Effect Of Chain Mutation Of FSHβ Gene Upstream Regulatory Region On Transcription In Bovine

Preliminary studies in our laboratory found chain mutation of FSHβgene in the upstream regulatory region in five Simmental bull, which include four single-base mutation (-1708 T-C,-1654 C-G,-1526 G-A,-1096 G-A) and an insert mutation (-1468,-1469 GG-TTAACT).In this study, chain mutation of FSHβgene in the upstream regulatory region in bovine as the object of research, research mainly for transcription regulation. In order to clarify the influence of chain mutation of FSHβgene in the upstream regulatory region in bovine on transcription regulation, it has vital significance to better understand the function of FSHβgene,provide important theory basis for making FSHβgene as candidate gene of bull semen quality and fertility and to do marker assisted selection.First ,design PCR primers in each mutation site which is contained in chain mutation respectively, PCR product detected by 1% agarose gel, the size of fragment as expected, Monoclonal sequencing results show that the above chain mutation do exist in FSHβgene upstream regulatory region. Secondly, detect weather each mutation change the binding of transcription factor by chemiluminescence EMSA. Test results indicate that insert mutation (-1468,-1469 GG-TTAACT) in the chain mutation of FSHβgene upstream regulatory region change the binding with transcription factor.Wild type bind with transcription factors in nonspecific mode, while mutant type bind with different type of transcription factors in specific mode, but other sites in the chain mutation don't change the transcription factor binding mode both before and after the mutation., they have no influence on the binding with transcription factor. Finally. Construct FSHβ-PGL3-Basic vector, transfect LβT2 cell, detect Luciferase activity and verdict weather the chain mutation in FSHβgene upstream regulatory region change gene transcription activity. Construct two length and three types of restructuring plasmid respectively, They are wild type, mutant type (including chain mutation), Site-directed mutagenesis type (only including insert mutation)-1864/+ 154 FSHβ-PGL3-Basic and -1864/+702 FSHβ-PGL3-Basic. Luciferase activity detection results indicate that the transcriptional activity of-1864/+ 702 FSHβ-PGL3-Basic is higher than-1864/+154 FSHβ-PGL3-Basic in corresponding types, we infer that+154/+ 702 may exist some transcriptional regulatory element participate in transcption. Mutant type and Site-directed mutagenesis type compared with wild type, transcription activity reduced but has no significant difference, and mutant type and Site-directed mutagenesis type keep the same transcription level. So we predict the chain mutation of FSHβgene upstream regulatory region will not significantly reduce gene transcription activity.and the insert mutation which is contained in chain mutation of FSHβgene upstream regulatory region play a role in reducing gene transcription activity. Overall results show that insert mutation (-1468,-1469 GG-TTAACT) in the chain mutation of FSHβgene upstream regulatory region change the binding with transcription factor.Wild type bind with transcription factors in nonspecific mode, while mutant type bind with different type of transcription factors in specific mode, but other sites in the chain mutation don't change the transcription factor binding mode both before and after the mutation, they have no influence on the binding with transcription factor. The chain mutation of FSHβgene upstream regulatory region will not significantly reduce gene transcription activity.and the insert mutation which is contained in chain mutation of FSHβgene upstream regulatory region play a role in reducing gene transcription activity.