Cloning Of Porcine Pam Nramp1Gene Promoter And Its Functional Verification Of Specific Expression

PAMs are the target cells infected by PRRSV in pigs. It is very important to screen the promoter of pig PAM-specific expression gene, which could iniciate efficient and specific expression of anti-PRRSV exogenous genes in target cells. Without Transcriptional factors, the polymerase can’t initiate transcripiton, and eukaryotic genes would not express. Only if the Transcriptional fators (protein) recognize and bind to the DNA sequences, the genes begin to transcript. Therefore, the investigation of specific gene promoter expression includes both definition of cis-acting elements regions and regulation of transcription factors in vitro.Accumulating evidence indicates that the natural resistance associated macrophage protein1(Nrampl) mRNA can be expressed in mononuclear macrophages with lung and spleen tissues, but the amount of expression is the highest in macrophages. In this study, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) approach was adopted to analyse the tissue expression profile of Nrampl gene and verified the specific expression of Nrampl gene in PAMs.The different length promoters of Nrampl gene were cloned and their function was verified in PAM, Preadipocyte, PK and PEF, by dual-luciferase reporter assay system. We obtained the core promoter region with specific regulatory function. The approaches are as follows:I. The Nrampl gene transcription start site is determined by rapid amplification of cDNA ends (RACE) technique.Nramp1gene transcription start site settles a basis for the study of transcriptional regulation of the gene. Downloading the Nramp1gene mRNA sequence from the NCBI, and gene specific primers were designed for5’RACE. According to Smart RACE kit, Nrampl gene5’untranslated region (UTR) was cloned and A was determined as its transcription start site, which located at90bp upstream of ATG. The sequence data were submitted to the GenBank databases under accession No. HM754433. 2. Deletion expression vector construction of Nrampl gene promoterFirst, the use of bioinformatics methods to find pig Nrampl gene5’regulatory sequences and predict the location of promoter as well as transcription factor binding sites; Second, application of PCR amplified different lengths of promoters fragments, which connected to the firefly luciferase reporter gene pGL3-Basic vector. Eight recombination expression vectors, such us pLUC1430, pLUC1136, pLUC840, pLUC751, pLUC487, pLUC379, pLUC274, pLUC179, were successfully constructed by restriction enzymes digestion and DNA sequencing.3. Activity analysis of deletion promoter and cell-specific verificationThe recombinant firefly luciferase expression vector and Renilla luciferase reporter vector as20:1were co-transfected alveolar macrophages. After24hours, the luciferase activity was detected by dual-luciferase reporter assay system, the value of pRL-TK luciferase activity as an internal control and pGL3-Basic luciferase activity as a basis value, pLUC487as core promoter region was screened by promoters activity analysis. The cell-specific core promoter was verified by transient transfection in other non-macrophages such as kidney cells, fat precursor cells and porcine fetal fibroblast cells, while we get some regulatory elements associated with cell-specific expression.The results showed that pLUC487[-1327～+86] is the core promoter region of Nramplgene specific expression and Sp1, AP-1, c-Ets, C/EBP, GR and other transcription factors were initially identified to play an important role in specific transcription in vitro, by bioinformatics analysis combined with experimental study. We can use the Nrampl gene core promoter and important transcription regulatory elements to start downstream target gene specific expression in PAM, accordingly realize new lines cultivation of transgenic pigs.