刨花润楠SRAP-PCR体系建立与优化

Establishment and Optimization of SRAP-PCR System for Machilus pauhoi

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Mg^(2+)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、Mg^(2+)2.0 mmol/L、d NTP 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, d NTP concentration, Mg^2＋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 Mg^2＋, 0.225 mmol/L d NTP, 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.