GPR143在绵羊皮肤组织中的表达及定位分析

doi:10.16288/j.yczz.16-068

遗传 ›› 2016, Vol. 38 ›› Issue (7): 658-665.doi: 10.16288/j.yczz.16-068

GPR143在绵羊皮肤组织中的表达及定位分析

陈天直¹, 赵兵令¹, 刘宇², 赵园园³, 王海东¹, 范瑞文¹, 王鹏超¹, 董常生¹

1. 山西农业大学动物科技学院,太谷 030801;
2. 东北农业大学动物科学技术学院,哈尔滨 150030;
3. 铜仁学院乌江农林经济学院,铜仁 554300

收稿日期:2016-02-26 出版日期:2016-07-20 发布日期:2016-07-20
作者简介:陈天直,硕士研究生,专业方向：基础兽医学。E-mail: chentianzhi15@163.com
基金资助:
国家高技术研究发展计划项目(“863”计划)（编号：2013AA102506）和国家公益性行业（农业）科研专项（编号：201303119）资助[Supported by the High Technology Research and Development Program of China (863 Program)(No.2013AA102506) and the Special Foundation for Agro-scientific Research in the Public Interest (No; 201303119)]

Expression and localization of GPR143 in sheep skin

Tianzhi Chen¹, Bingling Zhao¹, Yu Liu², Yuanyuan Zhao³, Haidong Wang¹, Ruiwen Fan¹, Pengchao Wang¹, Changsheng Dong¹

1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;
2. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
3. Wujiang River Institute of Agriculture and Forestry Economics, Tongren University, Tongren 554300, China

Received:2016-02-26 Online:2016-07-20 Published:2016-07-20

摘要/Abstract

摘要： G蛋白偶联受体143(G-protein coupled receptor143, GPR143)在黑素体的生物合成中起重要作用,本文旨在研究GPR143基因在不同毛色绵羊皮肤组织中的差异表达及定位,探索GPR143基因与毛色形成的相关性。通过qRT-PCR方法和免疫印迹方法分别检测不同毛色绵羊皮肤组织中GPR143基因mRNA水平和蛋白水平的表达差异;运用免疫荧光法对不同毛色绵羊皮肤组织中的GPR143基因进行定位并对结果进行光密度值分析。qRT-PCR结果显示,GPR143基因在黑色绵羊皮肤组织中mRNA相对表达量为白色绵羊的7.84倍,二者差异极显著(P<0.01);免疫印迹结果显示,黑色绵羊皮肤组织中GPR143蛋白表达量是白色绵羊的1.3倍,二者差异显著(P<0.05)。免疫荧光结果显示,GPR143蛋白的主要表达部位为绵羊皮肤组织毛囊外根鞘和表皮层,经光密度值分析后发现,GPR143在黑色绵羊皮肤毛囊外根鞘和表皮层的表达量显著高于白色绵羊。本研究结果表明不同毛色绵羊皮肤组织均能表达GPR143基因,但黑色绵羊皮肤组织中该基因的mRNA和蛋白水平都显著高于白色绵羊,说明GPR143的mRNA和蛋白在黑色绵羊皮肤组织中表达上调,在白色绵羊皮肤组织中表达下调。GPR143基因可能通过调控MITF水平和黑素体的数量、大小、运动和成熟进而参与绵羊毛色的形成过程。

关键词: 绵羊, 毛色, GPR143, qRT-PCR, 免疫印迹, 免疫荧光

Abstract: G-protein coupled receptor143 (GPR143) plays an important role in melanogenesis. In this study, we investigated the expression pattern and localization of GPR143 in skin of sheep with different coat colors and explored the correlation between GPR143 gene and coat color. The mRNA level and protein level of GPR143 in skin of sheep with different coat colors were detected by qRT-PCR and immunoblotting separately while the localization of GPR143 in sheep skin was detected by immunofluorescence assay following optical density analysis. The qRT-PCR results showed that the relative expression level of GPR143 mRNA in black sheep skin was 7.84 times of that in white sheep skin (P<0.01). Immunoblotting results demonstrated that the expression level of GPR143 protein in black sheep skin was 1.30 times of that in white sheep skin (P<0.05). Immunofluorescence assay revealed that GPR143 was primarily expressed in the outer root sheath of hair follicles and epidermal skin tissue. Optical density analysis showed that expression levels of GPR143 in the outer root sheath and epidermis of black sheep skin were significantly higher than that of white sheep skin. Our studies demonstrated that GPR143 is expressed in skin of sheep with different coat colors. However, the mRNA and protein levels of GPR143 in black sheep skin are significantly higher than that in white sheep skin, indicating that GPR143 mRNA and protein levels are upregulated in skin of black sheep while downregulated in skin of white sheep. GPR143 may participate in the formation of coat color by regulating the level of MITF and the number, size, motility and maturation of the melanosome.

