遗传 ›› 2018, Vol. 40 ›› Issue (8): 657-667.doi: 10.16288/j.yczz.18-038

• 研究报告 • 上一篇    下一篇

鸡PPARγ基因转录本3上游开放阅读框转录后的调控作用

褚衍凯1,2,3,靳艳飞1,2,3,邢天宇1,2,3,马广伟1,2,3,崔婷婷1,2,3,闫晓红1,2,3,李辉1,2,3,王宁1,2,3()   

  1. 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
  • 收稿日期:2018-02-07 修回日期:2018-05-31 出版日期:2018-08-16 发布日期:2018-07-10
  • 通讯作者: 王宁 E-mail:wangning@neau.edu.cn
  • 作者简介:褚衍凯,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail: chuyankai1220@163.com
  • 基金资助:
    国家自然科学基金项目(31572392);农业部产业体系项目资助(CARS-41)

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

Yankai Chu1,2,3,Yanfei Jin1,2,3,Tianyu Xing1,2,3,Guangwei Ma1,2,3,Tingting Cui1,2,3,Xiaohong Yan1,2,3,Hui Li1,2,3,Ning Wang1,2,3()   

  1. 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China
    3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
  • Received:2018-02-07 Revised:2018-05-31 Online:2018-08-16 Published:2018-07-10
  • Contact: Wang Ning E-mail:wangning@neau.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(31572392);the China Agriculture Research System(CARS-41)

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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, uORFs)。为了揭示该uORF转录后的调控作用,本研究构建了鸡PPARγ基因转录本3 (cPPARγ3)野生型5′UTR报告基因载体psiCHECK2-cPPARγ3- 5′UTR-WT和uORF突变(uATG突变为终止密码子TGA)的5′UTR报告基因载体psiCHECK2-cPPARγ3- 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hRluc活性及其mRNA表达。荧光素酶报告基因检测结果显示,在ICPA细胞中,psiCHECK2-cPPARγ3-5′UTR-Mut的hRluc报告基因活性极显著高于psiCHECK2- cPPARγ3-5′UTR-WT (P<0.01);在DF1细胞中,psiCHECK2-cPPARγ3-5′UTR-Mut的hRluc报告基因活性高于psiCHECK2-cPPARγ3-5′UTR-WT,但差异不显著(P>0.05)。qRT-PCR检测hRluc基因mRNA表达结果显示,与psiCHECK2-cPPARγ3-5′UTR-WT相比,在ICPA细胞中,psiCHECK2-cPPARγ3-5′UTR-Mut转染细胞的hRluc基因的mRNA表达水平极显著降低(P<0.01);在DF1细胞中,psiCHECK2-cPPARγ3-5′UTR-Mut转染细胞后,hRluc基因的mRNA表达水平也降低,但差异不显著(P>0.05)。为进一步分析该uORF对鸡cPPARγ3的转录后调控作用,本研究又分别构建了野生型cPPARγ3真核表达载体pcDNA3.1-cPPARγ3-WT和uORF突变的cPPARγ3真核表达载体pcDNA3.1-cPPARγ3-Mut。qRT-PCR检测cPPARγ3的mRNA表达水平,结果显示,在这两种细胞中,pcDNA3.1-cPPARγ3-Mut转染细胞的cPPARγ3 mRNA表达水平均显著低于pcDNA3.1-cPPARγ3-WT转染细胞(P<0.05),但Western blot结果显示,pcDNA3.1-cPPARγ3-Mut转染细胞的PPARγ蛋白表达水平极显著高于pcDNA3.1-cPPARγ3-WT转染细胞(P<0.01)。这些研究结果表明,5′UTR区的uORF抑制鸡cPPARγ3的翻译。

关键词: 鸡, PPARγ基因;, 上游开放阅读框, 翻译抑制

Abstract:

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript 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 PPARγ gene, 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 PPARγ transcript 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 PPARγ gene plus either wild-type or mutant uORF 5′UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5′UTR of the cPPARγ3 inhibits its translation.

Key words: chicken, PPARγ gene;, uORF, translational repression