利用正交设计法对叉叶苏铁ISSR-PCR反应体系的优化研究

比较CTAB法和SDS法提取叉叶苏铁（Cycas micholitzii）的总DNA，发现CTAB法是提取叉叶苏铁总DNA的较好方法；对叉叶苏铁ISSR-PCR反应体系中的4个主要因素dNTPs，Taq DNA聚合酶，引物及Mg²⁺进行最优条件筛选；采用单因素法探讨DNA模板量、退火温度和循环次数的最佳反应条件，建立了适合于叉叶苏铁的最佳ISSR-PCR反应体系 （20 μL体系）： 1×Buffer，75 ng DNA模板，dNTPs 250 μmol·L^-1， Taq酶1.0U，引物0.2 μmol·L^-1，Mg2+ 2.5mmol·L^-1，反应程序为：94℃预变性5 min，94℃变性1 min，52℃退火1 min，72℃延伸2 min，循环40次，72℃延伸10 min，4℃保存。本研究结果为苏铁纲植物的分子生物学和分子系统学研究提供理论参考。

Establishment of Cycas micholitzii ISSR-PCR Optimal Conditions with Orthogonal Optimization Method

CTAB method and SDS method were used to extract genomic DNA from Cycas micholitzii and CTAB method was testified to be more effective to C.micholitzii.The Orthogonal design was used to optimize ISSR-PCR amplification system in three levels of four factors dNTP, Taq DNA plymerase,Primer and Mg²⁺. Base on the pre-liminary experiment,other three factors（amount of DNA，anneal-ling temperature，circular times）were further optimized. A sutable ISSR-PCR reaction system was established, namely 20 μL reaction system contaning:1.0 U Taq DNA polymerase, 2.5 mmol·L^-1 Mg²⁺, 250 μmol·L^-1 dNTPs, 0.2 μmol·L^-1 Primer, 1×Buffer, 75 ng DNA. Programmed for an initial step of 5 min at 94℃, followed by 40 cycles of 1 min at 94℃, 1 min at 52℃ and 2 min at 72℃,and a final 10 min extension at 72℃. The result afford a reference to the research on molecular biology and molecular systematics of Cycadopcida.