冬虫夏草cDNA-AFLP银染法显带研究

Establishment and Optimization of the AFLP Reaction System in Cordyceps Sinensis Sacc.

目的探讨影响冬虫夏草扩增片断长度多态性(Amplified Fragment Length Polymorphism,AFLP)的主要因素,建立并优化冬虫夏草AFLP反应体系。方法变性裂解液法提取冬虫夏草基因组cDNA,分别用一步法和两步法进行酶切与连接,再进行预扩增、选扩后,聚丙烯酰胺凝胶电泳分离,银染检测。结果本研究建立适用于冬虫夏草的AFLP体系:用乙醇沉淀cD-NA,建立的模板不含PCR反应抑制剂及其他酶反应抑制剂,可被限制性内切酶MseI和EcoRI完全酶切;确定两步法进行酶切、连接,酶切时间为37℃3h,连接时间为16℃过夜,缓冲液选用NEB公司buffer2;选择性扩增后的银染显色在10℃以下。结论本研究建立的反应体系适用于冬虫夏草基因组cDNA的AFLP银染法显带研究。

Aim To study factors affecting Amplified Fragment Length Polymorphism （AFLP） in Cordyceps sinensis Sacc. and to optimize the AFLP reaction system. Methods The denaturation lysate method was used to extract the genomic eDNA in Cordyceps sinensis Sacc. cDNA was digested by restriction enzyme and ligated with onestep method or two-step method. Then the products underwent pre-amplification and selected amplification. Finally PAGE electrophoresis and silver-staining were performed to detect cDNA strips. Results The AFLP reaction system in Cordyceps sinensis Sacc. was as follows. The genomie DNA was deposited by alcohol using CTAB method, uncontaminated by any inhibitor, cDNA was digested by restriction enzyme and ligated with two-step method, and completely digested by restriction enzyme of MseI and EcoRI at 37 ℃ for 3 hours. Buffer 2 from NEB Company served as the specific buffer in the experiment. Furthermore, the strips in PAGE electrophoresis were under 10℃. Conclusion The AFLP reaction system established and optimized in this experiment is suitable for silver-stained AFLP in Cordyceps sinensis Sacc.