南瓜SRAP反应体系的建立与优化

Establishment and Optimization of SRAP Amplification System in Squash

以‘密本’南瓜作为筛选体系的材料,通过单因素试验对南瓜20μL SRAP-PCR扩增体系的Mg2+、dNTP、Taq酶、引物及DNA浓度和扩增程序的退火温度与循环次数进行优化,筛选出各组分的最佳浓度、最佳的退火温度和最佳循环次数。试验结果确立南瓜最佳的20μL SRAP体系为:0.2mmol·L-1dNTP,1.5U Taq酶,80ng DNA,0.16μmol·L-1的单条引物,Mg2+1.5mmol·L-1,2μL 10×Buffer;最佳扩增程序为:94℃预变性5min;94℃1min,35℃1min,72℃1min,5个循环;94℃1min,52℃1min,72℃1min 35个循环;最后72℃延伸10min。选用17个南瓜品种对确立扩增体系及扩增程序进行验证,检测结果表现为扩增产物条带清晰明亮、多态性丰富、特异性强、重复性好,表明本试验所确定的反应体系及反应程序适用于南瓜的SRAP分子标记。

In order to screen out the optimal concentrations of various components ,the best annealing temperature and the best cycle times ,we took an optimization experiment of 20 μL SRAP-PCR amplification system with single factor design which focused on the concentrated of Mg^ 2＋ ,dNTP ,Taq DNA polymerase ,primer ,template DNA and the amplification procedures of annealing temperature and cycle times by using the Squash ‘Miben’ as the material of filter system .Experimental results showed the optimal 20 μL SRAP amplification of Squash contained 0.2 mmol · L ^-1 dNTP ,1.5 U Taq DNA polymerase ,80 ng template DNA ,0.16 μmol · L^ -1 single primer ,1.5 mmol · L ^-1 Mg^2＋ ,2 μL 10 ＆#215; Buffer .And the best amplification procedure was pre-denaturation at 94℃ for 5 min . Next by 5 cycles of denaturation at 94℃for 1 min ,anneal at 35℃ for 1 min and the extension at 72℃ for 1min then 35 cycles of 94℃ for 1 min ,52℃ for 1 min and 72℃ for 1 min and a final extension at 72℃ for 10min . The amplification system and the amplification procedures were tested by using 17 variaties of Squash .The test results showed the amplification products which were clear and bright ,abundant polymorphism ,strong specificity and good repeatability .Accordingly ,it was suitable for SRAP analysis of Squash .