内蒙古绒山羊CRABP I基因的克隆及表达

doi:10.3864/j.issn.0578-1752.2012.13.019

摘要/Abstract

摘要： 【目的】克隆内蒙古绒山羊视黄酸结合蛋白Ⅰ（cellular retinoic acid binding proteinⅠ，CRABPⅠ）基因的cDNA，进行蛋白结构的预测和表达分析，为绒山羊毛和绒形成的分子机理奠定基础。【方法】利用RT-PCR方法克隆内蒙古绒山羊CRABPⅠ基因序列，利用生物信息学方法预测其蛋白结构，采用实时荧光定量PCR探讨该基因在绒山羊4个胎儿日龄皮肤中的表达。【结果】内蒙古绒山羊CRABP I基因cDNA长679 bp（JN936490），其开放阅读框为414 bp，氨基酸序列与其它物种相比具有较高的序列相似性。CRABP Ⅰ蛋白无明显的信号肽和跨膜区域，不存在N糖基化位点和O糖基化位点；蛋白二级结构主要由α 螺旋、β折叠和少量的转角及无规则卷曲构成。胎儿皮肤中CRABPⅠ基因在 90 d 的表达量明显高于100、120和130 d（P<0.05）。【结论】绒山羊的CRABPⅠ基因cDNA中的开放阅读框（open reading frame，ORF）在不同物种间较为保守，但种属特异性集中表现在第33和123氨基酸残基处，该基因在胎儿期的90 d表达量最大。

关键词: 绒山羊, CRABPⅠ基因, 克隆, 基因表达, 生物信息学

Abstract: 【Objective】The cDNA sequence of cellular retinoic acid binding protein I (CRABPⅠ) gene was cloned in Inner Mongolian cashmere goats, and the protein structure gene and expression were also analyzed. All these would establish a foundation for molecular mechanism of follicle and cashmere formation. 【Method】 The cDNA sequence of CRABP I gene was cloned by RT-PCR in Inner Mongolian cashmere goats. The protein structure was predicted through bioinformatics approach, the mRNA expression of the gene at four embryo ages in skin of cashmere goat were detected through real time PCR. 【Result】The length cDNA is 679 bp (JN936490) , its open reading frame (ORF) is 414 bp，which shares high similarity with other species. CRABPⅠprotein has no obvious signal peptide, transmembrane segments, N-glycosylation sites and O-glycosylation sites. The secondary structure of CRABPⅠprotein consisted of mainly β sheets, α helixes and loops, also few turn and coil. CRABPⅠ gene in cashmere goats was highly expressed on 90 d compared with on 100 d, 120 d and 130 d (P＜0.05). 【Conclusion】The open reading frame (ORF) of CRABPⅠgene is conserved among different species, but it has characteristics at 33 and 123 amino sites in cashmere goats. The level of mRNA expression was the highest on 90 d at four embryo stages, the polymorphism of this gene in different breeds and its regulation mechanism to sebaceous gland of hair follicle needs to be studied further.

Key words: cashmere goats, CRABPⅠgene, clone, gene expression, bioinformatics

李华, 苏立宁, 刘东军, 李雪峰, 旭日干. 内蒙古绒山羊CRABP I基因的克隆及表达[J]. 中国农业科学, 2012, 45(13): 2743-2750.

LI Hua, SU Li-Ning, LIU Dong-Jun, LI Xue-Feng, XU Ri-Gan. Cloning and Expression of Cellular Retinoic Acid Binding ProteinⅠGene in Inner Mongolian Cashmere Goats[J]. Scientia Agricultura Sinica, 2012, 45(13): 2743-2750.

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参考文献

[1]郭晓红, 储明星, 周忠孝. 视黄酸受体的分子生物学. 畜牧与兽医, 2007, 39(7): 61-64.

Guo X H, Chu M X, Zhou Z X. The molecular biology of retinoic acid receptors. Animal Husbandry and Veterinary medicine, 2007, 39(7): 61-64. (in Chinese)

[2]Nelson A M, Zhao W, Gilliland K L, Zaenglein A L, Liu W, Thiboutot D M. Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells. The Journal of Clinical Investigation, 2008, 118(4): 1468-1478.

[3]龚春华, 李军, 王大为. 视黄酸信号与胚胎心脏发育的关系. 心脏杂志, 2010, 22(1): 130-132. (in Chinese)

Gong C H, Li J, Wang D W. The relationship of retinoic acid signaling and embryonic development. Chinese Heart Journal, 2010, 22(1): 130-132.

[4]公维华, 张敬虎, 林莹, 张锐, 李奎. 猪细胞视黄醇结合蛋白基因 1 (CRBP1) 内含子 1 的克隆测序. 漳州师范学院学报: 自然科学版, 2009, 22(3): 143-145.

Gong W H, Zhang J H, Lin Y, Zhang R, Li K. Sequencing of the intron 1 of porcine cellular retinol binding protein gene 1 (CRBP1). Journal of Zhangzhou Normal University: Nature Science, 2009, 22(3): 143-145. (in Chinese)

[5]Shubeita H E, Sambrook J F, McCormick A M. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein. Proceedings of the National Academy of Sciences of the United States of America, 1987, 84(16): 5645-5649.

