山羊卵巢维持基因FOXL2启动子活性及调控区域分析

Activity and Regulation Region Analysis of Promoter of Goat FOXL2 Gene

旨在研究山羊卵巢维持基因FOXL2启动子活性以及探究该基因的调控机理。从NCBI数据库调取FOXL2基因启动子序列,用生物信息学软件对其核心启动子和转录因子进行预测分析。使用PCR技术克隆FOXL2基因启动子序列,并构建一系列缺失载体,瞬时转染293T和A375细胞,利用双荧光素酶基因检测仪测定相对荧光素酶活性值。结果表明,该基因启动子区域存在两个典型的CpG岛,分别位于(-920/+51(972bp))和(+125/+555(430bp))区域;经KpnⅠ和HindⅢ双酶切鉴定表明,重组载体质粒构建正确;在细胞中插入不同长度的FOXL2基因启动子片段,随着启动子5′端截短,荧光素酶转录活性先升高再逐渐降低。(-934/+324)区域存在转录活性,(-32/+324)区段包含了转录的基本元件;(-934/-456)区域在转录过程中对FOXL2基因起负调控作用,(-456/-192)区域为正调控区域。

The research was designed to study the activity of goat ovarian maintenance gene FOXL2 promoter and explore the gene's regulation mechanism.FOXL2 promoter sequence was retrieved from the NCBI database,bioinformatics software was adopted to predict its core promoter and transcription factors.PCR technology was used to clone FOXL2 gene promoter sequence and construct a series of deletion vectors,293 Tand A375cells were transiently transfected,dual luciferase gene detector was used to measure the relative luciferase activity value.The results indicated that there were 2typical CpG islands in the gene promoter region,which were located at(-920/+51(972bp))and(+125/+555(430bp))regions;the result of KpnI and HindⅢ dual enzyme digestion test suggested that the recombinant plasmid was constructed correctly;FOXL2gene promoter fragments with different lengths were inserted into the cells.When the promoter 5′was truncated,luciferase transcriptional activity firstly increased and then decreased.The result indicate that(-934/+324)region has transcriptional activity,(-32/+324)region contain the basic elements of transcription;(-934/-456)region negatively regulates FOXL2 gene during the transcription process,(-456/-192)region is a positive regulatory region.