山羊转录因子c-fos克隆、鉴定及功能分析

Cloning, identification and functional analysis of the goat transcription factor c-fos

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AP2和C/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.