摘要
用抑制性消减杂交技术（SSH）研究与鲤鱼（Cyprinus carpio）抗寒性状相关的基因。通过对荷包红鲤抗寒品系（Cyprinus carpio wuyanensis Tchang）和柏氏鲤（Cyprinus pellegnini Tchang）杂交F2代进行降温诱导实验，获得不同温度下的实验材料。利用抑制消减杂交技术构建了一个低温下差异表达基因的消减cDNA文库，共含有2000个克降。对其叶1480个克隆进行斑点杂交筛选，共得到60个阳性克隆，选取其中29个克隆测序。通过序列分析比对，13个克隆与已知基因序列高度同源，同源性为85％-98％；另外的13个克隆为未知新基因序列。在13个同源性较高的克隆序列巾，有brevican基因、AMP—acid连接酶、CFL2基因、催产素基因、主要组织兼容复合体（MHC）Ⅱβ链、锌指蛋白、细胞色素氧化酶、EPD-1基因、透明质酸结合蛋白多糖类、核受体／类视色素信号调节器等，其中9个基因上调表达，另有4个则抑制表达。这些基因的获得可为研究鱼类抗寒性状以及从分子水平上阐明其机制提供重要依据。
Suppression subtractive hybridization （SSH） is a powerful means to identify the differentially expressed genes including low, rare abundance transcripts. In this study, SSH was used to identify the cold tolerance-related genes in the brain of common carp （Cyprinus carpio ）. The materials were prepared by cooling the cross F2 of purse red carp（ Cyprinus carpio wuyanensis Tchang） cold-tolerant strains and Boshi carp（Cyprinus pellegnini Tchang） to different temperatures. After isolation of total RNA, double strand cDNAs were synthesized and digested with restriction enzyme RsaⅠ . The digested tester was then ligated to two different adaptors. Following the subtractive hybridization between the tester and the driver, a subtracted cDNA library, where enriched genes were expressed differentially under the lower temperature, was then constructed containing 2 000 clones. Among 480 clones detected by dot blotting, 60 positive clones were determined to differentially express, and 29 clones were sequenced and aligned on Internet, which showed higher identities among 13 clones. The major differentially expressed genes included brevican genes, AMP-acid ligase, muscle cofilin 2 （CFL 2） gene, isotocin gene, MHC class Ⅱβ chain, zinc finger protein, cytochrome oxydase, EPD-1 gene, sup-related nuclear receptor/retinoid signaling modulator and so on. Nine genes were induced up regulated, indicating the variation of regulating genes to adapt the lower temperature. Four genes were down regulated, indicating the reduced metabolic activities during the cooling process. This study plays an important role in researching the molecular genetic mechanism of fish cold tolerance at the molecular level.
出处
《中国水产科学》
CAS
CSCD
北大核心
2006年第2期193-199,共7页
Journal of Fishery Sciences of China
基金
国家"973"基础研究项目资助(2004CB117404)
关键词
抗寒机制
抑制消减杂交
消减cDNA文库
鲤鱼
cold tolerance mechanism
suppression subtractive hybridization （SSH）
subtracted cDNA library
Cyprinus carpio
