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

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210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c DNA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。

Heavy metals,such as cadmium（ Cd）,lead（ Pb）,can pose serious toxin to human and aquatic organisms.Suppression subtractive c DNA library of Gobiocypris rarus treated by Cd was constructed to investigate the underlying mechanism. The efficiency of subtractive c DNA was reach up to 210 folds. A total of 9 expressed sequence tags with average sequence length of 438 bp and enriched in the c DNA library were obtained. The complete coding sequence of ribosomal protein s18（ Rps18） from G. rarus was amplified by rapid amplification of c DNA 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.