利用单因子和正交设计双重实验法优化鹧鸪茶RAPD-PCR反应体系

为了建立鹧鸪茶RAPD-PCR的优化反应体系，首先通过单因素试验选定其各影响因子比较适宜的浓度范围，再利用正交试验设计方法，对影响鹧鸪茶RAPD-PCR反应的5种因素进行四水平优化试验。并运用SAS软件对试验结果进行了分析，最后确定优化的RAPD-PCR反应体系为：10×Buffer缓冲液2.5 μL+Mg2+ 2.5 mmol/L + dNTPs 0.2 mmol/L + TaqDNA聚合酶1.5 U + S28引物0.48 mmol/L + 80 ng模板，定容至25 μL。PCR扩增程序为：94℃预变性4 min，然后按94℃变性30 s，38℃退火45 s，72℃延伸120 s，进行45个循环，最后72℃延伸10 min；16℃保存。该优化的RAPD-PCR反应体系具有良好的稳定性和重现性，可应用于鹧鸪茶不同居群间亲缘关系和遗传多样性分析。

Optimization of RAPD-PCR Experimental System in Mallotus oblongifolius (Miq.) Muell.-Arg. Using the Dual Experiments of the Single Factor and the Orthogonal Design

In order to establish the optimized RAPD-PCR（randomly amplifiled polymorphic DNA-p olymerase chain reaction）experiment system for Mallotus oblongifolius（Miq.）Muell.-Arg.,the single factore xperiment was firstly involved to detect the suitable concentration ranges of different influential factors,andt hen the orthogonal design was involved to optimize RAPD-PCR amplification system of M.oblongifolius in 5f actors（Mg 2＋ ,dNTPs,primer,Taq polymerase,DNA template）at 4 levels,respectively.Meanwhile,theo btained data were analyzed by software SAS.An optimal RAPD reaction system was finally established.N amely,2.5μL 10×PCR buffer,2.5 mmol/L Mg 2＋ ,0.2 mmol/L dNTPs,1.5 U Taq polymerase,0.48 mmol/Lp rimers,and 80 ng DNA template were contained in 25μL reaction solution.The PCR amplification programw as pre-denaturing at 94℃for 4 min,then denaturing at 94℃for 30 s,annealing at 38℃for 45 s,extension at7 2℃for 120 s,for 45 cycles,at last extension at 72℃for 10 min.The productions were stored at 16℃.Theo ptimized RAPD-PCR reaction system was suitable for the study of relationship and genetic diversity of M.o blongifoliu among the different populations.