滩羊FGF21基因CDS区克隆表达及生物信息学分析
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摘要
为了探究滩羊卷曲被毛时空变化特征,本研究运用基因克隆技术对滩羊FGF21基因的编码区全长序列进行克隆测序,利用生物信息学方法进行序列分析,并对FGF21基因在滩羊不同生长时期皮肤中的表达模式进行分析。结果表明:滩羊FGF21基因的CDS区有1个630 bp的开放阅读框,编码209个氨基酸,其编码蛋白分子量和理论等电点分别为22645.8 ku和6.08 ku,该蛋白不存在跨膜结构,为膜外蛋白,且属于亲水性蛋白;二级结构由α-螺旋、延伸、β-折叠及无规则卷曲4种结构组成;滩羊FGF21基因序列与山羊、牛等物种间同源性较高,所构建系统发生树与其物种同源性关系吻合。半定量RT-PCR分析FGF21基因在滩羊不同组织中分布发现,FGF21仅在肝脏、肺脏、胃和皮肤中表达,在皮肤中表达量低于在肝脏中的表达量,但高于在胃中的表达量;不同时期滩羊皮肤组织荧光定量PCR结果显示,FGF21基因在幼龄组(1月龄)滩羊皮肤中表达量极显著高于成年组(48月龄)表达量(P<0.01),表明FGF21基因有可能是调控滩羊皮肤被毛卷曲差异的候选基因之一。本研究将为滩羊毛发生长的生物学机制研究提供一定的参考价值。
This study aimed to elucidate the structure, physiological function and the expression levels of FGF21 gene on Tan sheep. The FGF21 gene of Tan sheep was amplified through PCR using extracted RNA from skin samples Then the gene was sequenced and analyzed by bioinformatics tools. The CDS region length of cloned Tan sheep FGF21 gene was 630 bp encoded 209 amino acids with a molecular mass of 22645. 8 Da. The secondary of the protein were mainly composed of α-helix, β-turn, extended strand structure and random coli. Phylogenetic tree and sequence alignment showed the FGF21 gene of Tan sheep had a high homology with other species in nucleotide sequence and the gene was relatively conservation. Semi-quantitative RT-PCR analysis showedthat FGF21 gene only expressedin liver, lung, skin and stomach, but higher in liver, then in lungs and skin, lower in stomach. Further q-PCR data from skin showed that the expression of FGF21 gene at one-month-old was significantly higher than( P<0.01) that at 48-month-old. All the results suggest that FGF21 gene may play an important role in curly fleece formationmechanism in Chinese Tan sheep,and supply some value clues for further research on biology characterization of curly fleece growth.
This study aimed to elucidate the structure, physiological function and the expression levels of FGF21 gene on Tan sheep. The FGF21 gene of Tan sheep was amplified through PCR using extracted RNA from skin samples Then the gene was sequenced and analyzed by bioinformatics tools. The CDS region length of cloned Tan sheep FGF21 gene was 630 bp encoded 209 amino acids with a molecular mass of 22645. 8 Da. The secondary of the protein were mainly composed of α-helix, β-turn, extended strand structure and random coli. Phylogenetic tree and sequence alignment showed the FGF21 gene of Tan sheep had a high homology with other species in nucleotide sequence and the gene was relatively conservation. Semi-quantitative RT-PCR analysis showedthat FGF21 gene only expressedin liver, lung, skin and stomach, but higher in liver, then in lungs and skin, lower in stomach. Further q-PCR data from skin showed that the expression of FGF21 gene at one-month-old was significantly higher than( P<0.01) that at 48-month-old. All the results suggest that FGF21 gene may play an important role in curly fleece formationmechanism in Chinese Tan sheep,and supply some value clues for further research on biology characterization of curly fleece growth.
引文
[1]李殿启,杨甲梅,殷正丰.FGF-20:成纤维细胞生长因子家族成员[J].生命的化学,2006,26(5):445-448.
[2]Nishimura T,Nakatake Y,Konishi M,et al.Identification of a novel FGF,FGF-21,preferentially expressed in the liver[J].Biochim Biophys Acta,2000,1492(1):203-206.
[3]Harris L A,Skinner J R,Shew T M,et al.Perilipin 5-driven lipid droplet accumulation in skeletal muscle stimulates the expression of fibroblast growth factor 21[J].Diabetes,2015,64(8):2757-2768.
