正交设计优化太子参ISSR-PCR反应体系  

Optimization of ISSR-PCR System for Pseudostellaria Heterophylla

作　　者：王小燕[1] 叶祖云[1] 陈美霞[1] 史辉[1] 

机构地区：[1]宁德师范学院生物系,福建宁德352100

出　　处：《邵阳学院学报（自然科学版）》2015年第2期29-34,共6页Journal of Shaoyang University：Natural Science Edition

基　　金：宁德师范学院服务海西建设项目(2011HQ102)

摘　　要：利用正交设计实验对影响太子参ISSR-PCR扩增的重要因素(d NTPs、引物、Taq酶、模板DNA浓度)进行优化,以建立太子参ISSR-PCR的优化反应的最佳体系,25μL反应体系中包含d NTPs300μmol/L,引物500nmol/L,Taq酶1U及模板DNA 50ng.适宜的扩增程序为94℃预变性5min;94℃变性40s,46℃退火45s,72℃延伸2min,41次循环;最后于72℃延伸7min,4℃进行保存.该正交体系的建立,将为太子参种质资源鉴定及遗传多样性的研究提供技术支持.Through the orthogonal design experiments, the important factors of the inter simple sequence repeat PCR （ISSR- PCR） amplification system, such as dNTPs .primers .Taq polymerase.DNA template fit for Pseudostellaria heterophylla was optimized to establish the suitable PCR reaction system as follows： the 25μL ISSR-PCR system contains 50ng template DNA,300μmol/L dNTPs,50Onmol/L Primer, and 1UTaq DNA polymerase. The suitable PCR program was predenaturing at 94℃ for 5 minutes; denaturing at 94℃ for 40 seconds, annealing at 46℃ for 45 seconds, extending at 72℃ for 2 minutes,41 cycles, and extending at 72℃ for 7 minutes, then remained at g ℃. This optimal system would offer a technique of ISSR molecular marker suitable to PseudosteUaria heterophylla identification on germplasm and genetic diversity.

关 键 词：太子参 正交优化 ISSR—PCR 

分 类 号：R284.2[医药卫生—中药学]