正交优化法建立沙打旺ISSR-PCR最佳反应体系  

Establishment of Astragalus adsurgens' ISSR-PCR Optimal Conditions with Orthogonal Optimization Method

作　　者：陈志宏[1] 黄琳凯[2] 张新全[2] 王志刚[1] 

机构地区：[1]全国畜牧兽医总站畜禽牧草种质资源保存利用中心,北京100094 [2]四川农业大学动物科技学院草业科学系,四川雅安625014

出　　处：《安徽农业科学》2006年第13期2980-2982,共3页Journal of Anhui Agricultural Sciences

摘　　要：根据正交试验设计的原理,设计了探索性正交试验和细调性正交试验来确定沙打旺ISSR-PCR体系中各成分的浓度。得到既稳定又能扩出最多条带的适合沙打旺的ISSR-PCR最佳反应体系,即20μl的反应体系中含有1×buffer,dNTP 0.2 mmol/L,Taq酶1.0 U,引物0.3μmol/L,Mg2+2.5 mmol/L,DNA模板2.5 ng/μl。然后对沙打旺ISSR-PCR最佳反应体系进行梯度退火试验,得到这条引物的最佳退火温度为54.1℃。这一最佳体系的建立为今后利用ISSR标记技术,研究沙打旺的遗传多样性提供了标准化程序。Orthogonal design is applied to the ISSR-PCR conditions optimization in this paper. Two orthogonal experiments were made to investigate the amplification efficiency of different amplification conditions. As a result, a satisfactory ISSR technique system for Astragalus adsurgens with desirable repeatability and polymorphic bands was established.In a total volume of 20μl ISSR-PCR system, it contained 1 × buffer,0.2 mmol/L dNTP, 0.3 μmol/L Primer,2.5 mmol/L Mg^2＋ ,1U Taq DNA polymerase and 2.5 ng/μl template DNA. The optimal annealing temperature of this primer for ISSR-1PCR reaction was proposed by gradient PCR and it was 54.1℃. The result provided standardizing ISSR-PCR program for the analysis of genetic diversity of Astrugabus adsurgens.

关 键 词：沙打旺 ISSR 正交优化 反应体系 

分 类 号：Q946-33[生物学—植物学]