木麻黄SSR-PCR反应体系的建立与优化

Establishment and Optimization of SSR-PCR Reaction System for Casuarina

为得到木麻黄SSR-PCR反应的最佳体系,以4个种(短枝木麻黄、山地木麻黄、粗枝木麻黄、细枝木麻黄)的24个无性系为材料,采用L_(16)(4^(5))正交设计对影响木麻黄SSR-PCR反应的4个因素(Taq酶、dNTP、Mg^(2+)和引物)在4个水平上进行了优化,并利用直观分析和方差分析两种方法对PCR结果进行评价。结果表明:引物、Mg^(2+)和dNTP均对木麻黄SSR-PCR反应结果有极显著的影响(P<0.01),影响程度由大到小为:引物>Mg^(2+)>dNTP,而Taq酶对扩增结果无显著影响;确定了两种木麻黄SSR-PCR反应体系(体系6和体系15),体系6为1×PCR buffer、2ng模板DNA、0.5μmol·L^(-1)引物、1.5 mmol·L^(-1) Mg^(2+)、0.1 mmol·L^(-1) dNTP、0.5 U Taq酶;体系15为1×PCR buffer、2ng模板DNA、0.5μmol·L^(-1)引物、1.75 mmol·L^(-1) Mg^(2+)、0.2 mmol·L^(-1) dNTP、1.25 U Taq酶,反应体系共10μL,不足部分用ddH2O补足。从节约成本和降低非特异性产物的角度考虑可将体系6作为最佳体系,体系15为备用体系;本试验所选荧光引物M26、M36的退火温度在52~62℃均可扩增出清晰明亮的条带。为简化操作步骤和减少非特异性产物,可选择60℃作为引物M26、M36的最佳退火温度。

In order to obtain the optimal reaction medium of SSR-PCR from casuarina,24 somaclonal clones from 4 species including Casuarina equisetifolia,C.junghuhniana,C.glauca and C.cunninghamiana,were selected as experimental materials.L_(16)(4^(5))orthogonal design was used to determine the optimum concentrations of four factors(primer,Mg^(2+),dNTP,Taq polymerase)within four concentration levels in the SSR-PCR reaction system of Casuarina respectively,and visual analysis and analysis of variance were used as evaluation.The results showed as follows:The three factors(primer,Mg^(2+),dNTP)had significant differences on the SSR-PCR reaction systems(P<0.01)respectively,and the order of significance was primer>Mg^(2+)>dNTP,while Taq polymerase had no significant difference.The two optimal SSR-PCR reaction systems(10μL)of Casuarina were determined,including system 6,which consisted of 1×PCR buffer,2ng template DNA,0.5μmol·L^(-1)primer,1.5 mmol·L^(-1)Mg2+,0.1mmol·L^(-1)dNTP,0.5 U Taq polymerase,and system 15,which contained 1×PCR buffer,2 ng template DNA,0.5μmol·L^(-1)primer,1.75 mmol·L^(-1)Mg^(2+),0.2 mmol·L^(-1)dNTP,1.25 U Taq polymerase.The system 6 was better than system 15.60℃was determined as the optimal annealing temperature of the fluorescent primers M26 and M36.