作 者：
梁利群;李绍戊;常玉梅;高俊生;孙效文;雷清泉
单 位：
中国水产科学研究院黑龙江水产研究所;哈尔滨理工大学
关键词：
抗寒机制;抑制消减杂交;消减cDNA文库;鲤鱼
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 wuyanensisTchang)和柏氏鲤(Cyprinus pellegniniTchang)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2 000个克隆。对其中480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中,有brevican基因、AMP-acid连接酶、CFL 2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。
译 名：
Application of suppression subtractive hybridization(SSH) to study of cold tolerance in common carp(Cyprinus carpio)
作 者：
LIANG Li-qun~(1,2),LI Shao-wu~(2,3),CHANG Yu-mei~(2),GAO Jun-sheng~(2,4),SUN Xiao-wen~(2),LEI Qing-quan~(1)(1.Harbin University of Science and Technology,Harbin 150080,China;2.Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;3.College of Life Sciences and Technology,Shanghai Fisheries University,Shanghai 200090,China;4.College of Life Sciences and Technology,Dalian Fisheries College,Dalian 116023,China)
关键词：
cold tolerance mechanism;suppression subtractive hybridization(SSH);subtracted cDNA library;Cyprinus carpio
摘 要：
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 F_(2) 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 Rsa Ⅰ.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 2000 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 Ⅱ β 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.
