葡萄ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Grape(Vitis vinifera L.)

采用正交设计L9(34)对影响葡萄ISSR-PCR反应体系的4个因素(dNTP、TaqDNA聚合酶、引物、模板DNA)在3个浓度水平上进行试验,并通过直观分析初步确定其反应体系;在此基础上,通过单因素试验探讨了dNTP、TaqDNA聚合酶、引物、模板DNA、退火温度及循环次数等因素或条件对葡萄ISSR-PCR扩增结果的影响,确定最佳反应水平。最终建立了葡萄ISSR-PCR扩增的最佳反应体系:在25μL的反应体系中,dNTP浓度0.2 mmol/L,TaqDNA聚合酶的用量0.5 U,引物浓度0.4mmol/L,DNA模板用量40 ng。反应程序:94℃预变性5 min;94℃变性1 min,52℃退火1 min,72℃延伸1 min 30 s,40次循环;最后72℃延伸10 min,10℃保存。

The ISSR-PCR（Inter-simple sequence repeat-Polymerase chain reaction product） amplification system for grape in three levels of four factors（dNTP,Taq DNA polymerase,primer,DNA template） were investigated by using orthogonal design and was determined preliminarily through the result direct analysis.On this basis,the research discussed effect of components to the PCR amplification,including dNTPs,Taq DNA polymerase,primer,DNA template,annealing temperature and cycle number through ladder experiments.The suitable ISSR-PCR reaction system for grape was established ultimately,namely 25 μL reaction system containing 0.2 mmol/L dNTP,0.25 U Taq DNA polymerase,0.4 mmol/L primer,40 ng DNA template.Amplification conditions were performed as initial DNA denaturation at 94℃ for 5 min,followed by 40 cycles of 1 min denaturation at 94℃,1 min annealing at 52℃ and 1 min 30 s of extension at 72℃ with a final extension time at 72℃ for 10 min,preserved under 10℃.