鸡FBXO38转录剪接体1的克隆、表达和功能分析

Cloning, Expression and Function Analysis of Chicken FBXO38t1

F-box蛋白（F-box proteins）广泛存在于真核生物，并且成员众多，功能多样．FBXO蛋白是一类C端没有亮氨酸重复序列（leucine-rich repeats，LRRs）和WD重复序列（WD repeats）的F-box蛋白．本研究利用PCR和克隆测序的方法从鸡腹部脂肪组织获得一个未知全长编码区序列（coding sequence，CDS），序列分析显示，该序列是鸡FBXO38的一个转录体，本研究将其命名为鸡FBXO38基因转录本1（gallus gallus FBXO38 transcript variant 1，gFBXO38t1）．Real-time reverse transcriptase-PCR分析显示，gFBXO38t1广泛表达于肉鸡多种组织，其中胰腺、回肠和腹部脂肪组织中表达水平较高．进一步分析发现，gFBXO38t1在高、低脂系1～12周龄肉鸡腹部脂肪组织中均有表达，并且在3和4周龄时，低脂肉鸡腹部脂肪组织中gFBXO38t1的表达水平显著高于高脂肉鸡（P 〈 0.05），其余时间点两系间没有显著差异（P 〉 0.05）．在鸡前脂肪细胞（stromal- vascular cells，SV）诱导分化过程中，gFBXO38t1的表达量随着脂肪细胞分化而呈现下降趋势，此外，gFBXO38t1在前脂肪细胞中的表达量明显高于成熟脂肪细胞（fat cells，FC；P 〈 0.05），暗示gFBXO38t1可能是鸡脂肪组织形成的负调控因子．报告基因分析显示，过表达gFBXO38t1抑制CCAAT增强子结合蛋白α（CCAAT/enhancer-binding protein α，C/EBPα）、脂蛋白脂酶（lipoprotein lipase，LPL）、脂肪酸合成酶（fatty acid synthase，FASN）和脂肪酸结合蛋白4（fatty acid-binding protein 4，FABP4）基因的启动子活性（P 〈 0.05），从另一个角度提示了gFBXO38t1对鸡脂肪组织形成具有抑制作用．同时过表达鸡KLF7和gFBXO38t1并不能显著增强过表达gFBXO38t1对鸡LPL、FASN和FABP4基因启动子活性的调控作用，暗示gFBXO38t1对这些基因的调控不完全依赖于鸡转录因子KLF7．本研究为进一步研究鸡脂肪细胞分化和FBXO38的生物学功能提供参考．

F-box proteins are widely present in eukaryotes, and their biological functions are diverse in animals. In this study, the full-length coding sequence of an unnamed gene LOC416151 （GenBank accession XM_414482） was cloned from the chicken abdominal adipose tissue by reverse transcriptase （RT）-PCR, and the sequence analysis showed that the acquired sequence （GenBank accession. JX290204） is one of the transcript variants of chicken FBXO38, which was designated as gFBXO38t1. Real-time RT-PCR analysis showed that gFBXO38t1 was widely expressed in various chicken tissues, with a relatively higher expression level in the pancreas, ileum and abdominal fat tissue. In addition, gFBXO38t1 was expressed in all the chicken abdominal fat tissues used in the present study, and at 3 and 4 weeks of age, the gFBXO38t1 expression in lean males was significantly greater than that in fat males （P 〈 0.05） and no significant difference was observed at the other ages （P 〉 0.05）. The gFBXO38t1 expression decreased followed by the differentiation of chicken preadipocytes induced by oleate, and gFBXO38t1 expressed more highly in chicken preadipocytes than in mature adipocytes （P 〈 0.05）, suggesting that gFBXO38t1 might play a negative role in the chicken adipogenesis. Additionally, The luciferase reporter assay showed that gFBXO38t1 overexpression inhibited the promoter activities of chicken CCAAT/enhancer-binding protein α （C/EBPα）, lipoprotein lipase （LPL）, fatty acid synthase （FASN） and fatty acid-binding protein 4 （FABP4）. The combined overexpression of gKLF7 and gFBXO38t1 did not lead to an enhanced ability for gFBXO38t1 to regulate the promoter activities of chicken LPL, FABP4 and FASN, indicating that gFBXO38t1 may regulate promoter activities of these genes through a KLF7-independent manner. The current study provides evidence that the gFBXO38t1 is involved in chicken adipogenesis.