荷花SRAP-PCR反应体系的优化与确立  

Optimization and establishment of SRAP-PCR reaction system for Nelumbo nucifera Gaertn

作　　者：孙祖霞[1] 刘兆磊[1] 陈素梅[1] 陈发棣[1] 楼望淮[1] 郭海林[1,2] 

机构地区：[1]南京农业大学园艺学院,江苏南京210095 [2]江苏省中国科学院植物研究所,江苏南京210014

出　　处：《南京农业大学学报》2011年第6期53-58,共6页Journal of Nanjing Agricultural University

基　　金：国家公益性行业(农业)科研专项(200903020)

摘　　要：以荷花(Nelumbo nucifera Gaertn)品种‘新红’叶片为材料,采用L16(45)正交试验设计,对SRAP-PCR反应体系中的Mg2+、dNTPs、TaqDNA聚合酶、引物和模板DNA用量5因素进行了优化,并确立了适用于荷花SRAP-PCR的最佳反应体系。结果表明:荷花的SRAP-PCR最佳反应体系为:反应总体积10μL,包含2.0 mmol.L-1 Mg2+、300μmol.L-1 dNTPs、0.5 UTaqDNA聚合酶、4μmol.L-1上下游引物、50 ng DNA及10×PCR Buffer。各因素水平变化对反应体系影响由大到小依次为:TaqDNA聚合酶、Mg2+、引物、dNTPs、DNA。用48个荷花品种对优化的SRAP-PCR反应体系进行验证,均获得了条带清晰、多态性丰富的扩增图谱,证实了该体系的稳定性和适应性。Using Nelumbo nucifera Gaertn ‘Xinhong’ leaves as the experimental material,by the orthogonal experiment design L16（45）,five factors including concentration for Mg2＋ and dNTPs,dosage for TaqDNA polymerase,primer and template DNA in SRAP-PCR reaction system were optimized,and the optimization SRAP-PCR reaction system suitable for lotus were also established.The result showed that the optimal SRAP-PCR reaction system was as follows：total volume 10 μL,including 2.0 mmol·L-1 Mg2＋,300 μmol·L-1 dNTPs,0.5 U TaqDNA polymerase,4 μmol·L-1 primer,50 ng DNA and 10×PCR Buffer.The order of each factor in different levels affecting the result of PCR was：TaqDNA polymerase,Mg2＋,primer,dNTPs,DNA.The optimal SRAP-PCR reaction system was identified by means of genomic DNA from 48 varieties of N.nucifera Gaertn and the amplification pattern with clear band and rich polymorphism was obtained.It is concluded that the SRAP-PCR reaction system is steady and reliable.

关 键 词：荷花 SRAP-PCR 正交试验设计 反应体系优化 

分 类 号：S682.32[农业科学—观赏园艺]