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Cloning In The5’ Regulatory Region Of Tai Hu Goose CYP7A1Gene And Preliminary Identification Of P53Bingding Site

Posted on:2014-03-14Degree:MasterType:Thesis
Country:ChinaCandidate:X DuFull Text:PDF
GTID:2250330425950826Subject:Forest Protection
Abstract/Summary:PDF Full Text Request
Cholesterol7α-Hydroxylase (CYP7A1) is also called cholesterol7α-monooxygenase orcytochrome P4507a1enzymes. CYP7A1is a cytochrome P450heme enzyme, belongs only tothe endoplasmic reticulum memebrane of liver cells. CYP7A1catalyzes decomposition ofcholesterol to generate7α-sterol.7α-sterol becomes primary bile acid by sterol nuclear reduction,hydroxylation, side chain Chemical bonds broken and addition of coenzyme A. This enzyme isalso the key enzyme that can limit the speed of related metabolic pathways. Numerous studiesdemonstrated that the balance between cholesterol and bile acid is important to the body health,high blood cholesterol levels will cause diseases such as hypercholesterolemia, atherosclerosis orgallstones, and high bile acid levels will lead to the occurrence of gallbladder cancer orcolorectal cancer. The expression of CYP7A1gene is regulated by a cascade network oftranscription activation/inhibition, this network is constituted by many factors, including singlenucleotide polymorphisms of its own. Such a network maintains the body balance betweencholesterol and bile acid. Curently, in the field of livestock and poultry, the research ofCYP7A1gene is not enough in-depth, especially at the research of transcription level. Researchresults showed that there might be some kind of relationship between CYP7A1gene and p53protein in the process of liver cell proliferation, fatty degeneration and cell apoptosis. In thisstudy, we cloned the5’ promoter region sequence of CYP7A1gene, and did the preliminary studyon the binding sites of P53protein and the regulation.Using genome walking technology, our study cloned Goose CYP7A1genen5’control regionfor the first time. It includes2004bp. We analysised the sequence and forecasted the functionregion and transcriptiona factor binding sites by online softwares, the number of P53bindingsites is6.A series of deletion mutants of CYP7A1gene5’control region were made in this study.We constructed pGL vectors by combining the mutants and dual luciferase report geneexpression vector, and tranfected them to DF-1cells by liposome transfection, as well aspTK-Renilla plasmid and P53eukaryotic expression plasmid p3XFLAG-P53or pET-His.The P53binding site in CYP7A1gene5’ control region and the regulation of P53were initiallydetermined by detecting efficiency of the report gene in different recombinant.
Keywords/Search Tags:Goose, CYP7A1gene, Genome walking, promoter
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