对新疆美利奴细毛羊基因组MHC(Major histocompatibility complex)区段细菌人工染色体(BAC)文库的DNA序列进行测定, 经过序列比对分析, 首次预测了约130个新基因, 其中有8个CDS(Coding sequences)未在其他物种中发现其同源序列, 推测可能系绵羊所特有。在此基础上, 文章对绵羊MHC区段预测的3个新基因(分别命名为OaN2、OaN5、OaN6)进行了实验验证和表达分析。从绵羊肺组织中克隆到了OaN2的cDNA序列, 其长度为270 bp; 从肠系淋巴结中扩增得到 OaN5和OaN6的cDNA序列, 长度分别为309 bp和205 bp。上述3个基因的GenBank登录号分别为JF330782、JF330783和JF330784。利用Northern blotting技术进行转录本水平分析, 发现这3个新基因均在免疫器官肠系淋巴结中高表达。通过Western blotting和原位免疫组化技术对OaN2蛋白水平进行了表达谱分析, 结果表明OaN2蛋白在绵羊脾脏和肠系淋巴结等免疫器官中高表达, 在心、肝及胰脏中不表达。这是首次通过实验验证绵羊MHC区段的3个预测的新基因, 为其在绵羊免疫器官中的功能研究奠定了基础。
Previous DNA sequencing of BAC clones covering entire ovine MHC (OLA) region resulted in identification of approximately 130 functional genes in the region, of which 8 were predicted by computer software to be exclusively existed in sheep, but not in any other species known to date. In the present study, we successfully identified and cloned cDNA sequence of OaN2, OaN5, and OaN6 from representative sheep tissues, confirmed their existence in reality. The sequences obtained experimentally exactly identical to those predicted previously. The length of cDNA fragments for OaN2, OaN5, and OaN6 was 270 bp, 309 bp, and 205 bp, respectively, with GenBank accession number assigned as JF330782 (OaN2), JF330783 (OaN5), and JF330784 (OaN6). Northern analyses indicated that the mRNA transcripts of OaN2 were mainly seen in ovine mesenteric lymph nodes and spleen, while OaN5 was observed in only in mesenteric lymph nodes. In contrast, OaN6 transcripts were detected in all tissues except for liver and heart. Western blot showed that OaN2 protein expression level was detected in mesenteric lymph nodes, spleen, and liver, essentially consistent with that of mRNA transcripts. Immunohistochemistry analysis showed that OaN2 protein was highly expressed in ovine mesenteric lymph nodes, moderately expressed in, and not expressed in heart, liver, and pancreas, consistent with the results of Western blotting. The cloning and expression analysis of 3 novel genes provide a basis for revealing their specificities and would be helpful to further study of their expression profile and their potential functions.
[1] Gao J, Liu K, Liu H, Blair HT, Li G, Chen C, Tan P, Ma RZ. A complete DNA sequence map of the ovine major histocompatibility complex. BMC Genomics, 2010, 11(1): 466-474.
[2] Dawkins R, Leelayuwat C, Gaudieri S, Tay G, Hui J, Cattley S, Martinez P, Kulski J. Genomics of the major his-tocompatibility complex: haplotypes, duplication, retrovi ruses and disease. Immunol Rev, 1999, 167(1): 275-304.
[3] Kelley J, Walter L, Trowsdale J. Comparative genomics of major histocompatibility complexes. Immunogenetics, 2005, 56(10): 683-695.
[4] Liu H, Liu K, Wang J, Ma RZ. A BAC clone-based physical map of ovine major histocompatibility complex. Genomics, 2006, 88(1): 88-95.
[5] Margaron Y, Bostan L, Exposito JY, Malbouyres M, Trun-fio-Sfarghiu AM, Berthier Y, Lethias C. Tenascin-X in-creases the stiffness of collagen gels without affecting fibrillogenesis. Biophys Chem, 2010, 147(1-2): 87-91.
[6] Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu D, Hanrahan F, Pertea G, Van Tassell CP, Sonstegard TS, Marcais G, Roberts M, Subramanian P, Yorke JA, Salzberg SL. A whole-genome assembly of the domestic cow, Bos taurus. Genome Biol, 2009, 10(4): R42.
[7] Chessa B, Pereira F, Arnaud F, Amorim A, Goyache F, Mainland I, Kao RR, Pemberton JM, Beraldi D, Stear MJ, Alberti A, Pittau M, Iannuzzi L, Banabazi MH, Kazwala RR, Zhang YP, Arranz JJ, Ali BA, Wang ZL, Uzun M, Dione MM, Olsaker I, Holm LE, Saarma U, Ahmad S, Marzanov N, Eythorsdottir E, Holland MJ, Ajmone-Marsan P, Bruford MW, Kantanen J, Spencer TE, Palmarini M. Revealing the history of sheep domestication using retro-virus integrations. Science, 2009, 324(5926): 532-536.
[8] Liu K, Zhang P, Gao J, Liu H, Li G, Qiu Z, Zhang Y, Ren J, Tan P, Ma RZ. Closing a gap in the physical map of the ovine major histocompatibility complex. Anim Genet, 2010, 42(2): 204-207.
[9] Barcellos LF, May SL, Ramsay PP, Quach HL, Lane JA, Nititham J, Noble JA, Taylor KE, Quach DL, Chung SA, Kelly JA, Moser KL, Behrens TW, Seldin MF, Thomson G, Harley JB, Gaffney PM, Criswell LA. High-density SNP screening of the major histocompatibility complex in systemic lupus erythematosus demonstrates strong evidence for independent susceptibility regions. PLoS Genet, 2009, 5(10): e1000696.
[10] McKinnon E, Morahan G, Nolan D, James I. Association of MHC SNP genotype with susceptibility to type 1 dia-betes: a modified survival approach. Diabetes Obes Metab, 2009, 11(S1): 92-100.
[11] Vignal C, Bansal AT, Balding DJ, Binks MH, Dickson MC, Montgomery DS, Wilson AG. Genetic association of the major histocompatibility complex with rheumatoid arthritis implicates two non-DRB1 loci. Arthritis Rheum, 2009, 60(1): 53-62.
