鲤TRAP分子标记反应体系的建立与优化  

Establishment and Optimization of Reaction System for TRAP Markers in Common Carp (Cyprinus carpio)

作　　者：曲疆奇[1] 苏胜彦[2] 董在杰[1,2] 明俊超[1] 梁政远[1] 袁新华[1,2] 

机构地区：[1]南京农业大学无锡渔业学院,无锡214081 [2]中国水产科学研究院淡水渔业研究中心农业部淡水鱼类遗传育种和养殖生物学重点开放实验室,无锡214081

出　　处：《动物学杂志》2010年第3期72-78,共7页Chinese Journal of Zoology

基　　金：现代农业产业技术体系建设专项资金项目(No.nycytx-49);国家"十一五"科技支撑计划项目专题(No.2006BAD01A1208);农业部淡水鱼类遗传育种和养殖生物学重点开放实验室开放课题(No.BZ2009-06)

摘　　要：针对鲤(Cyprinus carpio)这一主要水产养殖品种设计了靶位区域扩增多态性(target region amplified polymorphism,TRAP)分子标记的反应体系,对影响TRAP反应体系的各参数,包括Mg2+、dNTPs、TaqDNA聚合酶、模板DNA和引物浓度进行了优化,建立了适合鲤的、稳定、可重复的TRAP-PCR反应体系。在15μlPCR反应体系中,Mg2+浓度为1.5mmol/L、dNTPs浓度为0.35mmol/L、两个随机引物浓度均为3pmol/L、固定引物浓度为10pmol/L、含DNA模板60ng、TaqDNA聚合酶1.0U。鲤的TRAP反应程序为:94℃4min,1个循环;94℃45s,35℃45s,72℃1min,5个循环;94℃45s,53℃45s,72℃1min,35个循环;72℃10min,1个循环。这一优化体系的建立为今后进行鲤群体遗传多样性、种质鉴定、遗传连锁图谱及亲缘关系分析等方面的研究提供了新的分子标记。The reaction system for TRAP makers of Common Carp （Cyprinus carpio）,one of the major cultured fishes,was designed. The factors of reaction system including Mg2 ＋ ,dNTPs,Taq DNA polymerase,DNA template and primer concentrations were optimized and a stable,repeatable TRAP-PCR system for Common Carp was established. The PCR was performed with a final volume of 15 μl reaction solution containing 1. 5 mmol /L of Mg2 ＋ ,0. 35 mmol /L of dNTPs,3 mmol /L of each unlabeled 700- and 800- arbitrary primers,10 pmol /L of the fixed primer,60 ng of DNA template and 1. 0 U Taq DNA polymerase. The TRAP reaction program consisted of a pre-denaturing of template DNA at 94℃ for 4 min,followed by 5 cycles of denaturing at 94℃ for 45 s,annealing at 35℃ for 45 s,prolonging at 72℃ for 1min,35 cycles of denaturing at 94℃ for 45 s,annealing at 53℃ for 45 s,prolonging at 72℃ for 1min,and a final prolonging at 72℃ for 10min. This optimized TRAP- PCR reaction system provided a new molecular marker for the future study of genetic diversity,germplasm identification,genetic linkage map construction and relationship analysis in Common Carp.

关 键 词：鲤 TRAP标记 体系 优化 

分 类 号：S917.4[农业科学—水产科学]