Key words: sheep, coat color, GPR143, qRT-PCR, immunoblotting, immunofluorescence

陈天直, 赵兵令, 刘宇, 赵园园, 王海东, 范瑞文, 王鹏超, 董常生. GPR143在绵羊皮肤组织中的表达及定位分析[J]. 遗传, 2016, 38(7): 658-665.

Tianzhi Chen, Bingling Zhao, Yu Liu, Yuanyuan Zhao, Haidong Wang, Ruiwen Fan, Pengchao Wang, Changsheng Dong. Expression and localization of GPR143 in sheep skin[J]. HEREDITAS(Beijing), 2016, 38(7): 658-665.

参考文献

[1] Bassi MT, Schiaffino MV, Renieri A, De Nigris F, Galli L, Bruttini M, Gebbia M, Bergen AAB, Lewis RA, Ballabio A. Cloning of the gene for ocular albinism type 1 from the distal short arm of the X chromosome. Nat Genet , 1995, 10(1): 13-19.
[2] Fukuda N, Naito S, Masukawa D, Kaneda M, Miyamoto H, Abe T, Yamashita Y, Endo I, Nakamura F, Goshima Y. Expression of ocular albinism 1 (OA1), 3, 4- dihydroxy- L-phenylalanine (DOPA) receptor, in both neuronal and non-neuronal organs. Brain Res , 2015, 1602: 62-74.
[3] Schiaffino MV, Baschirotto C, Pellegrini G, Montalti S, Tacchetti C, De Luca M, Ballabio A. The ocular albinism type 1 gene product is a membrane glycoprotein localized to melanosomes. Proc Natl Acad Sci USA , 1996, 93(17): 9055-9060.
[4] Schiaffino MV, d'Addio M, Alloni A, Baschirotto C, Valetti C, Cortese K, Puri C, Bassi MT, Colla C, De Luca M, Tacchetti C, Ballabio A. Ocular albinism: evidence for a defect in an intracellular signal transduction system. Nat Genet , 1999, 23(1): 108-112.
[5] Samaraweera P, Shen B, Newton JM, Barsh GS, Orlow SJ. The mouse ocular albinism 1 gene product is an endolysosomal protein. Exp Eye Res , 2001, 72(3): 319-329.
[6] Newton JM, Orlow SJ, Barsh GS. Isolation and characterization of a mouse homolog of the X-linked ocular albinism (OA1) gene. Genomics , 1996, 37(2): 219-225.
[7] Surace EM, Angeletti B, Ballabio A, Marigo V. Expression pattern of the ocular albinism type 1 ( Oa1 ) gene in the murine retinal pigment epithelium. Invest Ophthalmol Vis Sci , 2000, 41(13): 4333-4337.
[8] Vetrini F, Auricchio A, Du JY, Angeletti B, Fisher DE, Ballabio A., Marigo V. The microphthalmia transcription factor (Mitf) controls expression of the ocular albinism type 1 gene: link between melanin synthesis and melanosome biogenesis. Mol Cell Biol , 2004, 24(15): 6550-6559.
[9] Schiaffino MV, Tacchetti C. The ocular albinism type 1 (OA1) protein and the evidence for an intracellular signal transduction system involved in melanosome biogenesis. Pigment Cell Res , 2005, 18(4): 227-233.
[10] Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B. The metabolic and molecular bases of inherited disease (8th ed). New York, Montreal: McGraw-Hill, 2001.
[11] Garner A, Jay BS. Macromelanosomes in X-linked ocular albinism. Histopathology , 1980, 4(3): 243-254.
[12] Wong L, O'Donnell FE, Richard Green W. Giant pigment granules in the retinal pigment epithelium of a fetus with X-linked ocular albinism. Ophthalmic Paediatr Genet , 1983, 2(1): 47-65.
[13] O'Donnell Jr FE, Hambrick GW, Richard Green W, Iliff WJ, Stone DL. X-linked ocular albinism: an oculocutaneous macromelanosomal disorder. Arch Ophthalmol , 1976, 94(11): 1883-1892.
