蕙兰ISSR-PCR反应体系的建立和优化（英文）

目的]以蕙兰（Cymbidium faberi Rolfe）基因组DNA为模板,对影响ISSR-PCR扩增结果的因素进行筛选和优化,建立适合蕙兰的ISSR-PCR的最佳反应体系。方法]利用改良的CTAB法提取蕙兰基因组DNA,并对影响ISSR-PCR扩增结果的因素进行优化。结果]获得了高质量的蕙兰基因组DNA并建立了最适的蕙兰ISSR-PCR体系（25μl）,即2.5μl10×PCRbuffer,2.0mmol/LMgCl2,60ng模板DNA,160μmol/LdNTPs,1.25UTaqDNA聚合酶,0.4μmol/L引物,双蒸水15.85μl;最佳扩增程序为：94℃预变性5min;94℃变性30s,复性温度比引物的Tm值低2～3℃,30s,72℃延伸50s,40个循环;72℃延伸7min。结论]该优化体系的建立可为今后利用ISSR标记技术进行蕙兰遗传多样性研究提供依据。

Establishment and Optimization of ISSR-PCR Reaction System for Cymbidium faberi Rolfe

Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows：25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃,followed by 40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair,extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.