山羊转录因子c-fos克隆、鉴定及功能分析_胡婷婷.pdf

胡婷婷 等/山羊转录因子 c-fos 克隆、鉴定及功能分析 Chinese Journal of Biotechnology http:/ Apr.25,2023,39(4):1684-1695 DOI:10.13345/j.cjb.220708 2023 Chin J Biotech,All rights reserved 资助项目：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)This work was supported by the National Natural Science Foundation of China(32072723,31902154),the Key Project of Sichuan Applied Basic Research Program,China(2022JDTD0030),and the Fundamental Research Funds for the Central Universities,Southwest Minzu University,China(3300222092).*Corresponding author.E-mail: Received:2022-09-04;Accepted:2023-01-13 1684 生物工程学报 山羊转录因子 c-fos 克隆、鉴定及功能分析 胡婷婷1,2，王永1,2，陈定双1,2，龚成思1,2，李艳艳1,2，熊燕1,2，王建梅1,2，李志雄1,2，林亚秋1,2*1 西南民族大学畜牧兽医学院，四川 成都 610041 2 西南民族大学 青藏高原动物遗传资源保护与利用教育部/四川省重点实验室，四川 成都 610041 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.HU Tingting,WANG Yong,CHEN Dingshuang,GONG Chengsi,LI Yanyan,XIONG Yan,WANG Jianmei,LI Zhixiong,LIN Yaqiu.Cloning,identification and functional analysis of the goat transcription factor c-fosJ.Chinese Journal of Biotechnology,2023,39(4):1684-1695.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 0120 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(P0.05)，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(P0.01)；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/EBP 的相对表达水平(P0.01)，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 AP2 和 C/EBP 基因的表达来发挥作用。关键词：转录因子；c-fos；山羊；皮下脂肪；细胞分化 动物及兽医生物技术 胡婷婷 等/山羊转录因子 c-fos 克隆、鉴定及功能分析 ：010-64807509： 1685 Cloning,identification and functional analysis of the goat transcription factor c-fos HU Tingting1,2,WANG Yong1,2,CHEN Dingshuang1,2,GONG Chengsi1,2,LI Yanyan1,2,XIONG Yan1,2,WANG Jianmei1,2,LI Zhixiong1,2,LIN Yaqiu1,2*1 College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,Sichuan,China 2 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 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(P0.05),and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h(P0.01).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/EBP lipogenic marker genes(P0.01).Moreover,AP2 and C/EBP 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/EBP gene expression.Keywords:transcription factor;c-fos;goat;subcutaneous fat;cell differentiation 肌内脂肪(intramuscular fat,IMF)和皮下脂肪(subcutaneous fat,SAT)与肉类品质性状相关，适当的 IMF 含量可以提高肉产品的嫩度、多汁性和风味，而皮下脂肪沉积过多会造成异位脂肪沉积和分泌失调1-2。因此，提高羊肉肌内脂肪沉积程度和减少皮下脂肪沉积是当前肉羊产业的重要研究内容。动物脂肪沉积是脂肪细胞数量增加以及脂肪细胞肥大共同作用的结果，ISSN 1000-3061 CN 11-1998/Q 生物工程学报 Chin J