鸡腹部脂肪组织生长发育过程KLF7基因表达调控机制研究

项目名称： 鸡腹部脂肪组织生长发育过程KLF7基因表达调控机制研究

项目编号： No.31501947

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 畜牧学与草地科学

项目作者： 张志威

作者单位： 石河子大学

项目金额： 22万元

中文摘要： KLF7是调控肉鸡腹脂沉积的重要转录因子，在腹部脂肪生长发育不同阶段表达水平差异显著。本项目将利用重亚硫酸盐测序分析肉鸡腹部脂肪生长发育过程KLF7基因CpG岛的DNA甲基化变化情况。克隆鸡KLF7转录起始位点附近2 000 bp序列，利用载体构建、缺失突变和报告基因检测技术，鉴定KLF7核心启动子序列和重要顺式调控元件；预测调控鸡KLF7表达的转录因子，通过共转染和报告基因分析、表达分析、EMSA和ChIP等方法研究前脂肪细胞中预测转录因子对KLF7表达的调控作用。克隆鸡KLF7基因3'UTR序列，利用缺失突变和报告基因技术，研究3'UTR对KLF7表达的调控作用；预测3'UTR的互作miRNA，在前脂肪细胞通过过表达和Anti-miRNA技术验证miRNA对KLF7的调控作用；以期从DNA甲基化、转录调控、mRNA稳定性水平揭示鸡腹部脂肪组织生长发育过程KLF7表达的调控机制。

中文关键词： 鸡；腹部脂肪；Krüppel-like；factor；7；DNA甲基化；转录调控

英文摘要： Krüppel-like factor 7 (KLF7) is a candidate gene for chicken fatness traits selection. Previous function study showed that, KLF7 regulated the development of abdominal adipose tissue, and during the growth and development of chicken abdominal adipose, KLF7 expression level in the abdominal adipose was significantly different at various time points. To reveal the expression regulation model of KLF7 and accelerate the application of KLF7 on the lean broiler breeding, the methylation pattern of KLF7 gene during the growth and development of chicken abdominal adipose, and the effect of Cis-regulatory elements in the promoter region and 3'Untranslated Region (3'UTR) sequence on the expression of KLF7 in adipocytes from abdominal adipose tissue will be studied in the current study. In briefly, The Bisulphite sequencing will be used to analyze the methylation pattern of KLF7 gene during the growth and development of chicken abdominal adipose. In addition, the regulation function of 2 000 bp sequence upstream chicken KLF7 translation start site, which has been showed promoter activity in our previous study, will be further analyzed using luciferase assay and cut mutation analysis. Bioinformatics method will be used to predict transcription factor binding site on this sequence, and co-transfection, luciferase assay, expression analysis, EMSA and ChIP will be used to study the role of candidate transcription factors on the expression of KLF7 in preadipocytes. Additionally, the expression regulation function of 3'-UTR will be studied using luciferase assay and mutation analysis. Bioinformatics method will be used to predict miRNAs functioned in 3'-UTR, and miRNA overexpression and Anti-miRNA technology will be used to verify the regulation effect of candidate miRNAs on the expression of KLF7 in preadipocytes.

英文关键词： Chicken;Abdominal adipose ;Krüppel-like factor 7;DNA methylation;Transcription regulation