摘要
目的首次利用甲基化敏感扩增多态性(MSAP)技术开展广藿香表观遗传多样性研究,建立并优化广藿香MSAP技术分析的反应体系。方法对MSAP技术中关键的步骤,包括酶切反应中的酶切时间和基因组DNA酶切量,预扩增反应中连接产物的稀释倍数、选择性扩增中预扩增产物的稀释倍数等条件进行了优化。同时,利用优化的条件,对81对引物进行初步的多态性筛选。结果分步酶切,以1 000 ng DNA量进行EcoRⅠ(30 U)酶切5 h,纯化反应产物,再各取15μL的EcoRⅠ酶切纯化产物进行MspⅠ/HapⅡ(10 U)酶切5 h,可完全酶切。优化后的反应体系得到条带清晰、特异性好、背景涂布少的选择性扩增产物。结论建立了MSAP反应体系,保证MSAP图谱稳定、清晰;初步筛选出的32对引物组合,可用于后续广藿香DNA甲基化模式的相关研究。同时,也为其他药用植物提供甲基化敏感扩增多态性研究的依据。
Objective To investigate the diversity of epigenetic in Pogostemon cablin （Blanco） Benth., and establish the methylation sensitive amplified polymorphism （MSAP） system. Methods The essential factors, which influence the results of MSAP system, have been comparatively analyzed. For instance, reaction time of restricted enzymes, diluted multiples of the adapter-ligation DNA for pre-amplification products, and diluted factor of pre-amplification products for selective amplification were optimized. Meanwhile, the optimized conditions were used to conduct a preliminary polymorphism screening on 81 paris of primers. Results The digestion reactions were carried out separately. 1 000 ng DNA was digested with 30 units of EcoR I for 5 h. Then, 15 μL of EcoR Ⅰ digestion products were respectively taken to Msp Ⅰ and Hap Ⅱ restietion for 5 h in order to digest fully. The clear bands, specificity and low background of selective amplification product could be obtained in the reaction system. Conclusion Clear and stable MSAP natterns were achieved, and 32 pairs of primers were selected for the MSAP analysis. Those results provide fundamental referenee for further analysis of DNA methylation patterns on P. cablin and other medieinal plants.
出处
《广东药学院学报》
CAS
2014年第1期28-35,共8页
Academic Journal of Guangdong College of Pharmacy
基金
广东省科技计划项目(2011B031700041)
