鸭CD8α基因启动子的分析及其转录活性

doi:10.3864/j.issn.0578-1752.2012.12.016

摘要/Abstract

摘要： 【目的】对鸭CD8α基因启动子活性区域进行分析，为鸭CD8α基因功能和表达调控机理研究提供依据。【方法】利用前期基因组步移技术获得的鸭CD8α基因的启动子区序列，制备一系列启动子缺失突变体（-625/-1 bp，-1 110/-1 bp，-1 413/-1 bp，-2 151/-1 bp），定向亚克隆至荧光素酶表达载体pGL3-Basic 中，构建荧光素酶报告基因重组载体，采用 Lipofectamine 2000 将重组质粒瞬时转染DT40细胞，分析CD8α基因启动子系列缺失突变体在细胞内的转录活性。【结果】鸭CD8α基因 5′侧翼区长片段具有较强的启动子活性，-1110—-625启动子活性最强，且-625—-1和-625—-1 110 bp区域均存在正调控元件。【结论】成功构建了荧光素酶报告基因真核表达载体，确定了鸭CD8α基因调控区，为进一步研究其转录调控机制奠定了基础。

关键词: 鸭, CD8&alpha, 基因, 启动子, 转录活性

Abstract: 【Objective】 This experiment was conducted to explore the function determination and expression regulation of CD8α gene in ducks by analysis of transcriptional activity of its promoter. 【Method】 The promoter missing mutants were constructed（-625/-1 bp, -1110/-1 bp, -1413/-1 bp, -2151/-1 bp）accroding to the sequence of CD8α gene promoter, and then subcloned into pGL3 basic vectors to construct luciferase reporter gene vectors, respectively. The recombinant vectors were transfected into DT40 cells with Lipofectamine 2000, and the transcriptional activities were detected. 【Result】Results of the study indicated that CD8α gene promoter had obviously promoter activity. The sequence from -1 110 to -625 bp of 5′ flanking region had the strongest promoter activities, including two positive (-625/-1 bp and -1 110/-625 bp) regulatory domains. 【Conclusion】 The luciferase reporter gene eukaryotic expression vector with CD8α gene promoter sequences was constructed successfully using deletion mutation and its major regulatory regions were found, which play an important role for analyzing the promoter activity and transcriptional regulation mechanism.

Key words: duck, CD8&alpha, gene, promoter, transcriptional activity

徐琪, 陈阳, 黄正洋, 张扬, 陈昌义, 赵荣雪, 李秀, 段修军, 陈国宏. 鸭CD8α基因启动子的分析及其转录活性[J]. 中国农业科学, 2012, 45(12): 2468-2473.

XU Qi, CHEN Yang, HUANG Zheng-Yang, ZHANG Yang, CHEN Chang-Yi, ZHAO Rong-Xue, LI Xiu, DUAN Xiu-Jun, CHEN Guo-Hong. Analysis of Duck CD8α Gene Promoter and Its Transcriptional Activity[J]. Scientia Agricultura Sinica, 2012, 45(12): 2468-2473.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.12.016

https://www.chinaagrisci.com/CN/Y2012/V45/I12/2468

参考文献

[1]王洪梅, 张利博, 侯明海, 王长法, 王玲玲, 孙涛, 何洪彬, 仲跻峰. 牛 Nramp1 基因启动子的克隆及其活性分析. 中国农业科学, 2011, 44(5): 1022-1028.

Wang H M, Zhang L B, Hou M H, Wang C F, Wang L L, Sun T, He H B, Zhong J F. Cloning and activity analysis of bovine natural resistance associated macrophage protein 1 (Nramp1) gene promoter. Scientia Agricultura Sinica, 2011, 44(5): 1022-1028. (in Chinese)

[2]岳华, 黄兴, 杨发龙, 李明义, 范根成, 马莉, 汤承. 荧光定量RT-PCR检测鸡CD4、CD8基因表达水平. 畜牧兽医学报, 2008, 39(6): 784-790.

