| The Nramp1/Slc11a1 locus encodes a proton-coupled divalent cation transporter, expressed in late endosomes/ lysosomes of macrophages, that constitutes a component of the innate immune response to combat intracellular pathogens and it was investigated to play an important role in regulating inherent immunity. The studies demonstrated some high degree of polymorphism in the Nramp1 gene of different species. The previously identified Z-DNA forming polymorphic repeat (GT)n in the promoter region of the human Nramp1 gene does act as a functional polymorphism influencing gene expression. The INF-γ,TNF-α,IL-1βand bacteria LPS could increase the level of Nramp1 expression. To research the molecule mechanisms of the gene regulation and expression, the bovine Nramp1 5′flanking region(-1748～+769) has been cloned and the sequence has been analyzed by bioinformatic tools. Then the promoter activity was identified in RAW264.7 cell and 293T cell using the EGFP reporter gene. To find the core promoter and the cis-acting elements, deletion analysis of promoter was performed. And we detected the polymorphisms of the 5′flanking region by sequencing,CRS-PCR-RFLP motheds and did the analysis of the SNPs.In this research, we cloned the 2517bp DNA sequence of bovine Nramp1 5′flanking region by LA-PCR method. The analysis of this 2517bp fragment indicated that there was no canonical TATA box. Comparative sequence alignment across species showed that the proximal promoter region was the most conserved region, whereas the distal promoter region was more divergent. Enrichment in G+C dinucleotides with about 66% overall G+C content in the proximal promoter region(within 140 bp of nucleotide position -90 to +50) was observed.Two CpG island (-580bp～-460bp and -132bp～+111bp),two short interspersed nuclear elements (-951～-757 and -609～-495)and one DNA repetitive element (-1155～-855)within promoter region were also found. Of particular interest is the fact that several well-documented transcription factor consensus motifs are precisely conserved across species in the region immediately upstream of the Nramp1 exon 1, including binding sites for IRE-1,NF–IL/6, PU.1, SP1,c-Myb et al. Multiple of the binding sites for AP1,GR,IRE-2 were also found. pEGFP-N1/2517 plasmid has been constructed and transfetced into RAW264.7 and 293T cells by Lipofectamine 2000,expression of EGFP was obtained. It implies that the 2517bp of bovine Nramp1 5′flanking region is promoter functional.To further characterize the 5′flanking region, We constructed a set of luciferase reporter gene constructs containing successive 5′deletions of the bovine Nramp1 promoter. Promoter activity analysis was performed by dual-luciferase reproter assay system. The core promoter of the gene was located at the +58~-89bp. Some positive regulatory elements are located at -89～-205bp and -278～-1495bp. According to the results of Bioinformatic analysis of the squence, it suggested that some elements might play an important role in the activity of bovine Nramp1 promoter such as SP1,PU.1 AP-1. And the results also showed the presence of repressor elements in this region -205～-278bp,intron1 and -1495～-1748bp. The promoter activity of -1748～+58 treated by LPS for 30h was increased significantly in RAW264.7 cells. Further study of the LPS induction indicated that LPS-responsive regions were located within -1495～1748bp,-278～-205bp. And AP-1 was suggested a major mediator of effect of LPS.In this research, five SNPs including -18 C/A,-185T/C,-336 G /A,-451C/T,-679G/A were examined by sequencing, and -20C/T was reported by Martinez R(2008). Based on the bioinformatic analysis, the SNPs were further surveyed. The mutation of -18, -20, -451, and -336 position may play an important role because they can change the binding of the transcription factors including SP1, PU.1, GR, IBP-1 et al. The allelic and genotypic frequency of -20 C/T,-185 T/C,-451 C/T were detected by CRS-PCR-RFLP method. The mutation gene frequence were 4.2%,1.2%,1.9%,respectively. And the Chi-square test for goodness of fit showed that the frequencies of the Nramp1 genotypes in the population of the Chinese holstein cattle tested agreed with Hardy-Weinberg. Thus, the present study provides an initial effort to explore the molecular mechanism of transcriptional activation of the bovine Nramp1 gene and should facilitate further studies to decode the complex regulatory process and to the molecular breeding for disease resistance of bovine.
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