[14] Schnur RE, Gao M, Wick PA, Keller M, Benke PJ, Edwards MJ, Grix AW, Hockey A, Jung JH, Kidd KK, Kistenmacher M, Levin AV, Lewis RA, Musarella MA, Nowakowski RW, Orlow SJ, Pagon RS, Pillers DAM, Punnett HH, Quinn GE, Tezcan K, Wagstaff J, Weleber RG. OA1 mutations and deletions in X-linked ocular albinism. Am J Hum Genet , 1998, 62(4): 800-809.
[15] Schiaffino MV, Dellambra E, Cortese K, Baschirotto C, Bondanza S, Clementi M, Nucci P, Ballabio A, Tacchetti C, De Luca M. Effective retrovirus-mediated gene transfer in normal and mutant human melanocytes. Hum Gene Ther , 2002, 13(8): 947-957.
[16] Palmisano I, Bagnato P, Palmigiano A, Innamorati G, Rotondo G, Altimare D, Venturi C, Sviderskaya EV, Piccirillo R, Coppola M, Marigo V, Incerti B, Ballabio A, Surace EM, Tacchetti C, Bennett DC, Schiaffino MV. The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells. Hum Mol Genet , 2008, 17(22): 3487-3501.
[17] Cortese K, Giordano F, Surace EM, Venturi C, Ballabio A, Tacchetti C, Marigo V. The ocular albinism type 1 ( OA1 ) gene controls melanosome maturation and size. Invest Ophthalmol Vis Sci , 2005, 46(12): 4358-4364.
[18] Giordano F, Bonetti C, Surace EM, Marigo V, Raposo G. The ocular albinism type 1 (OA1) G-protein-coupled receptor functions with MART-1 at early stages of melanogenesis to control melanosome identity and composition. Hum Mol Genet , 2009, 18(23): 4530-4545.
[19] Piccirillo R, Palmisano I, Innamorati G, Bagnato P, Altimare D, Schiaffino MV. An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1. J Cell Sci , 2006, 119(10): 2003-2014.
[20] Basrur V, Yang F, Kushimoto T, Higashimoto Y, Yasumoto KI, Valencia J, Muller J, Vieira WD, Watabe H, Shabanowitz J, Hearing VJ, Hunt DF, Appella E. Proteomic analysis of early melanosomes: identification of novel melanosomal proteins. J Proteome Res , 2003, 2(1): 69-79.
[21] Falletta P, Bagnato P, Bono M, Monticone M, Schiaffino MV, Bennett DC, Goding CR, Tacchetti C, Valetti C. Melanosome-autonomous regulation of size and number: the OA1 receptor sustains PMEL expression. Pigment Cell Melanoma Res , 2014, 27(4): 565-579.
[22] Fitzpatrick TB, Breathnach AS. The epidermal melanin unit system. Dermatol Wochenschr , 1963, 147: 481-489.
[23] Slominski A, Wortsman J, Plonka PM, Schallreuter KU, Paus R, Tobin DJ. Hair follicle pigmentation. J Invest Dermatol , 2005, 124(1): 13-21.
[24] Cui J, Shen LY, Wang GC. Role of hair follicles in the repigmentation of vitiligo. J Invest Dermatol , 1991, 97(3): 410-416.
[25] Schiaffino MV. Signaling pathways in melanosome biogenesis and pathology. Int J Biochem Cell Biol , 2010, 42(7): 1094-1104.
[26] Vachtenheim J, Borovanský J. “Transcription physiology” of pigment formation in melanocytes: central role of MITF. Exp Dermatol , 2010, 19(7): 617-627.
[27] Steingrímsson E, Copeland NG, Jenkins NA. Melanocytes and the Microphthalmia transcription factor network. Annu Rev Genet , 2004, 38(1): 365-411.
[28] Burgoyne T, Jolly R, Martin-Martin B, Seabra MC, Piccirillo R, Schiaffino MV, Futter CE. Expression of OA1 limits the fusion of a subset of MVBs with lysosomes-a mechanism potentially involved in the initial biogenesis of melanosomes. J Cell Sci , 2013, 126(22): 5143-5152.
(责任编委: 蒋思文)

