鸡腹部脂肪组织生长发育过程KLF7基因表达调控机制研究

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.

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。

腹部脂肪过多蓄积降低肉鸡养殖利润。KLF7是肉鸡腹部脂肪形成重要调控基因。本课题从DNA甲基化、转录调控和mRNA稳定性入手对鸡KLF7的表达调控机制进行了研究。在DNA甲基化水平：采用黄羽快大型肉鸡为实验材料，用焦磷酸处理和质谱分析研究了鸡脂肪组织生长发育过程中KLF7 上2个CpG岛甲基化情况，并且研究了它们与KLF7 mRNA表达水平的相关性。结果显示KLF7启动子区的甲基化与KLF7的mRNA表达水平显著负相关（P<0.05），回归分析显示该因素对KLF7 mRNA表达水平的作用占了其表达水平均值的11.7%。相关性分析显示，腹部脂肪组织中KLF7表达水平与黄羽肉鸡血液中血糖和TG含量负相关(P<0.05）。但是没有发现KLF7启动子区甲基化水平与肉鸡血糖和TG含量存在直接统计学相关性(P>0.05）。在转录调控水平：利用荧光素酶报告基因和表达分析技术研究了多组顺式调控元件和反式作用因子对KLF7表达的调控。构建了5个不同长度的KLF7启动子荧光素酶报告基因质粒，并在鸡前脂肪细胞和鸡胚成纤维细胞DF1中对它们进行了活性分析，结果显示这5个不同长度的鸡KLF7启动子都有活性。此外，本课题组利用多种分子生物学手段对鸡前脂肪细胞中可能对KLF7表达进行调控的转录因子进行了初步研究，研究过的候选转录因子包括PPARγ、C/EBPα、C/EBPβ、C/EBPz、SREBP1、GATA2、GATA3、KLF2和KLF3等。结果显示GATA2和GATA3可能在脂肪细胞中调控KLF7表达。mRNA稳定水平：针对富含miRNA结合位点的KLF7 mRNA序列构建了3个pSi-Check2报告基因质粒，并在鸡前脂肪细胞中研究了它们的活性，结果显示获得的3个富含miRNA结合位点的序列均可调控报告基因活性（P<0.05）。本课题研究结果初步明确了KLF7启动子区甲基化对其mRNA表达的作用；鉴定出了一个长度约150 bp的KLF7核心启动子；找到了2个在鸡脂肪细胞中调控KLF7表达的转录因子（GATA2和GATA3），构建了3个能用于研究KLF7 mRNA稳定性的3’UTR荧光素酶报告基因载体。本课题的研究结果为进一步完善鸡腹部脂肪形成的分子调控网络和研究功能基因KLF7的表达调控机制提供了工具和研究基础。