Yue H, Huang X, Yang F L, Li M Y, Fan G C, Ma L, Tang C. Development of real-time RT-PCR for detecting the expression level of CD4 and CD8 mRNA in chicken. Acta Veterinaria et Zootechnica Sinaca, 2008, 39(6): 784-790. (in Chinese)

[3]Mancebo E, Moreno-Pelayo M A, Mencía A, de la Calle-Martín O, Allende L M, Sivadorai P, Kalaydjieva L, Bertranpetit J, Coto E, Calleja-Antolín S, Ruiz-Contreras J, Paz-Artal E. Gly111Ser mutation in CD8A gene causing CD8 immunode?ciency is found in Spanish Gypsies. Molecular Immunology, 2008, 45(2): 479-484.

[4]Kothlowa S, Mannesb N K, Schaerera B, Rebeski D E, Kaspers B, Schultzb U. Characterization of duck leucocytes by monoclonal antibodies. Developmental and Comparative Immunology, 2005, 29(8): 733-748.

[5]廖定为, 唐秀山, 蒋一男, 张念之, 夏春. 北京鸭CD8α分子克隆与表达及其多克隆抗体制备. 中国兽医杂志, 2008, 44(6): 5-7.

Liao D W, Tang X S, Jiang Y N, Zhang N Z, Xia C. Cloning, expression and polyclonal antibodies production of Peking duck CD8α. Chinese Journal of Veterinary Medicine, 2008, 44(6): 5-7. (in Chinese)

[6]李卫, 白家媛, 许媛媛, 谷长勤, 张万坡, 程国富, 陈敏, 胡薛英. 麻鸭和樱桃谷鸭的CD3ε、CD4、CD8α基因ORF克隆及序列分析. 中国农业科学, 2009, 42(12): 4428-4434.

LI W, Bai J Y, Xu Y Y, Gu C Q, Zhang W P, Cheng G F, Chen M, Hu X Y. Cloning and sequence analyses of genes coding for CD3ε, CD4, CD8α of Sheldrake and Cherry Valley cuck. Scientia Agricultura Sinica, 2009, 42(12): 4428-4434. (in Chinese)

[7]Picchietti S, Guerra L, Buonocore F, Randelli E, Fausto A M, Abelli L. Lymphocyte differentiation in sea bass thymus: CD4 and CD8-α gene expression studies. Fish and Shell?sh Immunology, 2009, 27(6): 50-56.

[8]Duncan L G, Nair S V, Deane E M. The marsupial CD8 gene locus: molecular cloning and expression analysis of the alpha and beta sequences in the gray short-tailed opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii). Veterinary Immunology and Immunopathology, 2009, 129(1/2): 14-27.

[9]Suetake H, Araki K, Akatsu K, Somamoto T, Dijkstra J M, Yoshiura Y, Kikuchi K, Suzuki Y. Genomic organization and expression of CD8α and CD8β genes in fugu Takifugu rubripes. Fish and Shell?sh Immunology, 2007, 23(5): 1107-1118.

[10]Xu S W, Wu J Y, Hu K S, Ping H L, Duan Z G, Zhang H F. Molecular cloning and expression of orange-spotted grouper (Epinephelus coioides) CD8a and CD8b genes. Fish and Shell?sh Immunology, 2011, 30: 600-608.

[11]Gao M H, Kavathas P B. Functional importance of the cyclic AMP response element-like decamer motif in the CD8 alpha promoter. The Journal of Immunology, 1993, 150(10): 4376-4385.

[12]Landry D B, Engel J D, Sen R. Functional GATA-3 binding sites within murine CD8 alpha upstream regulatory sequences. The Journal of Experimental Medicine, 2003, 178(3): 941-949.

[13]Feik N, Bilic I, Tinhofer J, Unger B, Littman D R, Ellmeier W. Functional and molecular analysis of the double-positive stage-specific CD8 enhancer E8III during thymocyte development. The Journal of Immunology, 2005, 174(3): 1513-1524.

