三叶木通MSAP反应体系的优化及引物筛选  

Optimization of MSAP reaction system for Akebia trifoliata and primers screening

作　　者：高寰[1] 张铮[1,2,3] 周婷[1] 曹向然[1] 陈阳洋[1] 王喆之[1,2,3] 

机构地区：[1]陕西师范大学生命科学学院,陕西西安710062 [2]陕西师范大学药用资源与天然药物化学教育部重点实验室,陕西西安710062 [3]西北濒危药材资源开发国家工程实验室,陕西西安710062

出　　处：《中草药》2012年第3期572-576,共5页Chinese Traditional and Herbal Drugs

基　　金：陕西省科技攻关项目(2010K17-05-02);2010年国家级大学生创新性实验计划资助项目(101071825)

摘　　要：目的研究三叶木通表观遗传多样性,建立并优化三叶木通甲基化敏感扩增多态性(methylation sensitive amplifiedpolymorphism,MSAP)反应体系。方法以三叶木通叶片为材料,利用正交试验建立三叶木通MSAP的预扩增和选择性扩增的最佳反应体系。结果建立的MSAP最佳反应体系为酶切反应:HpaII/MspI 10 U,EcoR I 10 U;预扩增反应:总体积20μL,连接产物2μL,E00、HM00各125 ng,dNTPs 0.312 5 mmol/L,Mg2+1.875 mmol/L,Taq DNA聚合酶2 U;选择性扩增反应:总体积20μL,稀释200倍的预扩增产物5μL,引物E-ACT、HM-CAT各50 ng,dNTPs 0.250 mmol/L,Mg2+1.250mmol/L,Taq DNA酶1 U。利用优化的体系,最终从MSAP的80对引物中筛选出有效引物6对。结论建立的MSAP反应体系可用于后续三叶木通DNA甲基化的相关研究。Objective To analyze and compare the cell growth and accumulation of its secondary metabolites,flavonoids,in loose and compact calluses suspension cell culture of Tetrastigma hemsleyanum.Methods The calluses induced from the leaves of T.hemsleyanum were subcultured in different culture media,to screen loose and compact calluses,then established two suspension cell systems.The time courses of cell growth and yields of flavonoids were studied.Results Suitable culture period of loose callus suspension cell was 8 d,however,the suitable culture period of compact callus suspension cell was 20 d.The highest multiple proliferation was 3.68,however,the highest multiple proliferation of compact callus suspension cell was 2.65.The highest content of flavonoids was 11.7 mg/g,increased by 34.5%,compact callus suspension cell was 40.9 mg/g,increased by 212.2%,loose callus suspension cell had a high level of browning and varietal vitality decline after 4-5 subcultures.There was no significant changes in the subculture of the multicell suspension cultures.Conclusion Comparing with loose callus,the compact callus suspension cell shows a remarkable high content of flavonoids,and is preferable for subculture.

关 键 词：三叶木通 MSAP 遗传多样性 体系优化 引物筛选 酶切反应 

分 类 号：R282.12[医药卫生—中药学]