红芪ISSR-PCR体系的建立及优化  

Establishment and Optimization of ISSR-PCR Reaction System for Hedysari Radix

作　　者：强正泽 王燕 李硕 王明伟 李成义 

机构地区：[1]甘肃中医药大学药学院,兰州730000

出　　处：《分子植物育种》2017年第8期3117-3128,共12页Molecular Plant Breeding

基　　金：国家自然科学基金项目(81360621)资助

摘　　要：通过单因素与中心复合设计相结合的方法建立优化红芪ISSR-PCR反应体系,并筛选引物,优化电泳时间及扩增循环数。建立的红芪ISSR-PCR反应体系为:25μL反应体系中10×PCR Buffer(Mg^(2+))3.5μL、模板DNA(30 ng/μL)2μL、PCR扩增引物2μL、Taq DNA聚合酶为1.25 U、d NTPs为2μL,dd H_2O 14.5μL,建立的扩增程序:94℃预变性5 min,开始34个循环;94℃变性30 s,后据不同退火温度的引物复性45 s,72℃延伸2 min,循环结束后72℃延伸7 min。本研究筛选出红芪扩增的引物17条;PCR反应产物电泳时间为120 min,最佳循环数为34,建立了稳定的体系,为进一步研究红芪的遗传多样性及遗传结构提供了帮助。Through combination of single factor experiment and central composite design to establish and optimize the ISSR-PCR reaction system for Hedysari Radix, screen the primers, and to optimize the electrophoresis time and the amplification cycle numbers. The established ISSR-PCR reaction system as： a 25 μL volume of with10×PCR Buffer（Mg2＋） of 3.5 μL, DNA of 2 μL, primer of 2 μL, Taq DNA polymerase of 1.25 U, d NTPs of 2.0 μL,dd H2 O of 14.5 μL. The established amplification procedures as： denaturation for 5 min at 94℃ after 34 cycles;denaturation for 30 s at 94℃, then according to different annealing temperature primer annealing for 45 s,extension for 2 min at 72℃, extension for 7 min after the end of the cycle at 72℃. In this research, 17 primers of Hedysari Radix were screened. The research showed that the best electrophoresis time was 120 min, and the optimal cycle number was 34. It established a stable system, and provided help for the further study on the genetic diversity and genetic structure of Hedysari Radix.

关 键 词：红芪 ISSR-PCR体系 单因素 中心复合设计 

分 类 号：S567.239[农业科学—中草药栽培]