正交优化设计天冬ISSR反应体系的研究

Study on Optimization for ISSR Reaction System of Asparagus cochinchinensis(Lour.) Merr.with Orthogonal Design

根据正交试验设计的原理,设计了探索性正交试验和精致正交试验。以Mg2+、dNTP、引物和TaqDNA酶浓度4种因素3个水平对天冬ISSR-PCR反应体系进行研究,得到适合天冬的20μl最佳反应体系:1×PCR buffer,1.75 mmol/LMg2+,200μmol/L dNTPs,0.3μmol/L引物,1.2 U TaqDNA酶,50 ng模板DNA。为今后利用ISSR标记技术研究天冬的遗传多样性打下基础。

Orthogonal design was applied to the ISSR conditions optimization in this paper. Two orthogonal experiments were made to investigate the amplification efficiency of Asparagus cochinchinensis （Lour.）Merr. in four factors （ the concentration of Mg^2＋ , dNTP, Primers and Taq DNA polymerase） at three levels respectively. The result showed that 1×PCR buffer, 1.75 mmol/L Mg^2＋ ,200μmol/L dNTP,0.3μmol/L Primer, 1.2 U Taq DNA polymerase and 50 ng template DNA in a total volume of 20μL were suitable for ISSR amplification system of Asparagus cochinchinensis （Lour.） Merr.. This study provided a flundation for utilization ISSR-PCR labelling technique to study genetic diversity of Asparaguss cochinchinensis （Lour.） Merr.