Study On The Expression And Regulation Of CYP3A88and PRRSV Mediator Genes In Pigs

Porcine Reproductive and Respiratory Syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease, characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets. The Chinese Dapulian pigs (DPL) are more resistant to PRRSV than Duroc x Yorkshire x Landrace (DLY) commercial pigs. We analyzed the mRNA expression of five PRRSV mediator genes. We cloned the partial region of954bp which encodes NMMHC ⅡA protein region including23amino acid residue in the C-terminal that bound to PRRSV and analyzed the mRNA expression of NMMHC ⅡA genes in different tissues.The former study has suggested that difference in the mRNA expression of CYP3A88gene in the lung tissues exsit between DLY and DPL pigs. By genome walker we amplifyied the5’regulatory region of porcine CYP3A88gene. Six vectors carrying different CYP3A88promoter, i.e. P2801, P1871, P694, P282, P95and P27respectively, were constructed. Their effect on gene expression was analyzed in vitro by transient transfection of dual luciferase reporter system into Marc-145cells, and important mutation site was identified.The results are summarized as follows:1). The partial region of954bp was highly conserved among mammalian species, being higher in nucleotide and amino acid identity to those of human, rat, mouse, cattle, monkey and dog, respectively. Alignment of the nucleotide sequence of this gene fragment among Laiwu, Dapulian and DurocxYorkshirexLandrace crossbred commercial pigs revealed three changes, however, the amino acid sequence was identical, implying that the region of NMMHC-ⅡA protein responsible for binding to PRRSV is not different between DPL and DYL pigs;2). Analysis of mRNA expression of five PRRSVmediator genes (SIGLEC1, NMMHC-IIA, CD163, VIM and HSPG2) in the lung tissues indicated differences in expression between DLY and DPL pigs. In uninfected porcine lung tissues, the levels of SIGLEC1, NMMHC-IIA, CD163and VIM genes were significantly higher in DLY than in DPL pigs (P<0.05); in PRRSV-infected pigs, the expression levels of NMMHC-IIA and CD163mRNA were significantly higher in DPL pigs compared to uninfected ones (P<0.05), whereas these levels were not different in DLY pigs or between infected DPL and DLY pigs;3). The segment of-282--94bp in the upstream of5’ regulatory region of CYP3A88gene could stimulate transcription. One nuclear receptor (PPARα)binding site in this segment was predicted by TESS. The mutation (-78T>A) in the important regulatory region likely caused the binding of transcription factor YY1. When YY1binding site is mutated, the promoter activity in driving transcription was reduced significantly (P<0.05).In conclusion, our preliminary study revealed that the different expression of PRRSV mediator genes is likely related to pig resistance to PRRSV. No structural difference in the porcine NMMHC-IIA protein exists among Laiwu, Dapulian and Duroc×Yorkshire×Landrace crossbred commercial pigs. A fragment of2939bp in the5′regulatory region of porcine CYP3A88gene was obtained and YYlcould stimulate transcription of CYP3A88gene.