鸡PPARγ基因转录本3上游开放阅读框转录后的调控作用  

Post-transcriptional regulation of chicken PPARγ transcript variant 3 by upstream open reading frame

作　　者：褚衍凯 靳艳飞 邢天宇 马广伟[1,2,3] 崔婷婷 闫晓红[1,2,3] 李辉 王宁 Yankai Chu;Yanfei Jin;Tianyu Xing;Guangwei Ma;Tingting Cui;Xiaohong Yan;Hui Li;Ning Wang(Key Laboratory of Chicken Genetics and Breeding,Ministry of Agriculture,Harbin 150030,China;Key Laboratory of Animal Genetics,Breeding and Reproduction,Education Department of Heilongjiang Province,Harbin 150030,China;College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]农业部鸡遗传育种重点实验室,哈尔滨150030 [2]黑龙江省普通高等学校动物遗传育种与繁殖重点实验室,哈尔滨150030 [3]东北农业大学动物科学技术学院,哈尔滨150030

出　　处：《遗传》2018年第8期657-667,共11页Hereditas（Beijing)

基　　金：国家自然科学基金项目(编号:31572392);农业部产业体系项目(编号:CARS-41)资助~~

摘　　要：过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)是脂肪生成的关键调控因子。本实验室前期研究发现,与人和鼠等哺乳动物PPARγ基因的转录本不同,鸡PPARγ基因的多个转录本5′UTR区存在上游开放阅读框(upstream open reading frames,u ORFs)。为了揭示该u ORF转录后的调控作用,本研究构建了鸡PPARγ基因转录本3(c PPARγ3)野生型5′UTR报告基因载体psi CHECK2-c PPARγ3-5′UTR-WT和u ORF突变(u ATG突变为终止密码子TGA)的5′UTR报告基因载体psi CHECK2-c PPARγ3-5′UTR-Mut。将这两个报告基因载体分别转染永生化鸡前脂肪细胞(immortalized chicken pre-adipocytes,ICPA)和鸡胚成纤维细胞DF1,检测海肾荧光素酶报告基因h Rluc活性及其m RNA表达。荧光素酶报告基因检测结果显示,在ICPA细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut的h Rluc报告基因活性极显著高于psi CHECK2-c PPARγ3-5′UTR-WT(P<0.01);在DF1细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut的h Rluc报告基因活性高于psi CHECK2-c PPARγ3-5′UTR-WT,但差异不显著(P>0.05)。q RT-PCR检测h Rluc基因m RNA表达结果显示,与psi CHECK2-c PPARγ3-5′UTR-WT相比,在ICPA细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut转染细胞的h Rluc基因的m RNA表达水平极显著降低(P<0.01);在DF1细胞中,psi CHECK2-c PPARγ3-5′UTR-Mut转染细胞后,h Rluc基因的m RNA表达水平也降低,但差异不显著(P>0.05)。为进一步分析该u ORF对鸡c PPARγ3的转录后调控作用,本研究又分别构建了野生型c PPARγ3真核表达载体pc DNA3.1-c PPARγ3-WT和u ORF突变的c PPARγ3真核表达载体pc DNA3.1-c PPARγ3-Mut。q RT-PCR检测c PPARγ3的m RNA表达水平,结果显示,在这两种细胞中,pc DNA3.1-c PPARγ3-Mut转染细胞的c PPARγ3 m RNA表达水平均显著低于pc DNA3.1-c PPARγ3-WT转染细胞(P<0.05),但Western blot结果显示,pc DNA3.1-c PPARγ3-Mut转染细胞的PPARγ蛋白表达水平极显著高于pc DNA3.1-c PPARγ3-WT转染细胞(P<0.0Peroxisome proliferator-activated receptor gamma(PPARγ)is a critical regulator of adipogenesis.Our previous study showed that unlike human and mouse PPARγtranscripts,several chicken PPARγtranscript variants contain upstream open reading frames(uORFs)in their 5′untranslated region(5′UTR).To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγgene,we constructed wild-type(psiCHECK2-cPPARγ3-5′UTR-WT)and a uORF mutant(the upstream ATG(uATG)was mutated to stop codon TGA)5′UTR reporters(psiCHECK2-cPPARγ3-5′UTR-Mut)of chicken PPARγtranscript variant 3(cPPARγ3).These two reporters were individually transfected into immortalized chicken pre-adipocytes(ICPA)and DF1 cells,and the renilla luciferase(hRluc)activity and mRNA expression level were detected by reporter assay and qRT-PCR.The results showed that the hRluc activity of the mutated 5′UTR was significantly higher than that of the wild-type 5′UTR in ICPA cells(P<0.01),and the hRluc activity of the mutated 5′UTR tended to be higher than that of the wild-type 5′UTR in DF1 cells,but this difference did not reach statistical significance(P>0.05).The qRT-PCR analysis showed,in ICPA cells,the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5′UTR construct than in the cells transfected with the wild-type 5′UTR construct(P<0.01).In DF1 cells,the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5′UTR construct than in the cells transfected with the wild-type 5′UTR construct,but this difference did not reach statistical significance(P>0.05).To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF,we constructed the expression plasmids bearing the full-length coding region of chicken PPARγgene plus either wild-type or mutant uORF 5′UTR(pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut).These two constructed PPARγexpression plasmids were individually transiently transfected into both ICPA and DF1 cells,and PPARγmRNA

关 键 词：鸡 PPARΓ基因 上游开放阅读框 翻译抑制 

分 类 号：S831.2[农业科学—畜牧学]