枇杷属植物ISSR反应体系的建立和优化

Establishment and optimization of ISSR reaction system for species of Eriobotrya

首次通过正交实验,对影响枇杷属植物ISSR反应较大的Mg2+、Taq酶、dNTPs、引物、模板DNA浓度进行筛选,并对扩增反应程序进行优化。优化后的反应体系为:25μL反应体系中,含10×buffer2.5μL,Mg2+浓度2.0mmol·L-1,Taq酶1.5U,引物0.3μmol·L-1,模板DNA60ng,dNTPs0.15mmol·L-1。反应程序为94℃预变性5mim;94℃变性1mim,退火温度70s,72℃延伸1.5mim,40次循环;72℃延伸7mim,4℃保存。

The concentration of Mg^2＋, Taq polymerase, dNTPs, primer and template DNA, greatly influencing ISSR-PCR of loquat,were optimized by orthogonal design in this study, and the reaction procedure was also improved. Thus an optimized ISSR reaction system was established for the first time for loquat. The total 25 μL contained 10xbuffer 2.5 μL,2.0 mmol ·L^-1 Mg^2＋, 1.5 U Taq polymerase, 0.3 μmol ·L^-1 primer, 60 ng template DNA, and 0.15 mmol ·L^-1 dNTPs. The reaction procedure was： 94℃for 5 min ,followed by 40 cycles of 94℃ for 1 min ,annealing temperature for 70 s ,72℃ for 1.5 mim ,and was terminated with a 7 min extension step at 72 ℃.