南瓜SRAP扩增体系与扩增程序的优化  

Optimization of SRAP Reaction System in Squash

作　　者：刘泽发[1,2] 孙小武[1,2] 罗伏青[2] 张龑[1] 寇明明[1] 董亚静[2] 

机构地区：[1]湖南农业大学园艺园林学院,湖南长沙410128 [2]湖南省瓜类研究所,湖南邵阳422001

出　　处：《西北农业学报》2008年第6期148-152,183,共6页Acta Agriculturae Boreali-occidentalis Sinica

摘　　要：利用L45正交设计对影响南瓜SRAP反应体系的MgCl2浓度、dNTPs浓度、Taq酶含量、引物浓度及模板DNA浓度等5个因素进行了筛选,快速得到稳定性和重复性好的南瓜SRAP扩增体系。对复性温度、PCR循环次数等影响南瓜SRAP扩增结果的重要因素进行了优化。最终优化的南瓜SRAP反应体系为25μL反应液中:10×buffer2.5μL,0.2 mmol/L dNTPs,引物0.2μmol/L,1.5 mmol/LMgCl2,DNA模板6 ng/μL,Taq酶1.5 U。本研究最终确立的PCR反应程序为:94℃预变性5 min,94℃变性40 s,35℃退火40 s,72℃延伸90 s,进行5个循环,94℃变性40 s,50℃退火40 s,72℃延伸90 s,进行32个循环,最后72℃延伸5 min。在此条件下得到的SRAP标记可为南瓜遗传多样性、分子标记及辅助育种等研究提供稳定有效的手段。The optimum reaction system of SRAP in squash was studied in order to ensure stability and product bility of SRAP. After testing some important influencing factors of SRAP in squash such as MgCl2 ,dNTPs, Taq DNA polymerase, primer, template DNA, the temperature for annealing and the number of cycles,the results showed that in 25 uL SRAP reaction system , the optimum concentration were 1.5 mmol/L MgCl2, 0. 2 mmol/L dNTPs, Taq DNA polymerase 1. 5 U, primer 0.2 ng/uL, template DNA 6 ng/uL. The PCR amplification procedure used in this study was as follow： pre-denature at 94℃ for 5 min, then 94℃ 40s,35℃ 40s, 72℃ 90s,for 5 cycles, and then 94℃ 40s,50℃ 40s, 72℃ 90s, for 32 cycles. Finally extended at 72℃ for 5 rain. The results also indicated that the optimized conditions of SRAP could provide an effective means for the research of genetic diversity, molecular marker and auxiliary breeding in squash.

关 键 词：南瓜 SRAP 反应体系 

分 类 号：S642.1[农业科学—蔬菜学]