山羊转录因子c-fos克隆、鉴定及功能分析
作者:
基金项目:

国家自然科学基金(32072723,31902154);四川省应用基础研究计划重点项目(2022JDTD0030);西南民族大学中央高校基本科研业务费专项资金(3300222092)


Cloning, identification and functional analysis of the goat transcription factor c-fos
Author:
  • HU Tingting

    HU Tingting

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • WANG Yong

    WANG Yong

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • CHEN Dingshuang

    CHEN Dingshuang

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • GONG Chengsi

    GONG Chengsi

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • LI Yanyan

    LI Yanyan

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • XIONG Yan

    XIONG Yan

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • WANG Jianmei

    WANG Jianmei

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • LI Zhixiong

    LI Zhixiong

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • LIN Yaqiu

    LIN Yaqiu

    College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, Sichuan, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education, Sichuan Province, Southwest Minzu University, Chengdu 610041, Sichuan, China
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  • 摘要
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  • 参考文献 [28]
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    摘要:

    转录因子c-fos在细胞增殖、分化和肿瘤形成中发挥着重要作用。本研究旨在获得山羊c-fos基因序列并阐明其生物学性质,进一步利用过表达手段揭示c-fos在山羊皮下脂肪细胞分化过程中的调控作用。本试验以简州大耳羊为研究对象,用逆转录PCR (reverse transcription-polymerase chain reaction, RT-PCR)技术克隆获得c-fos基因序列并分析其生物学性质;用实时荧光定量PCR (quantitative PCR, qPCR)技术检测c-fos基因在山羊心、肝、脾、肺、肾、皮下脂肪和背最长肌7个组织以及皮下脂肪细胞诱导分化的0-120 h的表达情况;用基因重组方法构建山羊pEGFP-c-fos过表达载体并转染至皮下前体脂肪细胞诱导分化后,用油红O和Bodipy染色从形态学上观察其对脂滴积聚的影响,用qPCR技术在mRNA水平上检测过表达c-fos对成脂分化标志基因的影响。结果表明,克隆的山羊c-fos基因长1 477 bp[编码区(coding sequence, CDS) 1 143 bp],编码380个氨基酸,亚细胞定位预测该基因主要分布于细胞核中;表达蛋白具有一个碱性亮氨酸拉链结构;c-fos基因在山羊皮下脂肪组织相对表达水平较高(P<0.05),在皮下前体脂肪细胞诱导分化后48 h的相对表达水平最高(P<0.01);过表达c-fos可抑制山羊皮下脂肪细胞的脂滴形成,并极显著降低成脂标志基因AP2和C/EBPβ的相对表达水平(P<0.01),且启动子结合位点预测存在多个结合位点。综上所述,c-fos是山羊皮下脂肪细胞分化的负调控因子,通过调控AP2C/EBPβ基因的表达来发挥作用。

    Abstract:

    C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P<0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P<0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P<0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

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胡婷婷,王永,陈定双,龚成思,李艳艳,熊燕,王建梅,李志雄,林亚秋. 山羊转录因子c-fos克隆、鉴定及功能分析[J]. 生物工程学报, 2023, 39(4): 1684-1695

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  • 收稿日期:2022-09-04
  • 录用日期:2023-01-13
  • 在线发布日期: 2023-04-14
  • 出版日期: 2023-04-25
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