摘要
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.
出处
《中国药房》
CAS
CSCD
北大核心
2011年第31期2889-2891,共3页
China Pharmacy
基金
成都市科技计划项目(06GGYB860SW-030)