Analysis Of The Effect Of Variation In Porcine AQP3 Gene Promoter On Its Expression Level And PEDV Resistance

Porcine epidemic diarrhea(PED)is caused by Porcine epidemic diarrhea virus(PEDV),it had the characteristics of high infectivity,high pathogenicity and high mortality in pigs,which seriously threatens the healthy development of pig industry all over the world.In recent years,many pig farms had been vaccinated with PEDV vaccine and the feeding and management level had been improved,the infection rate and mortality rate of PED V had decreased,but the invasion of PEDV has not been truly solved.It has a broad application prospect to select the key genes related to PEDV infection from the genetic point of view and enhance the resistance of piglets to PEDV by means of molecular breeding.In this study,4 confirmed pigs infected with PEDV and 4 normal piglets were identified based on the previous evaluation criteria.Their jejunal tissues were selected to screen important differentially expressed genes using RNA-seq technology,and part of the DEGs were verified on individuals.According to literature mining and bioinformatics analysis,AQP3 gene,an important candidate gene whose biological function is closely related to PEDV infection,was selected to explore the molecular mechanism of its expression regulation from the perspective of methylation and promoter region variation.This study provides a new basis for further study of the genetic regulation mechanism of PEDV infection and its resistance breeding.The main results are as follows:1.Confirmed PEDV infection and normal piglets were obtained by PCR amplification,sequencing alignment and pathological section observation according to the previous research.The Confirmed individual jejunal tissues of piglets were selected to extract total RNA for RNA-seq,and candidate genes associated with PEDV infection were validated by RT-qPCR and screened based on literature mining,bioinformatics analysis,and proteomics sequencing results on PEDV-infected individuals and cells.The results showed that a total of 290 differential genes were selected,104 genes were up-regulated and 186 were down-regulated in PEDV infected group.290 DEGs were mainly concentrated in 3 functions:chemokine activity(GO:0008009),chemokine receptor binding(GO:0042379),and G protein coupled receptor binding(GO:0001664),and they were mainly enriched in 16 signaling pathways,such as fat digestion and absorption pathway,chemokine signaling pathway and metabolic pathway.A total of 15 candidate genes closely related to PEDV infection were selected by comparing with the proteomic results and biological function mining.The expression patterns of these genes in RNA-seq were highly consistent with those in RT-qPCR,indicating the high accuracy of transcriptome data.Based on bioinformatics analysis and literature mining,AQP3 gene was identified as an important candidate for the next step of functional validation and mechanistic analysis.2.The regulatory role of promoter methylation on AQP3 gene in PEDV infected and normal piglets was revealed by a series of experiment such as RT-qPCR,methylation sequencing,ChIP-PCR,and transcription factor interference.The results showed that the expression of AQP3 gene in the intestine tissues of PEDV infected piglets decreased significantly,and the decline was most obvious in jejunum.The overall methylation level of the 2 CpG islands in the A QP3 gene promoter region of diarrhea piglets increased but no significant,while the mC-20 site of CpGl and mC-10 site of CpG2 were significant negative correlation with gene expression.ChIP-PCR demonstrated that there were binding regions between the two methylation sites significantly negatively correlated with AQP3 gene expression and the transcription factor Spl,and in the Spl-interfered IPEC-J2 cell line,the expression of AQP3 gene was extremely reduced.These results indicated that PEDV infection can increase methylation degree of mC-20 and CpG2 of CpG1 in AQP3 promoter region,inhibiting the binding of TF Spl and AQP3 promoter to reduce the expression of AQP3 gene.3.There was an insertion sequence in the promoter of AQP3 gene identified by a series of experiment such as PCR amplification,fragment separation and sequencing alignment,construction of wild-type and mutant luciferase plasmid,dual-luciferase reporter detection and plasmid co-transfection.The results showed that there was an insertion sequence’GGGCGGGGTTGCGGGC’ with a length of 16 bp between-89～-88 bases of promoter region of AQP3 gene,and it could significantly improve the promoter region activity of AQP3 gene.ChIP-PCR confirmed there was a binding region between this insertion sequence and transcription factor C/EBPα.Compared with the non-co-transfection group,the luciferase activity of co-transfection pcDNA3.1-C/EBPa and pGL3-basic-insert plasmid was significantly higher than other groups,which showed the insertion sequence could interact with C/EBPα.