锦带花ISSR-PCR反应体系的建立与优化

以锦带花幼叶为试材,提取锦带花基因组的DNA,并对其纯度、浓度进行检测,建立适合锦带花的ISSR-RCR反应体系.结果表明:DNA扩增效果良好,完全能满足进一步试验的需要.同时对锦带花ISSR-PCR反应体系中各个影响因素进行优化,建立了适合锦带花ISSR分子标记的最佳反应体系,即在20μL的反应体系中,含稀释浓度为3倍的DNA模板,0.20 mmol/LdNTP,稀释10倍体积高保真PCR缓冲液含(Mg2+)3.0 μL;1.00 μmol/L引物,1.25 U Taq酶.扩增程序为:94℃预变性5 min；94℃变性30 s,52.0℃退火45 s,72℃延伸1.50 min,45个循环；最后72℃延伸5 min,4℃保存.应用该ISSR体系对3份锦带花种质进行了扩增,证实了该体系的适用性和稳定性.

Establishment and Optimization of ISSR-PCR Reaction System in Weigela florida

Extracting DNA from young leaves of Weigela florida and the purity,concentration of the DNA were tested ISSR-PCR reaction system a suitable Weigela florida were established.The results showed that the DNA could be well amplified,which was fully able to meet the demand of further experiments.At the same time,impacting factors of Weigela florida ISSR-PCR reaction system were opti-mized,and established the best suitable reaction system for Weigela florida.That was in 20 μL reaction system,DNA template with 3 times of dilution concentration,0.20 mmol/L dNTP,10×Pfu PCR Buffer(Mg2+)3.0 μL,1.00 μmol/L primerand and 1.50 U Taq enzyme.The optimal PCR amplification process was:5 minutes at 94℃ for predenaturation,then followed by 45 cycles,each with 30 seconds at 94℃ for denaturation,45 seconds at 52.0℃ for annealing,1.50 minute at 72℃ for extension,finally extension at 72℃ for 5 minutes and holding the samples at 4℃.The system was applied in the amplification of three varieties of Weigela florida indicating the suitability and stability of the system.