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

GPR143在绵羊皮肤组织中的表达及定位分析

Expression and localization of GPR143 in sheep skin

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	赵志达,张莉. 基因组选择在绵羊育种中的应用[J]. 遗传, 2019, 41(4): 293-303.
[2]	胡广东,郝科兴,黄涛,曾维斌,谷新利,王静. 绵羊高效转基因通用型piggyBac转座子载体构建及功能验证[J]. 遗传, 2018, 40(8): 647-656.
[3]	夏青, 刘秋月, 王翔宇, 胡文萍, 李春艳, 贺小云, 储明星, 狄冉. 绵羊季节性繁殖分子机制及休情季节诱导绵羊发情配种技术[J]. 遗传, 2018, 40(5): 369-377.
[4]	赵若阳, 赵一萍, 李蓓, 格日乐其木格, 张心壮, 陶克涛, 图格琴, 旭仁其木格, 青柏, 李超, 白东义, 芒来. 马毛色遗传机理研究进展[J]. 遗传, 2018, 40(5): 357-368.
[5]	赵永欣, 李孟华, 赵要风. 中国绵羊起源、进化和遗传多样性研究进展[J]. 遗传, 2017, 39(11): 958-973.
[6]	王伟, 王玉霜, 黄兰兰, 简子健, 王新华, 刘守仁, 皮文辉. siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率[J]. 遗传, 2016, 38(9): 831-839.
[7]	高磊,沈敏,甘尚权,杨井泉,张译元. 绵羊CCNG1基因克隆及表达分析[J]. 遗传, 2015, 37(4): 374-381.
[8]	孙新华, 贾攀, 张继超, 潘洪玉. 四翅滨藜盐胁迫应答基因AcPsbQ1的克隆及其在酵母中的功能分析[J]. 遗传, 2015, 37(1): 84-90.
[9]	邢万金,莫日根. 小鼠毛色遗传的控制机制及其在遗传学教学中的应用[J]. 遗传, 2014, 36(10): 1062-1068.
[10]	王春玲孟晨玲曹少先张俊孟春花王慧利方永飞朱冬冬茆达干. 湖羊、东湖杂种羊POMC基因外显子3单核苷酸多态性及其与生长性状的关联分析[J]. 遗传, 2013, 35(9): 1095-1100.
[11]	庞有志许永飞. 白色獭兔蓝眼突变体的发现与遗传分析[J]. 遗传, 2013, 35(6): 786-792.
[12]	高磊，甘尚权，杨井泉，杨剑波，梁耀伟，阿不都艾尼·努拉洪，沈敏. 绵羊发情周期不同组织Cry1 mRNA转录水平相对定量研究[J]. 遗传, 2013, 35(1): 85-92.
[13]	杨小亮，白大章，邱巍，董慧琴，李大全，陈芳，马润林，Hugh T Blair，高剑峰. 以绵羊MHC区段BAC克隆酶切片段为探针杂交筛选绵羊混合组织cDNA文库[J]. 遗传, 2012, 34(7): 887-894.
[14]	杨剑波，甘尚权，杨永林，张红琳，宋天增，冯静，杨井泉，高磊，石国庆，沈敏. 绵羊ILK基因的克隆及其在毛囊生长期的表达[J]. 遗传, 2012, 34(6): 719-726.
[15]	李洪涛何新周志勇赵松华张文祥刘钢赵宗胜贾斌. Slc7a11基因在不同被毛颜色哈萨克羊皮肤组织中的表达分析[J]. 遗传, 2012, 34(10): 1314-1319.