involved in PRRSV immune response and antiviral pathway regulationThe LncRNA was differentially expressed after PRRSV infection as described above,and the candidate LncRNAs involved in the host immune response to PRRSV were screened by three pathways.The first approach,using weighted gene co-expression network analysis(WGCNA)to construct a co-expression network and module for differential expression of LncRNA and differentially expressed mRNA after PRRSV infection,introduce viral load information,and focus on PRRSV immunity.Respond to LncRNAs with high viral load correlation in key modules.A total of 59 candidate LncRNAs were obtained by this screening;T he second approach focused on the extreme differential LncRNA of the top 10% of the differential expression folds after PRRSV infection,and the extreme difference of PRRSV infection in the spleen and inguinal lymph node of Tongcheng pig and Large White pig was selected.A total of 109 LncRNAs;The third pathway,based on the genes and miRNAs reported in the literature that are involved in the host immune response to PRRSV,reversely reverses the LncRNA associated with it.Through this screening,four LncRNAs co-localized with PRRSV immune response-related genes were obtained,and 71 LncRNAs with binding sites for PRRSV immune response-related miRNAs were obtained.By analyzing the candidate LncRNA,it was found that LncRNA ALDB4471 can be screened by three different routes.ALDB4471 overlaps with the anti-PRRSV gene RSAD2,and the expression level is positively correlated with viral load.It is the top 10% of LncRNA in the brown module associated with viral load.ALDB4471 is extremely up-regulated in spleen and inguinal lymph node after infection with PRRSV in Tongcheng pigs and Large White pigs.3.Functional study of ALDB4471A new transcript of RSAD2 was discovered by RACE experiments.ALDB4471 overlapped with the 3’ end of new RSAD2 transcript.PRRSV infection experiments were performed after overexpressing ALDB4471 in cells,and it was found that overexpression of ALDB4471 significantly inhibited the expression of RSAD2 in cells and significantly promoted PRRSV replication.To investigate the direct targeting relationship between ALDB4471 and RSAD2,Poly(I:C)was used to stimulate the expression of RSAD2 in PK-15 cells,and then overexpressing ALDB4471 revealed that ALDB4471 significantly inhibited the expression of RSAD2.By predicting the binding of ALDB4471 to the protein by RPIseq and catRAPID website,it was found that ALDB4471 can bind to the transcription factor p53.It is reported that p53 can promote the expression of RSAD2,so it is hypothesized that ALDB4471 can compete with RSAD2 for binding to transcription factor p53.RSAD2 expression is inhibited.This study mainly completed the identification and differential expression analysis of LncRNAs in spleen and inguinal lymph node after PRRSV infection in Tongcheng pigs and Large White pigs;Identification and analysis of differentially expressed LncRNAs between Tongcheng pigs and Large White pigs without PRRSV infection;Screening and functional verification of LncRNAs involved in host immune response to PRRSV.These studies led us to gain a deeper understanding of the differences in LncRNA levels in the PRRSV immune response between Tongcheng pigs and Large White pigs.At the same time,LncRNA ALDB4471,which is involved in the immune response of PRRSV,was found to provide a new molecular target for disease resistance breeding of PRRSV. |
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