摘要
以牡丹叶片DNA为模板,对SRAP-PCR反应程序进行研究,确定适合的反应程序,即94℃预变性5 min;94℃变性1 min,33℃退火1 min,72℃延伸1 min,共5个循环;随后94℃变性1 min,52℃退火1 min,72℃延伸1 min,共35个循环;最后72℃延伸10 min。基于毛细管电泳技术,采用正交设计L18(37)对牡丹SRAP-PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTP,引物)3个水平进行了优化,构建了牡丹SRAP最佳反应体系:模板DNA 50 ng,dNTP 0.25 mmol/L,Mg2+浓度2.5 mmol/L,引物浓度0.4μmol/L,TaqDNA聚合酶0.5 U,总体积为25μL。各因素对扩增结果影响程度均不同:dNTPs>引物>DNA模板>Mg2+>Taq酶。运用该体系从756个SRAP引物组合中筛选出多态性好、条带清晰的26个引物组合,并证明了该体系稳定可靠。该体系的建立以及引物组合的确定为今后利用SRAP分子技术进行牡丹的相关研究奠定了科学基础。
In this study, the procedure of SRAP - PCR was determined ： an initial denaturing step was performed at 94 ℃for 5 min ; followed by 5 cycles at 94℃ for 1 min, 33℃ for 1 min and 72℃ for 1 min ; followed by 35 cycles at 94℃ for 1 min, 50℃ for 1 min, 72 ℃ for 1 rain, and a final extension at 72℃ for 10 rain. The orthogonal design was used to optimize SRAP-PCR system with five factors at three levels respec- tively based on capillary electrophoresis. The results showed that the optimized SRAP reaction mixtures for Paeonia suffruticosa Andr. ： （ total volume 25 μL） 50 ng DNA template, 0.25 mmolL dNTP, 2.5 mmol/L Mg2＋, 0.4 p, mol/Lprimer, 0.5 U Taq DNA polymerase. The concentration of dNTP had the greatest effect and DNA template had the least effect on the result. And twenty-six pairs of primers were selected with abun- dant polymorphism above 70% from 756 pairs of primers. The optimized SRAP-PCR system and pairs of prim- ers could be applied to related research of Paeonia suffruticosa Andr.
出处
《江西农业大学学报》
CAS
CSCD
北大核心
2012年第1期158-164,共7页
Acta Agriculturae Universitatis Jiangxiensis
基金
国家林业局林业公益性行业科研专项(200904050)
关键词
牡丹
SRAP
毛细管电泳
体系优化
引物筛选
Paeonia suffruticosa Andr.
SRAP marker
capillary electrophoresis
optimization
selection of primers
