用正交设计法优化辣椒ISSR-PCR反应体系

Optimization for ISSR Reaction System of Pepper by Orthogonal Design

采用正交设计L16(45)对辣椒ISSR-PCR反应体系的5个因素(Taq酶、Mg2+、模板DNA、dNTPs、引物浓度)在4水平上进行优化试验。结果表明,最佳反应体系为:在25μl总反应体系中,含1×PCR缓冲液、2.0 mmol Mg2+、1.5 unitTaqDNA聚合酶、0.25 mmoldNTP、0.48μmol引物、120 ng模扳DNA。扩增程序为:95℃预变性5 min;94℃变性45 s,51℃退火1 min,72℃延伸2min,进行38个循环;最后72℃延伸10 min。新优化的辣椒ISSR-PCR反应体系和程序有很好的重复性和稳定性。此研究为今后利用ISSR技术对辣椒进行种质资源分类、遗传图谱构建和基因定位提供了一个较理想的应用方案。

In this research,the orthogonal design was used to optimize ISSR-PCR system on pepper in for four levels of five factors（dNTPs,primer concentration Mg2＋,Taq DNA polymerase and DNA template） respectively.The results showed that the orthogonal experiment design was a practicable method and in a total volume of 25 μl SRAP-PCR system,The optimum reaction system of ISSR-PCR was 1×PCR buffer,2.0 mmol Mg2＋,1.5 unit Taq DNA polymerase,0.25 mmol dNTP,0.48 μmol primer and 120 ng template DNA in total 25 μl reaction volume complemented with ddH2O.Amplification procedure by PCR was pre-denaturation at 95 ℃ for 5 min,38 cycles of denaturation at 94 ℃ for 45 s,annealing at 51 ℃ for 1 min and extension at 72 ℃ for 2 min,and a final extension at 72 ℃ for 10 min.The new optimized ISSR-PCR system and procedure of pepper had good repeatability and stability.This work would provide basis for the analysis of diversity,genetic map construction and gene localization by using ISSR technique of pepper in the future.