以刨花润楠(Machilus pauhoi)1.5 a生小苗幼嫩叶片为试材,对影响刨花润楠SRAP-PCR扩增的模板DNA量、引物、dNTP和Mg2+体积摩尔浓度、Taq DNA聚合酶、退火温度6个主要因素进行优化.结果表明,SRAP-PCR的最佳反应体系为:25μL的SRAP-PCR反应体系中,2.5μL 10×PCR buffer、模板DNA量60 ng、Mg2+2.0 mmol/L、dNTP 0.225 mmol/L、引物0.3μmol/L和Taq DNA聚合酶1.25 U.对优化的反应体系和扩增程序的验证结果表明,优化的刨花润楠SRAP-PCR反应体系和扩增程序是稳定可行的.
Machilus pauhoi is a tree specie with variety of economic value and development prospects. This study aimed to establish an optimized SRAP-PCR system for M. pauhoi, and the young leaves of the 1.5 years old seedlings were used as test materials. Six quality factors including the template DNA, primer concentration, dNTP concentration, Mg2+concentration, Taq DNA polymerase, and annealing temperature were optimized for M. pauhoi SRAP-PCR assay. The obtained results suggested a optimized reaction system of SRAP-PCR (total 25 μL) involving 2.5 μL 10×PCR buffer, 60 ng DNA, 2.0 mmol/ L Mg2+, 0.225 mmol/L dNTP, 0.3 μmol/L primer, 1.25 U Taq DNA polymerase. The verification results showed that the optimized SRAP-PCR reaction system and amplification program were stable and feasible.