均匀设计优化黑木相思ISSR-PCR体系

The Uniform Design Optimization of ISSR-PCR System in Acacia melanoxylon

采用均匀设计，对影响黑木相思ISSR-PCR体系的引物、Mg^2＋、dNTP、Taq DNA聚合酶和模板DNA浓度等进行5因素5水平和5因素3水平两轮优化，建立了适合于黑木相思ISSR-PCR的体系：在20μL反应体系中，含引物0.30μmol·L^-1，Mg^2＋ 3.00mmol·L^-1，dNTP 0.15mmol·L^-1，Taq DNA聚合酶0.75U，模板DNA 2.00ng·μL^-1。在此基础上对扩增程序中的循环次数、退火温度、退火时间进行筛选，获得的扩增程序为：94℃预变性5min；接着进行33个循环：94℃变性35s，52-61.5℃退火52s，72℃延伸90s；循环结束后，72℃延伸10min。同时筛选得到10个扩增稳定、多态性丰富的ISSR引物。

Uniform design was used to optimize ISSR-PCR system in Acacia melanoxylon. Five influencing factors on ISSR-PCR, including primer, Mg^2＋, dNTP, Taq DNA polymerase and DNA template concentration were tested by uniform design. A suitable ISSR-PCR system for A. melanoxylon was established as follows ： each 20μL ISSR-PCR amplification reaction solution was consisted of 0.30μmol·L^-1 primer, 3.00mmol·L^-1 Mg^2＋, 0.15 mmol·L^-1 dNTP, 0.75U Taq DNA polymerase , 2.00ng·L^-1 DNA template. The suitable ISSR-PCR procedure was 1 cycle of pre-denaturing for 5 min at 94℃, 33 cycles of denaturing for 35 s at 94℃, annealing for 52 s at 52-61.5℃, extending for 90 s at 72℃ and the last cycle of extending for 10 min at 72℃. And 10 primers with stable amplification and rich polymorphism for ISSR were screened. Meanwhile, 10 suitable ISSR primers have been obtained.