栝楼ISSR-PCR体系的正交优化

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

Establishment of ISSR Reaction System of Trichosanthes kirilowii Maxim

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, MgCl_2, 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) Mg~(2+), 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.