根据牛的成纤维细胞内生长因子5(Fibroblast growth factor 5, FGF5)基因cDNA序列设计引物, PCR扩增得到绵羊FGF5基因cDNA 的开放阅读框序列, 并比较和其他6种高等哺乳动物的序列同源性; 同时研究该基因在绵羊多种组织的表达情况, 以及研究以细胞模型RNA干扰下的表达情况。结果表明, 绵羊FGF5基因ORF全长为813 bp, 编码270个氨基酸, 分子量约为29.58 kDa, 理论等电点10.59。绵羊FGF5基因cDNA序列与牛、人、小鼠、大鼠、犬和猫的对应序列同源性高度保守, 预测氨基酸序列同源性同样具有高度保守性。RT-PCR分析表明FGF5在绵羊皮肤、小肠、肾脏、心脏、肝脏、脾脏、胰脏和肺中均有表达, 皮肤中表达量最高。构建该基因的原核表达载体和RNAi载体, IPTG诱导在大肠杆菌中融合表达获得55 kDa的蛋白条带, 设计的RNA干扰片段能显著抑制FGF5基因的表达。文章为进一步阐明绵羊FGF5的功能尤其是在羊毛生长发育中的作用提供了理论和实验基础。
The cDNA of fibroblast growth factor 5 (FGF5) gene in sheep was cloned, and the nucleotides sequence homology of FGF5 was compared with other six mammal. In addition, the expression of FGF5 in different tissues was analysed. Gene FGF5 was then recombined into prokaryotic expression vector (pGEX-4T-2) and RNA interference vector (pSilencer 5.1 H1) to study its pression in fibroblast cell lines. Results showed that the open reading frame (ORF) of cDNA in sheep consisted of 813 nucleotide acids encoding 270 amino acids, with the molecular mass of 29.58 kDa and theoretical pI of 10.59. The amino acids sequence of FGF5 gene in sheep shared high identity with those in cow, human, mouse, rat, dog, cat and rabbit. In addition, analysis on tissue expression showed that FGF5 expressed in skin, heart, kidney, liver, pancreas, spleen, lung, and small intestine, especially presenting high levels in skin. The ex-pression of FGF5 in E. coli was induced with IPTG, which produced a protein band with the expected size of 56 kDa on SDS-PAGE, while the expression of FGF5 in sheep fibroblast cell line was knocked down remarkably with the help of integrated RNAi vector.
[1] 杨建青. 国际羊毛市场状况与中国细毛羊发展分析. 农业展望, 2009, 5(2): 28-31.
[2] 黄立新. Optim纤维及产品的开发与应用. 纺织学报, 2004, 25(2): 101-102.
[3] Pennycuik PR, Raphael KA. The angora locus (go) in the mouse: hair morphology, duration of growth cycle and site of action. Genet Res, 1984, 44(3): 283-291.
[4] Hébert JM, Rosenquist T, Götz J, Martin GR. FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations. Cell, 1994, 78(6): 1017-1025.
[5] Suzuki S, Kato T, Takimoto H, Masui S, Oshima H, Ozawa K, Suzuki S, Imamura T. Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5s protein in hair follicle: possible involvement of two Fgf-5 gene products in hair growth cycle regulation. J Invest Dermatol, 1998, 111(6): 963-972.
[6] Ito C, Saitoh Y, Fujita Y, Yamazaki Y, Imamura T, Oka S, Suzuki S. Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5. J Cell Physiol, 2003, 197(2): 272-283.
[7] 高爱琴, 李宁, 赵兴波, 李金泉. 不同绵羊品种FGF5基因的多态性分析. 畜牧兽医学报, 2006, 37(4): 326-330.
[8] Bates B, Hardin J, Zhan X, Drickamer K, Goldfarb M. Biosynthesis of human fibroblast growth factor 5. Mol Cell Biol, 1991, 11(4): 1840-1845.
[9] Haub O, Drucker B, Goldfarb M. Expression of the murine fibroblast growth factor 5 gene in the adult central nervous system. Proc Natl Acad Sci USA, 1990, 87(20): 8022-8026.
[10] Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu D, Hanrahan F, Pertea G, van Tassell CP, Sonstegard TS, Marçais G, Roberts M, Subramanian P, Yorke JA, Salzberg SL. A whloe-genome assembly of the domestic cow, Bos taurus. Genome Biol, 2009, 10(4): R42.
[11] Hébert JM, Basilico C, Goldfarb M, Haub O, Martin GR. Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev Biol, 1990, 138(2): 454-463.
[12] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucelotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836): 449-498.
[13] Sui GC, Soohoo C, Affarel B, Gay F, Shi YJ, Forrester WC, Shi Y. A DNA vector based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA, 2002, 99(8): 5515-5520.
[14] Brantl S. Antisense-RNA regulation and RNA interference. Biochim Biophys Acta, 2002, 1575(1-3): 15-25.
