蒙古黄芪MSAP反应体系的建立与优化

Establishment and Optimization of MSAP Reaction System of Astragalus Mongolica

目的利用甲基化敏感扩增多态性(MSAP)技术开展蒙古黄芪表观遗传多样性研究,建立并优化蒙古黄芪MSAP技术的反应体系。方法以蒙古黄芪(Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao)为材料,采用单因素与正交设计法优化蒙古黄芪甲基化敏感扩增多态性(MSAP)技术各反应体系的关键影响因素,建立蒙古黄芪MSAP反应最佳体系。结果20μL双酶切反应体系中,加入EcoRⅠ和MspⅠ/Hpa II各0.5μL,于37℃水浴酶切6 h可酶切完全;最佳连接体系25μL,包含10μL酶切产物、0.2μL T4 DNA Ligase、1μL EcoR I-adapter、1μL Hpa II/Msp I-adapter、2.5μL 10×T4 Buffer;最佳预扩增反应体系25μL,包含2.5μL 10×PCR buffer、0.5μL dNTP、2.0μL连接产物、0.3μL Taq DNA polymerase、上下游引物各1.0μL;最佳选择性扩增体系25μL,包括2μL稀释20倍的预扩增产物,1.0μL 10×PCR buffer,1.0μL dNTP,0.2μL Taq DNA polymerase,0.8μL引物。结论优化后的MSAP体系经聚丙烯酰胺凝胶电泳检测显示扩增产物多态性好、条带清晰,表明该体系稳定、可重复,为后续进行蒙古黄芪DNA甲基化分析奠定了基础。

Objective To study epigenetic diversity of Astragalus mongolicus by methylation-sensitive amplification polymorphism(MSAP)technique,and to establish and optimize the response system of MSAP technique applicable to Astragalus Mongolicus.Methods Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao was used as material.The single factor and orthogonal design method were used to optimize the key influencing factors of the reaction system of Astragalus Mongolian methylation-sensitive amplification polymorphism(MSAP)technique,and the best reaction system of Astragalus Mongolian MSAP was established.Results 0.5μL each of EcoR I and Msp I/Hpa II were added to the 20μL double-enzyme digestion reaction system,and the digestion was completed in a water bath at 37℃for 6 h to complete the digestion;the optimal ligation system was 25μL,including 10μL digestion product,0.2μL T4 DNA Ligase,1μL EcoR I-adapter,1μL Hpa II/Msp I-adapter,2.5μL 10×T4 Buffer;25μL optimal pre-amplification reaction system,including 2.5μL 10×PCR buffer,0.5μL dNTP,2.0μL ligation product,0.3μL Taq DNA polymerase,1.0μL upstream and downstream primers;25μL optimal selective amplification system,including 2μL 20-fold diluted pre-amplification product,1.0μL 10×PCR buffer,1.0μL dNTP,0.2μL Taq DNA polymerase,0.8μL primers.Conclusion The optimized MSAP system was detected by polyacrylamide gel electrophoresis,which showed that the polymorphism was good,the system was stable,the bands were clear and repeatable,which laid the foundation for the subsequent MSAP analysis of Astragalus mongolica.