Hedgehog(Hh)信号通路对动物脂肪沉积具有抑制作用, 并且从果蝇到脊椎动物具有高度保守性, 但在家猪研究中鲜见报道。文章选择家猪Hh通路的转录激活因子Gli1进行研究, 通过RT-PCR结合RACE技术, 首次获得家猪Gli1基因cDNA全长, 利用Real-time PCR对家猪Gli1基因在不同组织中的表达丰度进行了分析, 并构建了真核表达载体和脂肪组织特异性表达载体。结果表明：猪Gli1基因cDNA全长3 576 bp, 基因组序列全长10 715 bp, 共12个外显子, 编码1 106个氨基酸。生物信息学分析表明, 猪Gli1为不稳定亲水性蛋白, 不具有跨膜结构域和信号肽序列, 但具有锌指结构与核定位序列。对7个物种的Gli1蛋白序列和基因组序列相似性进行分析, 发现各物种间序列相似性均在80%以上, 说明Gli1在物种间高度保守。组织表达谱分析表明, Gli1仅在成体猪舌组织中表达; 在家猪脂肪组织发育进程中, Gli1仅在出生1周的猪脂肪组织中检测到微弱表达, 但1月龄及3月龄猪脂肪组织中均检测不到表达, 由此推断猪Gli1表达与脂肪组织发育呈负相关。最后, 将猪Gli1编码区克隆到真核表达载体pIRES2-EGFP, 体外转染实验证明该载体能够正确表达猪Gli1, 另外还构建了脂肪组织特异性表达载体, 为构建脂肪组织特异性转基因动物奠定基础。
The Hedgehog (Hh) signaling pathway inhibits fat accumulation, which is conserved in a wide variety of organisms from Drosophila to vertebrates, but few reports about its effect on pigs are available. In this study, pig Gli1 gene was cloned for the first time by rapid amplification of cDNA ends (RACE) and RT-PCR. Pig Gli1 expression profiles were then studied in different tissues and in different developmental stages of the adipose tissue of pigs using real-time PCR. Finally, the eukaryotic expression vector and the adipose tissue specific expression vector were constructed. The results showed that the full pig Gli1 cDNA length was 3 576 bp, the genomic sequence contained 10 715 bp with 12 exons, and 1 106 amino acids were encoded. Pig Gli1 was predicted as an unstable hydrophilic protein without a tans-membrane structure or a signal peptide. The C2H2 zinc finger domain and a nuclear localization sequence were found in pig Gli1. A homology analysis of the Gli1 amino acids and the genomic sequences among seven species showed that the identities were all greater than 80%, which indicates that Gli1 is highly conserved among different species. Tissue expression profile analysis showed that pig Gli1 was only expressed in the tone tissue of adult pigs. Analysis of the pig adipose tissue developmental process showed that Gli1 was detected in the adipose tissue of one-week-old pigs, but not in one-month-old and three-month-old pigs. Finally, a pig Gli1 eukaryotic expression vector was constructed and properly expressed with cell transfection. An adipose tissue specific expression vector was constructed for transgenic animal studies.
[1] Perrimon N, Pitsouli C, Shilo BZ. Signaling mechanisms controlling cell fate and embryonic patterning. Cold Spring Harb Perspect Biol, 2012, 4(8): 1101-1110.
[2] Cohen MM Jr. The hedgehog signaling network. Am J Med Genet, 2003, 123A(1): 5-28.
[3] Østerlund T, Kogerman P. Hedgehog signalling: how to get from Smo to Ci and Gli. Trends in Cell Biol, 2006, 16(4): 176-180.
[4] Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O′Brien SJ, Wong AJ, Vogelstein B. Identification of an amplified, highly expressed gene in a human glioma. Science, 1987, 236(4797): 70-73.
[5] Pavletich NP, Pabo CO. Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. Science, 1993, 261(5129): 1701-1707.
[6] Shin SH, Kogerman P, Lindström E, Toftgárd R, Biesecker LG. GLI3 mutations in human disorders mimic Drosophila Cubitus interruptus protein functions and localization. Proc Natl Acad Sci USA, 1999, 96(6): 2880-2884.
[7] Mullor JL, Dahmane N, Sun T, Ruiz i Altaba A. Wnt signals are targets and mediators of Gli function. Curr Biol, 2001, 11(10): 769-773.
[8] Ruiz i Altaba A, Sánchez P, Dahmane N. Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat Rev Cancer, 2002, 2(5): 361-372.
[9] Markku M, Jussi T. Hedgehog: functions and mechanisms. Genes Dev, 2008, 22(18): 2454-2472.
[10] Suh JM, Gao XH, McKay J, McKay R, Salo Z, Graff JM. Hedgehog signaling plays a conserved role in inhibiting fat formation. Cell Metab, 2006, 3(1): 25-34.
[11] Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lu-cas PC, Opp MR, MacDougald OA. Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem, 2004, 279(34): 35503-35509.
[12] 刘岩, 童佳, 张然, 汤波, 彭云乾, 王淑辉, 李宁. 转基因动物育种研究的现状与趋势. 中国医药生物技术, 2009, 4(5): 329-334.
[13] Pursel VG, Bolt DJ, Miller KF, Pinkert CA, Hammer RE, Palmiter RD, Brinster RL. Expression and performance in transgenic pigs. J Reprod Fertil Suppl, 1990, 40: 235-245.
[14] Whyte J J, Prather R S. Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev, 2011, 78(10-11): 879-891.
[15] Cousin W, Fontaine C, Dani C, Peraldi P. Hedgehog and adipogenesis: Fat and fiction. Biochimie, 2007, 89(12): 1447-1453.
[16] Zhang XJ, Abreu JG, Yokota C, MacDonald BT, Singh S, Coburn KL, Cheong SM, Zhang MM, Ye QZ, Hang HC, Steen H, He X. Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation. Cell, 2012, 149(7): 1565-1577.
[17] McMahon AP, Ingham PW, Tabin CJ. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol, 2003, 53: 111-114.
[18] Hartman TR, Zinshteyn D, Schofield HK, Nicolas E, Okada A, O'Reilly AM. Drosophila Boi limits Hedgehog levels to suppress follicle stem cell prolifera-tion. J Cell Biol, 2010, 191(5): 943-952.
[19] Feng YH, Zhu YF, Sun WJ. Hedghog and tumorigenesis. Cancer Prevent Cure Res, 2005, 32(12): 796-799.
[20] Pospisilik JA, Schramek D, Schnidar H, Cronin SJF, Nehme NT, Zhang XY, Knauf C, Cani PD, Aumayr K, Todoric J, Bayer M, Haschemi A, Puviindran V, Tar K, Orthofer M, Neely GG, Dietzl G, Manoukian A, Funovics M, Prager G, Wagner O, Ferrandon D, Aberger F, Hui CC, Esterbauer H, Penninger JM. Drosophila genome-wide obesity screen reveals Hedgehog as a determinant of brown versus white adipose cell fate. Cell, 2010, 140(1): 148-160.
[21] Du M, Yin JD, Zhu MJ. Cellular signaling pathways regulating the initial stage of adipogenesis and marbling of skeletal muscle. Meat Sci, 2010, 86(1): 103-109.
[22] Ruiz i Altaba A. Gli proteins encode context dependent positive and negative functions: implications for development and disease. Development, 1999, 126(14): 3205-3216.
[23] Zhang YY, Mao FF, Lu Y, Wu W
