重金属胁迫下稀有鲫抑制消减杂交文库的筛选与分析
Construction and Analysis of the Subtractive cDNA Library of Gobiocypris rarus Treated by Cd and Pb

中文摘要：重金属包括镉和铅等污染严重威胁人类健康。为从基因水平研究鱼类应答重金属胁迫的分子机理,本研究利用抑制消减杂交技术构建稀有鲫 Gobiocypris rarus对镉处理反应的正、反向抑制消减文库。文库质量检测表明消减效率达2 10倍,通过对正、反向文库中部分表达序列标签进行序列测定,获得9条表达丰度较高的表达序列标签,平均长度为438 bp。利用快速扩增cDNA末端技术克隆获得核糖体蛋白s18(Rps18)基因的完整编码区,序列长度为525 bp,其中编码区459 bp,编码152个氨基酸,3'非翻译区38 bp,5'非翻译区28 bp。并利用实时荧光定量技术对Rps18基因在铅胁迫下的表达谱进行了研究,结果表明稀有鲫肝组织中Rps18基因的表达量变化较明显。
英文摘要：Heavy metals, such as cadmium (Cd), lead (Pb), can pose serious toxin to human and aquatic organisms. Suppression subtractive cDNA library of Gobiocypris rarus treated by Cd was constructed to investigate the underlying mechanism. The efficiency of subtractive cDNA was reach up to 2 10 folds. A total of 9 expressed sequence tags with average sequence length of 438 bp and enriched in the cDNA library were obtained. The complete coding sequence of ribosomal protein s18(Rps18) from G. rarus was amplified by rapid amplification of cDNA ends technology, and it is found that this gene comprised of 5'-UTR (28 bp), ORF region (459 bp) that encoded 152 amino acids (aa) and 3'-UTR (38 bp). Then the expression of Rps18 in G. rarus after exposure to Pb was determined by real-time PCR, and the results showed that the expression difference of Rps18 was more obvious in liver than in muscle after Pb treatment. 2016,(35): 372-377 收稿日期：2015-12-17 DOI：10.11984/j.issn.1000-7083.20150403 分类号：Q9594;Q78 基金项目：国家自然科学基金项目(30900154);中国科学院水生生物研究所青年人才创新项目(Y15E011101);中国长江三峡集团公司项目(0799522) 作者简介：王春伶(1984-),女,博士,实验师,主要从事鱼类遗传学方面的研究,E-mail:chwang@ihb.ac.cn *通讯作者：张富铁,男,博士,副研究员,主要研究方向为鱼类保护遗传学、分子生态学及分子生物学,E-mail:futiezhang@ihb.ac.cn 参考文献： 陈宏, 姜浩, 谢兆霞. 2005. 核糖体蛋白在肿瘤中的改变及作用[J]. 国际病理科学与临床杂志, 25(5):393-396. 李小光, 宋伦, 袁胜涛. 2013. 核糖体蛋白参与调控p53诱导活化的分子机制[J]. 生物技术通讯, 24(3):423-426. 李英华, 胡振林, 张俊平. 2012. 核糖体蛋白S3a在肿瘤细胞增殖分化和凋亡调控作用的研究概况[J]. 药学实践杂志, 30(3):165-167. Amsterdam A, Sadler KC, Lai K, et al. 2004. Many ribosomal protein genes are cancer genes in zebrafish[J]. PLoS Biology, 2:690-698. Cao L, Huang W, Shan X, et al. 2012. Tissue-specific accumulation of cadmium and its effects on antioxidative responses in Japanese flounder juveniles[J]. Environmental Toxicology and Pharmacology, 33:16-25. Diatchenko L, Lau C, Campbell AP, et al. 1996. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Proceedings of the National Academy of Sciences of the United States of America, 93:6025-6030. James R, Thampuran N, Lalitha KV, et al. 2010. Differential gene expression profile of the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus by suppression