Cloning In The Promotor Of Goose PIT-1Gene And Preliminary Identificate Regulatory Region

PIT-1(pituitary transcription factor1) gene acts as a positive regulatory factor of GH,PRL and TSH-β, and plays an important role in the initiation of expression of related genes and regulation of early growth in mammal. Therefore, it is selected as a candidate gene for growth trait. In this study, a3.0kb PIT-1promoter region was cloned firstly, then Research from the transcription regulation.1. Cloning and sequence analysis of the promoter region of Goose PIT-1geneA3.0kb PIT-1promoter region was cloned firstly by Gemone Walking approach from Taihu goose genomic template and sequenced then. The result had been submitted to Genebank (No. EU924269). Then the sequence was analyzed with biology software and alignmented with the PIT-1promoter of others animal to construct the phylogenetic tree.The result revealed that the cloned region contained typical TATA box and CAAT box like sequence,and many Transcription Element were calculated, for example, myogenin, TFIID, GATA-1, AP-1, GR, HOXD10, TCF-4E, PRA, Sp1, POU1Fla, POU3F2, GATA-4, Foxj2. The alignment results showed that the conservative region spaned from-200to-1000bp of upstream of transcription start site, and the-200bp upstream of transcription start site was strongly conservative and it was potential core promoter. The phylogenetic tree constructed by MLAGAN program of Vista represneted the evolution relationship among different species.2. The research of PIT-1gene promoter region function identification and transcription regulationWe have cloned promoter region sequence the goose PIT-1gene by genome walking. Using series deletion method to construct a series of the promoter fragment deletion (-2995/-1bp,-1485/-1bp,-1293/-lbp,-1014/-1bp,-775/-1bp,-561/-1bp,-353/-1bp,-206/-1bp), To verify whether the promoter region sequence has promoter activity, we constructed the luciferase reporter vector and used the transient transfection analysis to research the determination of promoter activity and transcriptional regulations preliminarily. and it was found that different segment all had promoter activity, compared with the negative control. In addition, there were differences between promoter activity of fragment deletion of the series promoter, and promoter activity of-561/-1bp segment was strongest. We can deduce that the segment is the core promoter region of this gene preliminarily, as well as this region (353/-1bp-561/-Ibp) contains the cis-acting element of the regulation of gene expression,it maybe TFIID, HOXD10, Spl, POU1F1a, POU3F2, The study provided the basis for further research on the mechanism of transcriptional regulations.