[4]Lee C H,Hui E Y,Woo Y C,et al.Circulating fibroblast growth factor 21 levels predict progressive kidney disease in subjects with type 2 diabetes and normoalbuminuria[J].J Clin Endocrinol Metab,2015,100(4):1368-1375.
[5]Lu P,Yan J,Liu K,et al.Activation of aryl hydrocarbon receptor dissociates fatty liver from insulin resistance by inducing fibroblast growth factor 21[J].Hepatology,2015,61(6):1908-1919.
[6]Menzies M,Stockwell S,Brownlee A,et al.Gene expression profiles of BMP4,FGF10 and cognate inhibitors,in the skin of foetal Merino sheep,at the time of secondary follicle branching[J].Exp Dermatol,2009,18(10):877-879.
[7]Nesterova A P,Nizamutdinov I I,Koniukhov B V.Interaction of mutant genes Fgf5(go-Y),we,and wal changes the duration of hair growth cycles in mice[J].Ontogenez,2012,43(1):60-65.
[8]Housley D J,Venta P J.The long and the short of it:evidence that FGF5 is a major determinant of canine‘hair’-itability[J].Anim Genet,2006,37:309-315.
[9]Kang X,Liu G,Liu Y,et al.Transcriptome profile at different physiological stages reveals potential mode for curly fleece in Chinese tan sheep[J].PLo S One,2013,8(8):e71763.
[10]Ornitz D M,Itoh N.Fibroblast growth factors[J].Genome Biol,2001,2(3):1-12.
[11]Price C.Mechanisms of fibroblast growth factor signaling in the ovarian follicle[J].J Endocrinol,2016,228(2):R31-R43.
[12]Kawano M,Suzuki S,Suzuki M,et al.Bulge-and basal layerspecific expression of fibroblast growth factor 13(FHF-2)in mouse skin[J].J Invest Dermatol,2004,122:1084-1090.
[13]Higgins C A,Petukhova L,Harel S,et al.FGF5 is a crucial regulator of hair length in humans[J].Proc Natl Acad Sci USA,2014,111(29):10648-10653.
[2]Nishimura T,Nakatake Y,Konishi M,et al.Identification of a novel FGF,FGF-21,preferentially expressed in the liver[J].Biochim Biophys Acta,2000,1492(1):203-206.
[3]Harris L A,Skinner J R,Shew T M,et al.Perilipin 5-driven lipid droplet accumulation in skeletal muscle stimulates the expression of fibroblast growth factor 21[J].Diabetes,2015,64(8):2757-2768.
[4]Lee C H,Hui E Y,Woo Y C,et al.Circulating fibroblast growth factor 21 levels predict progressive kidney disease in subjects with type 2 diabetes and normoalbuminuria[J].J Clin Endocrinol Metab,2015,100(4):1368-1375.
[5]Lu P,Yan J,Liu K,et al.Activation of aryl hydrocarbon receptor dissociates fatty liver from insulin resistance by inducing fibroblast growth factor 21[J].Hepatology,2015,61(6):1908-1919.
[6]Menzies M,Stockwell S,Brownlee A,et al.Gene expression profiles of BMP4,FGF10 and cognate inhibitors,in the skin of foetal Merino sheep,at the time of secondary follicle branching[J].Exp Dermatol,2009,18(10):877-879.
[7]Nesterova A P,Nizamutdinov I I,Koniukhov B V.Interaction of mutant genes Fgf5(go-Y),we,and wal changes the duration of hair growth cycles in mice[J].Ontogenez,2012,43(1):60-65.
[8]Housley D J,Venta P J.The long and the short of it:evidence that FGF5 is a major determinant of canine‘hair’-itability[J].Anim Genet,2006,37:309-315.
[9]Kang X,Liu G,Liu Y,et al.Transcriptome profile at different physiological stages reveals potential mode for curly fleece in Chinese tan sheep[J].PLo S One,2013,8(8):e71763.
[10]Ornitz D M,Itoh N.Fibroblast growth factors[J].Genome Biol,2001,2(3):1-12.
[11]Price C.Mechanisms of fibroblast growth factor signaling in the ovarian follicle[J].J Endocrinol,2016,228(2):R31-R43.
[12]Kawano M,Suzuki S,Suzuki M,et al.Bulge-and basal layerspecific expression of fibroblast growth factor 13(FHF-2)in mouse skin[J].J Invest Dermatol,2004,122:1084-1090.
[13]Higgins C A,Petukhova L,Harel S,et al.FGF5 is a crucial regulator of hair length in humans[J].Proc Natl Acad Sci USA,2014,111(29):10648-10653.