垂穗披碱草ISSR反应体系的正交优化

Optimization of the ISSR reaction system of Elymus nutans by orthogonal design

以垂穗披碱草(Elymus nutans)基因组DNA为模板,对ISSR-PCR反应体系中5个因素(TaqDNA聚合酶、Mg2+、模板DNA、dNTPs、引物)进行优化试验,建立垂穗披碱草稳定的ISSR-PCR反应体系及最佳扩增程序。在25μL反应体系中,最佳反应体系为2.5μL 10×buffer(不含Mg2+)、1.0 U Taq酶、2.0 mmol/L Mg2+、30～120ng模板DNA、0.25 mmol/L dNTPs、0.25μmol/L引物。在优化体系的基础上筛选出11条多态性丰富,扩增稳定的引物,并通过设置退火温度梯度、循环次数梯度、延伸时间梯度,得到垂穗披碱草ISSR-PCR最佳扩增程序。反应程序:94℃预变性2 min,94℃变性1 min,51℃退火1 min(视不同引物而定),72℃延伸1.5 min,共41个循环,72℃延伸10 min。通过对ISSR-PCR最佳反应体系和最佳扩增程序的验证,发现该反应体系和扩增程序具有较高的稳定性和较好的重复性。

Five factors（DNA polymerase, Mg^2＋, template DNA, dNTPs, primer） in ISSR-PCR reaction system were carried on the optimized experiment by taking the genomic DNA of Elymus nutans as tem- plate. The optimum reaction system and reaction process of E. nutans ISSR-PCR analysis were established. The results of this study showed that the volume of reaction system was 25 μL. It included 2.5 μL 10）〈buffer （except Mg^2＋）, 1.0 U TapDNA polymerase, 2.0 mmol/L Mg2＋, 30-120 ngtemplate DNA, 0.25 mmol/L dNTP and 0.25 μmol/L primer. Eleven primers with stable amplification and rich polymorphism were obatined based on the optimal PCR reaction system. The reaction program was devised for 2 min at 9℃in the first circle, 1 rain at 94℃, 51℃ for 1 rain （depended on different primer）, 72℃ for 1.5 rain for 41 cycles, and 72℃ for 10 min for extending,which was obatined across the gradient PCR experiments, including temperature gradient,cycle gradient and extending time gradient experiments. Validation of the optimistic ISSR-PCR reaction system and amplification program showed that the reaction system and amplification program had high stability and good reproducibility.