【摘要】 目的:通过优化草鱼TRAP-PCR反应体系,将新型分子标记-靶位区域扩增多态性(target region amplified polymorphism,TRAP)引用到草鱼遗传多样性研究中。方法:以草鱼DNA为材料,分析了模板DNA、Mg2+、dNTPs、引物浓度,以及循环参数、退火温度对TRAP-PCR扩增结果的影响。结果:确立了稳定性强、重复性好的草鱼TRAP-PCR最佳反应体系和扩增参数:在25μl的PCR反应体系中,含约50ng模板DNA,1UTaq酶,1×PCR缓冲液,2.0mmol/L MgCl2,4种dNTPs各0.2mmol/L,固定引物与随机引物各15pmol;首先使模板在94℃变性3min;然后94℃变性1min,38℃退火1min,72℃延伸lmin进行5个循环;接着94℃变性45s,55℃退火45s,72℃延伸lmin再进行35个循环,最后72℃延伸7min。结论:TRAP-PCR反应体系稳定可靠,该新型分子标记可应用于草鱼遗传多样性研究中。更多还原

【Abstract】 Objectives: In order to apply a new kind of molecular marker,which is target region amplified polymorphism（TRAP）,to grass carp genetic analysis.In this paper,the influencing factors of target region amplified polymorphism（TRAP） were analyzed.Methods: By using genomic DNA of grass carp（Ctenopharyngodon idella）,adjusting the concentration of template DNA,Mg²⁺,dNTPs,primers and the parameters of cycles to select the best optimization.Results: The stable and reproducible optimum reaction system of TRAP-PCR amplification parameters were established for grass carp.The optimal system was in 25 μl reaction volume containing about 50 ng template DNA,1U Taq polymerase,1×PCR Buffer,2.0mmol/L MgCl₂,0.2mmol/L dNTPs and 15pmol of each primer.The amplification program was after 1 cycle initial denaturation at 94℃ for 3 min,each 5 cycles consisted of 94℃ denaturation for 1 min,38℃ annealing for 1 min,72℃ extension for 1 min,then each 35 cycles consisted of 94℃ denaturation for 45s,55℃ annealing for 45s,72℃ extension for 1 min and finally extension was at 72 ℃for 7 min.Conclusion: The TRAP-PCR amplification was stable and reproducible,this new kind of molecular was useful for grass carp genetic diversity analysis.更多还原