[14]袁舟. 稳定表达鸡CD4、CD8α细胞系的建立与鉴定[D]. 江苏: 扬州大学, 2008.

Yuan Z. Construction and identification of CD4, CD8α cell line stably expressing[D]. Jiangsu: Yangzhou University, 2008. (in Chinese)

[15]Huang Y H, Li N, Burt D W, Wu F. Genomic research and applications in theduck (Anas platyrhynchos). World’s Poultry Science Journal, 2008, 64(3): 329-341.

[16]张晓, 罗军, 李建华, 赵旺生, 王伟. 西农萨能奶山羊脂肪酸合酶基因启动子的克隆及活性测定. 中国农业科学, 2010, 43(3): 640-647.

Zhang X, Luo J, LI J H, Zhao W S, Wang W. Cloning and activity determination of fatty acid synthase (FAS) gene promoter of Xinong Saanen Dairy goat. Scientia Agricultura Sinica, 2010, 43(3): 640-647. (in Chinese)

[17]Kawach Y, Otsuka F, Nakauchi H. Characterization of the mouse CD8 beta chain-encoding gene promoter region. Immunogenetics, 1996, 44(5): 358-365.

[18]Rath B, Pandey R S, Debata P R, Maruyama N, Supakar P C. Molecular characterization of senescence marker protein-30 gene promoter: identification of repressor elements and functional nuclear factor binding sites. BMC Molecular Biology, 2008, 43(9): 1471-1480.

[19]张宁波, 井文倩, 李　奎. 猪SelS基因启动子区的克隆及序列分析. 畜牧兽医学报, 2011, 42(6): 759-764.

Zhang N B, Jing W Q, Li K. Cloning and sequence analysis of the promoter region of porcine SelS gene. Acta Veterinaria et Zootechnica Sinica, 2011, 42(6): 759-764. (in Chinese)

[20]余瑞元, 王燕峰, 徐长法. CREB研究进展. 中国生物工程杂志, 2003, 13(1): 39-43.

Yu R Y, Wang Y F, Xu C F. Progress in the studies on CREB. Journal of Chinese Biotechnology, 2003, 13(1): 39-43. (in Chinese)

[21]Wen A Y, Sakamoto K M, Miller L S. The role of the transcription factor CREB in immune function. Journal of Immunology, 2010, 185(11): 6413-6419.

[22]Landry D B, Engel J D, Sen R. Functional GATA-3 binding sites within murine CD8α upstream regulatory sequences. Journal of Experimental Medicine, 1993, 178: 941-949.

[23]Liu W W, Zhang P J, Chen W W, Yu C X, Cui F A, Kong F, Zhang J Y, Jiang A L. Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription. BMC Molecular Biology, 2008, 9(45): 471-481.

[24]Winslow M M, Dayton T L, Verhaak R G W, Kim-Kiselak C, Snyder E L, Feldser D M, Hubbard D D, Dupage M J, Whittaker C A, Hoersch S, Yoon S, Crowley D, Bonson R T, Chiang D Y, Meyerson M, Jacks T. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature, 2011, 473: 101-104.

[25]张澄, 王萍. 转录因子CREB对细胞周期调控机制的研究进展. 中国细胞生物学学报, 2011, 33(5): 577-583.

Zhang C, Wang P. Progress in the regulation of transcription factor CREB on cell cycle. Chinese Journal of Cell Biology, 2011, 33(5): 577-583. (in Chinese)

[26]Hang M H, Chou C M, Hsieh Y C, Lu I C, Devi M K N, Chang J P, Kuo T F, Huang C J. Identification of 5′-upstream region of pufferfish ribosomal protein 129 gene a strong constitutive pmmoter to drive GFP expression in zebrafish. Biochemical and Biophysical Research Communications, 2004, 314: 249-258.