[6]Vaezeslami S, Jia X, Vasileiou C, Borhan B, Geiger J H. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein Ⅱ addresses the relationship between structural integrity and ligand binding. Acta Crystallographica Section D: Biological Crystallography, 2008, 64(12): 1228-1239.

[7]Boylan J F, Gudas L J. Overexpression of the cellular retinoic acid binding protein-I (CRABP-Ⅰ) results in a reduction in differentiation- specific gene expression in F9 teratocarcinoma cells. The Journal of Cell Biology, 1991, 112(5): 965-979.

[8]Georges S, Isabelle T, Dominique C G, Stefano J, Erikssont U, Saurat J H. Expression of CRABP-Ⅰ and -Ⅱ in human epidermal cells. Biochemical Journal, 1992, 287: 383-389.

[9]Propping C, Mönig B, Luksch H, Mey J. Distribution of the cellular retinoic acid binding protein CRABP-Ⅰ in the developing chick optic tectum. Brain Research, 2007, 1168: 21-31.

[10]Uhrig M, Brechlin P, Jahn O, Knyazev Y, Weninger A, Busia L, Honarnejad K, Otto M, Hartmann T. Upregulation of CRABP 1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ 42 reduces their differentiation potential. BMC Medicine, 2008, 6(1): 38-50.

[11]屠军波, 姚天华, 杨壮群, 张引成. 激素在毛囊皮脂腺单位发育中的作用. 中国美容医学, 2002, 11(2): 188-191.

Tu J B, Yao T H, Yang Z Q, Zhang Y C. The role of hormone in pilosebaceous unit dedevelopment. Chinese Journal of Aesthetic Medicine, 2002, 11(2): 188-191. (in Chinese)

[12]Tang X H, Vivero M, Gudas L J. Overexpression of CRABP I in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid. Experimental Cell Research, 2008, 314(1): 38-51.

[13]张燕军, 尹俊, 李金泉, 李长青. 内蒙古阿尔巴斯绒山羊毛囊结构及形态发生过程研究. 中国农业科学, 2007, 40(5): 1017-1023.

Zhang Y J, Yin J, Li J Q, Li C Q. Study on hair follicle structure and morphogenesis of the Inner Mongolian Arbas Cashmere goat. Scientia Agricultura Sinica, 2007, 40(5): 1017-1023. (in Chinese)

[14]Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. β-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell, 2001, 105(4): 533-545.

[15]王宏博, 高雅琴. 绒山羊皮肤毛囊结构及其与产绒量关系的研究进展. 安徽农业科学, 2008, 36(29): 12701-12703.

Wang H B, Gao Y Q. Research progress in skin follicle structure of cashmere goat and its relationship with cashmere yield. Journal of Anhui Agricultural Sciences, 2008, 36(29): 12701-12703. (in Chinese)

[16]刘志红, 任立明, 郭英, 胡晓湘, 崔文涛, 李宁, 尹俊, 李金泉. 毛囊的周期性变化和分子调控及其在绒山羊上的研究进展. 中国畜牧兽医, 2009, 36(4): 103-107.

Liu Z H, Ren L M, Guo Y, Hu X X, Cui W T, Li N, Yin J, Li J Q. The cyclical variation and regulation of hair follicles and the study on cashmere goat. China Animal Husbandry and Veterinary Medicine, 2009, 36(4): 103-107. (in Chinese)

[17]周汝真, 李若瑜, 余进. 白念珠菌抗氧化基因体外表达研究. 中国真菌学杂志, 2009, 4(6): 325-329.

Zhou R Z, Li R Y, Yu J. Anti-oxidant gene expression of candida albicans in vitro culture. Chinese Journal of Mycology, 2009, 4(6): 325-329. (in Chinese)

[18]Krishnan V V, Sukumar M, Gierasch L M, Cosman M. Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding. Biochemistry, 2000, 39(31): 9119-9129.

[19]Sjoelund V, Kaltashov I A. A transporter-to-trap conversion: a disulfide bond formation in CRABPⅠ mutant triggered by retinoic acid binding irreversibly locks the ligand inside the protein. Biochemistry, 2007, 46(46): 13382-13390.

[20]Park S W, Huang W H, Persaud S D, Wei L N. RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation. Nucleic Acids Research, 2009, 37(21): 7085-7094.

[21]Roos T C, Jugert F K, Merk H F, Bickers D R. Retinoid metabolism in the skin. Pharmacological Reviews, 1998, 50(2): 315-333.

[22]吴江鸿, 闫祖威, 胡斯乐, 张文广, 李金泉. Hoxc13 在毛囊发育中的作用. 遗传, 2010, 32(7): 656-662.

Wu J H, Yan Z W, Hu S L, Zhan W G, Li J Q. Hoxc13 and the development of hair follicle. Hereditas, 2010, 32(7): 656-662. (in Chinese)

[23]Everts H B, Sundberg J P, King L E, Ong D E. Immunolocalization of enzymes, binding proteins, and receptors sufficient for retinoic acid synthesis and signaling during the hair cycle. Journal of Investigative Dermatology, 2007, 127(7): 1593-1604.