钩栗ISSR-PCR反应体系的建立与优化

Establishment and optimization of Castanopsis tibetana Hance ISSR-PCR reaction system

本研究以钩栗叶片为实验材料,采用单因素和L16(45)正交实验探讨了DNA模板用量、Mg2+浓度、引物用量、Taq DNA聚合酶、d NTP用量以及不同退火温度对钩栗ISSR-PCR扩增的影响,建立了钩栗ISSR-PCR反应体系。研究结果显示,优化后的最佳反应体系为,20μL反应总体系中,含30 ng模板DNA,3 mmol/L Mg2+,0.4 mmol/L d NTPs,0.75 U Taq DNA聚合酶,0.3μmol/L引物;对钩栗DNA样品材料进行ISSR-PCR扩增体系的检测结果显示,该优化体系扩增的产物条带清晰,稳定性高,本研究为钩栗种质ISSR分子标记遗传结构分析提供技术基础。

By taking the Castanopsis tibetana Hance leaves as the tested materials, and adopting single factor orthogonal test, the effects of template DNA, Mg2＋ concentration, primers amount, Taq DNA polymerase, d NTPs dosage and different annealing temperature on ISSR-PCR amplification of C. tibetana were investigated, and further the C. tibetana ISSR-PCR reaction system was set up. The results show that the optimized reaction system was consisted as follows： in the total reaction system with amount of 20 μL, there were 30 ng template DNA, 3 mmol/L Mg2＋, 0.4 mmol/L d NTPs, 0.75 U Taq DNA polymerase, 0.3 μmol/L primers, respectively. The obtained DNA ISSR-PCR amplification system of C. tibetana the DNA sample materials were detected, and the findings suggested that the products＇ bands were clear with high stability, thus laying a foundation for ISSR markers genetic diversity researches of C. tibetana germplasm.