【摘要】 目的:建立栝楼最佳ISSR-PCR正交优化体系,为开展栝楼ISSR分子标记奠定技术基础。方法:采用正交试验设计对影响栝楼ISSR-PCR扩增的重要参数(DNA模板、MgCl2、dNTPs、引物、TaqDNA聚合酶)进行优化试验,同时进行不同温度梯度试验和ISSR体系筛选。结果:最佳的栝楼ISSR-PCR的反应体系(20μl)为:30ng模板DNA,2.0mmol/L MgCl2,0.3mmol/L dNTPs,0.5μmol/L引物,0.5U Taq DNA聚合酶;退火温度为52℃~55℃;扩增反应程序为:94℃预变性5min;94℃变性30s,52℃退火1min,72℃延伸2min,35个循环;72℃延伸7min;4℃保存。结论:建立了栝楼的最佳ISSR反应体系,为栝楼种质鉴定提供了更客观可靠更多还原

【Abstract】 Objective:The optimal orthogonal ISSR-PCR of Trichosanthes lay a foundation for Trichosanthes ISSR molecular marker technology in this study.Method:The optimal experiments of the importmant parameters(DNA template,MgCl2,dNTPs,primers,TaqDNA polymerase) of Trichosanthes ISSR-PCR amplification through the orthogonal experimental design method,and different PCR temperatures,were studied.Result:The suitable ISSR-PCR reaction system(20μl) was established as follows:30ng DNA templet,2.0mmol·L-1 Mg2+,0.3mmol·L-1 dNTPs,0.5U TaqDNA polymerase,0.5μmol·L-1 primer;the suitable PCR reactions were predenaturing at 94℃for 5min,35 cycles of denaturation at 94℃ for 30s,annealing at 52℃-55℃ for 1 minute and extension at 72℃ for 2 minutes,with 7 min final extension at 72℃,and then saved at 4℃.Conlucsion: It provided a more objective and more reliable method of the best Trichosanthes ISSR reaction system for identification Trichosanthes germplasm.更多还原