Preliminary Identifying Regulatory Elements In The Promotor Of Chicken Piwil1

PIWI(P-element-induced wimpy testis)is a branch of the Argonatute family mainly existed in germlines, and plays an important role in germline stem cell maintenance and spermatogenesis. Since PIWI has been first discovered in Drosophila, it subsequently found in mouse, human and other animals, but related studies on poultry has been rarely reported. Previous studies have found differences in temporal and spatial expression level of Piwill transcription. To investigate the mechanism of transcriptional regulation in poultry, Langshan chickens were selected as model animals. According to the Piwill gene promoter sequence, a series of promoter missing mutants were directly subcloned into pGL3-Basic vector.The transcriptional activity of the recombinant plasmids was detected by Dual-Glo(?) Luciferase Assay System after transfecting COS-7and GC-1cells, aiming to the core promoter. Subsequently, we predicted the transfactor with point mutants and Dual-Glo(?) Luciferase Assay System, and identified the effect of the binding site on the chicken Piwill gene core promoter activity. Langshan testis extract nucleoprotein conducted to determine the binding of transcription factors and Piwill genes by EMSA experiments. Separated PGCs, SSCs, spermatogonia of langshan chiken, and handled spermatogonial cells with colchicine, then extracted nuclear protein of the four kinds of cells and take gel mobility shift assay (EMSA) experiment.The results were presented as follows:1. According to the Piwill gene promoter sequences, we constructed seven series deletion vector successfully and the deletion express vector Dual luciferase reporter found that, M1-M5series deletion express vector were expressed in COS-7and GC-1cell line, and the relative luciferase activity of M4series deletion express vector was the highest in both cell lines, while M6-M7hardly expressed, which indicated that:-268/-221bp was the core promoter region of the Piwill.2. In the basis of the core promoter region determination, we conducted point mutagenesis to the possibly existed transcription binding sites of the core promoter region:the mutant recombinant plasmid was constructed successfully and dual luciferase reporter gene show that none mutant recombinant plasmids and mutant recombinant plasmid were both expressed in COS-7and GC-1cell lines, and the former relative luciferase activity was significantly higher than the latter, which futher indicated that:the transcription factor binding sites existed in this region, and played a key role to the promoter activity.3.The results of EMS A trail showed:the designed and labeled probe successfully first, and gel migration trail showed that, transcription factor could bind to the DNA sequence; and super EMSA experiment further showed, transcription factor NF-Y actually existed in the testicular tissue, and could bind with the target DNA fragment specially.4.The gel shift experiments results of the four kinds of cells showed that, transcription factor NF-Y existed in all of the four cells, and combines with the DNA-239/-221sequence regions in Piwill gene promoter. Gray values showed that:in colchicine-treated cells, NF-Y content was the highest; followed spermatogonia and SSCs, while PGCs was the lowest.It can be concluded that:Piwill gene is present throughout the sperm development in poultry, and plays an important role in regulating the differentiation of spermatocytes in spermatogonia.