花椒RAPD-PCR反应体系的建立与优化

Establishment and Optimization of RAPD-PCR Reaction System for Zanthoxyli Pericarpium

目的:建立并优化花椒随机扩增的多态性DNA-聚合酶链式反应(RAPD-PCR)体系。方法:以汉源花椒DNA为材料,采用正交试验对Mg2+、dNTP、DNA、引物、TaqE等反应条件进行优化。结果:花椒的PCR反应体系为25μL,包括ddH2O13.5μL、25mmol·L-1的MgCl22.0μL、2.5mmol·L-1的dNTPs20μL、10×Buffer30μL、10ng·μL-1的引物2.0μL、DNA2.0μL、5IU TaqE0.5μL;PCR反应程序为95℃预变性3min,94℃预变性2min,94℃变性50s,43.8℃退火1min,72℃延伸1min20s,35个循环,72℃最后延伸5min。用所建立的方法对21个产地的花椒样品进行测定,均可获得清晰、稳定的PCR扩增条带。结论:本研究可为RAPD分析应用于花椒的遗传多样性研究打下良好的基础。

OBJECTIVE：To establish and optimize the RAPD-PCR reaction system for Zanthoxyli Pericarpium.METHODS：The reaction conditions,such as Mg2＋,dNTP,DNA,primer and Taq E,was optimized by orthogonal test using DNA of Zanthoxyli Pericarpium in Hanyuan as experimental material.RESULTS：Total volume of PCR reaction system was 25 μL,including 13.5 μL ddH2O,2.0 μL 25.0 mmol·L-1 MgCl2,2 μL 2.5 mmol·L-1 dNTPs,3.0 μL 10×Buffer,2.0 μL 10 ng·μL-1 primer,2 μL DNA and 0.5 μL 5 IU TaqE.The PCR reaction sequence was to pre-denature at 95 ℃ for 3 min and 94 ℃ for 2 min,followed by denaturing at 94 ℃ for 50 s,annealing at 43.8 ℃ for 1 min,extending at 72 ℃ for 1 min 20 s,cycling 35 times,last extending at 72 ℃ for 5 min.Zanthoxyli Pericarpium from 21 habitats were determined by this method.The PCR amplification straps of these individuals were clear and stable.CONCLUSION：The study lays a good foundation for application of RAPD in genetic diversity study of Zanthoxyli Pericarpium.