观光木ISSR-PCR反应体系的建立及优化

Establishment and optimization of Tsoongiodendron odorum ISSR-PCR reaction system

利用正交设计实验对影响观光木ISSR-PCR扩增的主要因素(模板DNA、引物、dNTPs、Mg2+的浓度,TaqDNA聚合酶的用量以及退火温度)进行优化,建立观光木ISSR-PCR反应的最佳体系。结果表明:最佳反应体系(20μL)为:模板DNA50 ng,引物0.4μmol/L,dNTPs 0.25 mmol/L,MgCl22.5 mmol/L,Taq DNA聚合酶1.25 U。扩增反应程序为:94℃预变性5 min;94℃变性1 min,55℃退火30 s,72℃延伸90 s,35个循环;72℃延伸7 min;4℃保存。该反应体系的建立,为观光木遗传多样性分析提供了客观可靠的方法。

Through the orthogonal design experiments, the important factors of the inter-simple sequence repeat PCR （ISSR-PCR） amplification system, such as DNA template, primers, dNTPs, Mg2＋ concentration, dose of TaqDNA polymerase and annealing temperature fit for Tsoongiodendron odorum were optimized. The results show that the optimal reaction system by the volume of 20μL was established as follows： 50 ng DNA template, 0.4 9mol/L primers, 0.25 mmol/L dNTPs, 2.5 mmol/LMgCl2 1.25 U Taq DNA polymerase. The PCR procedure was： pre-denaturing at 94℃ fbr 5 minutes, 35 cycles of denaturation at 94 ℃ for 1 minute, annealing at 55 ℃ for 30 seconds and extension at 72℃ for 90 seconds, with 7 minutes final extension at 72℃ and then stored at 4℃. The orthogonal-based 1SSR-PCR reaction system provided a reliable method for genetic diversity analysis in Tsoongiodendron odorum.