作 者：
辛晓萍;王家迎;张爱玲;钟玉宜;何颖婷;陈赞谋;张哲;张豪;李加琪;袁晓龙
单 位：
广东省农业动物基因组学与分子育种重点实验室/国家生猪种业工程技术研究中心/华南农业大学动物科学学院
关键词：
猪;卵巢颗粒细胞;Kiss1基因;CEBPα;p53
摘 要：
[目的]预测母猪Kiss1(GenBank Gene ID:100145896)上游区域潜在的转录因子结合位点,并验证部分转录因子在猪卵巢颗粒细胞中对Kiss1基因的调控作用,为研究Kiss1基因在母猪卵巢颗粒细胞中的分子调控机制提供理论基础.[方法]参考NCBI数据库中Kiss1基因上游区域的序列,通过生物信息学网站预测的Kiss1基因上游区域潜在转录因子结合的位点,结合文献阅读与资料查询,在转录因子CEBPα和p53与Kiss1基因上游区域潜在结合位点附近设计引物,利用染色质免疫共沉淀(ChIP)技术,验证转录因子CEBPα和p53与Kiss1基因上游区域的结合情况;参照NCBI数据库相关转录因子CEBPα和p53的mRNA序列,使用软件Primer Premier5设计引物,PCR分别扩增转录因子CEBPα(带有Kpn I和Xho I限制性内切酶的酶切位点)和p53(带有Kpn I和Hind III限制性内切酶的酶切位点)的CDS区序列,并进行测序鉴定,连接到真核表达载体pcDNA3.1(+)上,构建含有潜在转录因子CEBPα和p53 CDS区序列的真核表达载体,并获得无内毒素质粒,分别命名为pcDNA3.1-CEBPα和pcDNA3.1-p53;化学合成目标转录因子CEBPα和p53的干扰siRNA片段.屠宰场采集猪的卵巢,快速分离并培养原代猪的卵巢颗粒细胞,阳离子脂质体转染法分别将转录因子CEBPα和p53真核表达载体(pcDNA3.1-CEBPα和pcDNA3.1-p53)或siRNA片段(CEBPα-siRNA和p53-siRNA)转染进母猪卵巢颗粒细胞,使用实时荧光定量PCR和Western Blot技术分别验证预测的转录因子CEBPα和p53对Kiss1基因mRNA水平和蛋白水平的影响.[结果]生物信息学预测结果表明,Kiss1基因上游区域(-850-+221)存在p53(肿瘤抑制蛋白,tumor protein p53,p53)、CCAAT增强子结合蛋白(CCAAT/enhancer binding protein, CEBP)、信号传导及转录活化家族Stat4(signal transducer and activator of transcription 4, Stat4)等转录因子的潜在结合位点,其中转录因子p53(GenBank Gene ID:397276,NM213824.3)可能结合在Kiss1基因上游区域-533--523 bp处,转录因子CEBPα(GenBank Gene ID:397307,XM003127015.4)可能结合在Kiss1基因上游区域-744--733bp处;ChIP结果表明,转录因子p53和CEBPα可以特异性的结合在Kiss1基因上游区域-533--523 bp和-744--733 bp处;在母猪卵巢颗粒细胞中超表达转录因子p53或CEBPα后,Kiss1基因的mRNA的表达水平显著下降(P<0.05),蛋白水平显著下降(P<0.05);在母猪卵巢颗粒细胞中,干扰转录因子p53或CEBPα后,Kiss1基因的mRNA的表达水平显著升高(P<0.05),蛋白水平显著升高(P<0.05).[结论]在猪卵巢颗粒细胞里,转录因子p53和CEBPα能结合到Kiss1基因的上游区域降低其启动子转录活性,进而降低其mRNA和蛋白表达水平.
作 者：
XIN XiaoPing;WANG JiaYing;ZHANG AiLing;ZHONG YuYi;HE YingTing;CHEN ZanMou;ZHANG Zhe;ZHANG Hao;LI JiaQi;YUAN XiaoLong;Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science,South China Agricultural University;Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute,Guangdong University of Education;
单 位：
XIN XiaoPing%WANG JiaYing%ZHANG AiLing%ZHONG YuYi%HE YingTing%CHEN ZanMou%ZHANG Zhe%ZHANG Hao%LI JiaQi%YUAN XiaoLong%Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science,South China Agricultural University%Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute,Guangdong University of Education
关键词：
pig;;ovarian granulosa cells;;Kiss1;;CEBPα;;p53
摘 要：
【Objective】The objectives of this study were to predict the potential transcription factors in the upstream region of Kiss1, and then to verify the regulatory role of these transcription factors on the expression of Kiss1 in the ovarian granulosa cells of pigs. This study could provide the basic data for the molecular mechanism of Kiss1 in granulosa cells of pigs. 【Method】 By using the bioinformatic software, we predicted the potential binding site of transcription factors in the upstream region of Kiss1 in NCBI database. In addition, we reviewed a lot of relevant papers and references, and then CEBPα and p53 were selected as potential binding transcriptional factors in the upstream region of Kiss1 gene. The primers were designed near the potential binding sites in the upstream region of Kiss1 genes, and verified the binding of transcriptional factors CEBPα and p53 to the upstream region of Kiss1 gene by ChIP. According to the m RNA sequences of CEBPα and p53 in NCBI database, Primer Premier 5 software was used to design primers, and CDS regions of CEBPα(containing Kpn I and Xho I sites) and p53(containing Kpn I and Hind III sites) were amplified by PCR and identified by sequencing. Then the CDS regions were connected to the eukaryotic expression vector pcDNA3.1, and the constructed plasmids were extracted with endotoxin-freely, named pcDNA3.1-CEBPα and pcDNA3.1-p53. The interfering siRNA fragments of CEBPα and p53 were synthesized through the chemical method. Pig ovaries were collected from the slaughterhouse, and the ovarian granulosa cells were isolated and cultured. The eukaryotic expression vector or siRNA fragment was transfected into ovarian granulosa cells by cationic liposome, and the effects of p53 and CEBPα on the expression of Kiss1 were verified by the real-time fluorescence quantitative PCR and Western Blotting, respectively. 【Result】 The bioinformatic prediction indicated that there were putative binding sites of p53(tumor protein p53, p53), CEBP(CCAAT/enhancer binding protein, CEBP),Stat4(signal transducer and activator of transcription 4, Stat4) and other potential of transcription factor binding sites in the upstream(-850-+221) of Kiss1. And the putative binding site of CEBPα(GenBank Gene ID: 397307, XM_003127015.4) were found in-744—-733 bp region and the putative binding site of p53(GenBank Gene ID: 397276, NM_213824.3) were found in-533—-523 bp region. The results of ChIP showed that p53 and CEBPα were bounded at-533—-523 and-744—-733 of Kiss1, respectively. After overexpression of p53 or CEBPα, both mRNA and protein expression level of Kiss1 significantly decreased(P<0.05). Furthermore,both mRNA and the expression level of Kiss1 increased significantly(P<0.05) by interfering p53 or CEBPα. 【Conclusion】 In pigs,p53 and CEBPα could bind at the upstream region of Kiss1 to inhibit its expression in ovarian granulosa cells.
