金鱼头瘤形成相关基因差减文库的构建与分析  

Construction and analysis of subtractive cDNA library of goldfish wen-related genes

作　　者：张运超[1,2] 何川[1,2] 顾泽茂[2,3] 

机构地区：[1]北京市水产技术推广站,北京100021 [2]华中农业大学水产学院,武汉430070 [3]淡水水产健康养殖湖北省协同创新中心,武汉430070

出　　处：《华中农业大学学报》2014年第1期85-91,共7页Journal of Huazhong Agricultural University

基　　金：现代农业产业技术体系北京市观赏鱼创新项目(pxm2012-036237-000139)

摘　　要：采用差减抑制杂交技术（SSH）以1龄红狮头金鱼（Carassiusauratus）的头瘤组织为检测子（Tester），40日龄红狮头金鱼幼鱼的头顶上皮组织为驱动子（Driver），构建金鱼头瘤相关基因的差减cDNA文库。以鲫管家基因octubilin为参照检测差减效率，结果表明该文库差减效率达210倍，效率较高。随机挑选阳性克隆进行接头特异引物的PCR检测，结果表明插入片段均在0．1～2．0kb之间。利用斑点杂交对PCR检测结果为单一条带的阳性克隆进行筛选并测序，获得高差异性的有效序列93条。通过生物信息学方法对文库序列进行比对、分类和注释，发现若干结缔形成基因和原癌／抑癌基囚等与金鱼头瘤彤成相关的功能基因，表明cDNA文库构建成功。Goldfish （Carassius auratus auratus） are the most important ornamental fish and have closely genetic relationship with Carassius auratus. Some goldfish have obvious and beautiful oranda wen. However,how the oranda wen forms and grows is remained unknown. In this study, suppression subtractive hybridization （SSH） strategy was employed to construct a cDNA library that represented genes related to the wen. Wen tissue of 1-year oranda goldfish was treated as the tester and epithelial tis- sue on the head of 40-days oranda fry was treated as the driver. The housekeeping gene o：-tubilin was used to detect the efficiency of the library and the octubilin mRNA expression decreased by up to 210- fold in the subtracted libraries compared with the umsubtracted samples, indicating a high subtractive effi- ciency. The result of nested PCR with adapter specific primers showed that the length of the inserts ranged from 100 bp to 2 000 bp. A total of 93 effective sequence with high otherness were identified by dot blot screen and sequencing. ESTs in the library were compared, classified and annotated to common data base by bioinformatics analysis, and a set of special genes such as connective related genes and tumor-related genes,which could be involved in wen formation process of oranda goldfish, were identi- fied,indicating that the cDNA library was constructed successfully.

关 键 词：金鱼 头瘤 差减抑制杂交 CDNA文库 

分 类 号：Q785[生物学—分子生物学]