小菜蛾ISSR-PCR反应体系的建立与正交设计优化

摘要

To establish and optimize ISSR-PCR reaction system for Plutella xylostella , based on the genomic DNA of P. xylostella, the factors influencing ISSR system were explored with the L16 (45) orthogonal design. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. The optimal conditions for ISSR reaction system (20 μL) were determined as follows: 1. 5 mmol/L of Mg2+ , 2. 5U of Taq DNA polymerase, 0.2 mmol/L of dNTPs mixture, 1.25 μmol/L of each primer, and 20ng of template DNA. The reaction program was as followed: initial denaturation for 5.0 min at 94 ℃ , 35 cycles of denaturation for 45s at 94 ℃, annealing for 1.0 min at 40-61℃ , extension for 1.5 min at 72℃ , with a final extension of 10.0 min at 72 ℃. The clear and reproducible DNA bands were obtained by the established ISSR-PCR reaction system.%以小菜蛾(P lutella ylostella)基因组DNA为模板,采用L16(45)正交试验设计方法,建立小菜蛾的ISSR最佳反应体系.通过梯度退火试验,确定不同引物的最适退火温度.优化得到的反应体系(20 μL)为:Mg2+浓度1.5 mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2 mmol/L、引物浓度1.25 μmol/L、模板DNA量20 ng.反应程序为:94℃预变性5.0min;94℃变性45.0 s,40～61℃(不同引物退火温度各异)退火1.0 min,72℃延伸1.5 min,40个循环；72℃延伸10.0min;10℃保存.利用所建立的ISSR-PCR反应体系,获得了清晰、重复性好的DNA谱带.