桉树的ISSR反应体系建立及优化

Establishment and Optimization of ISSR Reaction System of Eucalyptus

以改进的CTAB法提取桉树嫩叶总DNA，进行ISSR分析，分别测试了退火温度、模板DNA浓度、Mg^2＋浓度、dNTPs浓度、TaqDNA聚合酶用量对反应结果的影响。桉树ISSR—PCR分析较适宜的扩增条件是：25μL PCR反应体积中，buffer（10mM Tris—HCl，pH9．0，50mM KCl，0．1％Triton X-100），1．25U TaqDNA聚合酶，4种dNTPs各0．2mM，0．4μM引物，2．0mM MgCl2，50ng模板DNA。最佳扩增程序为：94℃预变性5min，然后进行45个循环：9412变性45s，52℃～55℃复性45s，72℃延伸90s，循环结束后72℃延伸7min。

Eucalyptus genomic DNA extracted from its tender leaves by improved CTAB method was used as ISSR - PCR amplification template. The factors which affected the ISSR amplification such as annealing temperature, template DNA dosage, Mg^2＋ concentration, dNTP concentration and unit of Taq DNA polymerase were selected and optimized. The results showed that the conditions being suitable for ISSR - PCR of Eucalyptus were as follows ： 1 × Taq buffer （ 10 mM/L Tris - HCl , pH9.0 50 mM/L KCl and 0.1% Triton X- 100） , 1.25 U Taq DNA polymerase , 0.2 mM 4× dNTP, 0.4μM primers, 2.0 mM/L MgCl2 and 50 ng template DNA in total 25μL reaction volume. The suitable ISSR-PCR procedure was： predenaturation at 94℃ for 5 min, denaturation at 94℃ for 45 s, annealing at 52℃-55℃ for 45 s, extension at 72℃ for 90 s, reaction of 45 cycles and extension at 72℃ for 7 min.