怀地黄SRAP扩增体系的建立与引物的筛选

Establishment of SRAP amplification system for Rehmannia glutinosa and primer screening

关键词:  怀地黄  分子标记  扩增体系优化  SRAP  引物的筛选

Key words:  Rehmannia glutinosa  molecular marker  optimization of amplification protocol  SRAP  primer screening

摘要:

为建立适合怀地黄SRAP PCR分子标记技术体系,通过单因子实验分别研究了DNA模板浓度、TaqDNA聚合酶浓度、Mg2+浓度、引物浓度以及dNTP浓度对怀地黄SRAP扩增反应的影响,确立了适合怀地黄SRAP最佳反应体系为：在25 μL的反应体系中,模板DNA量20 ng/25 μL、2.5 mmol／L Mg2+、0.32 μmol／L的上下游引物、0.30 μmol／L的dNTP以及2.5 U Taq酶,并利用确定的体系从88个引物组合中筛选出12对适合怀地黄SRAP PCR反应的引物。

Abstract:

The single factor design was used to optimize SRAP amplification system of 22 Rehmannia glutinosa lines in five factors(the concentration of template DNA,Taq DNA polymerase,primer,Mg2+and dNTP). SRAP amplification system was established as follows:2.5 mmol/L Mg2+,0.30 mmol/L dNTP mixture,2.5 U Taq DNA polymerase,0.32 μmol／L each primer,20 ng template DNA and 2.5 μL 10 X PCR buffer in 25 μL SRAP reaction system were the best suitable PCR system. 12 primers were collected from 88 primers using the optimized amplification system above.