巨竹ISSR反应体系的建立与优化

Establishment and optimization of ISSR amplification system in Gigantochloa levis

以巨竹叶片提取的基因组DNA为材料,用引物UBC810(序列为GAG AGA GAG AGA GAG AT)研究了PCR反应体系的主要成分、退火温度及循环次数对该种植物ISSR扩增结果的影响。结果表明,20μL的反应体系含40 ng模板DNA、0.6μmol.L-1引物,1.0 U Taq DNA聚合酶,2.5 mmol.L-1Mg2+,0.25 mmol.L-1dNTPs,1×Buffer。PCR扩增程序为:94℃预变性5 min;94℃变性45 s,54.5℃复性30 s,70℃延伸90 s,循环40次;72℃延伸10 min,置4℃保存。

Taking genomie DNA extracted from Gigantochloa levis leaves as the template material, the concentrations of PCR compo- nents, annealing temperature and cycles, whicll affected the ISSR amplification, were optimized with the primer UBC810 （primer sequence ： GAG AGA GAG AGA GAG AT）. The results showed that the 20 μL ISSR reaction system included 40 ng template DNA, 0.6 μmol ·L^-1 primer, 1.0 U Taq DNA polymerase, 2.5 mmol·L^-1 Mg^2＋, 0.25 mmol·L^-1 dNTPs,1×Buffer. The optimal PCR amplification process was 5 minutes at 94 ℃ for predenaturation, followed by 40 cycles ,each with 45 seconds at 94 ℃ for denaturation, 30 seconds at 54.5 ℃ for annealing, 90 seconds at 72℃ for extension,finally extension at 72 ℃ for 10 minutes and holding the samples at 4 ℃.