润楠ISSR-PCR优化反应体系建立及引物筛选

Optimization of ISSR -PCR reaction system and primers screening in Machilus pingii

以润楠基因组总DNA为材料,利用单因素试验设计方法,对影响ISSR-PCR扩增的模板DNA、Mg2+、dNTPs、引物、Tag DNA聚合酶以及去离子甲酰胺等试验因子进行了优化试验,筛选出16条有效引物并确定了它们的退火温度,并检测了体系的重复性。优化后的反应体系为:20μL PCR反应体积中,10×PCR Buffer 2μL,2.0mmol/L Mg2+1.6μL,0.2 mmol/L dNTPs 2μL,0.4 mmol/L引物0.8μL,0.5U Taq DNA聚合酶0.1μL,40 ng DNA模板1.0μL,1.5%去离子甲酰胺0.3μL,ddH2O 12.2μL。扩增程序为:94℃预变性5 min,94℃变性30 s,据不同引物的退火温度复性45 s,72℃延伸90 s,反应37个循环,最后在72℃延伸7 min。润楠ISSR-PCR反应体系的建立为利用ISSR分子标记技术研究润楠的遗传多样性奠定了良好的基础。

In this paper,the different levels of concentration of template DNA,Mg2＋,dNTPs,primer,Taq DNA polymerase and formamide deionized were trailed by single factor experiment using Machilus pingii DNA,sixteen ISSR primers were selected out,and their annealing temperatures were also established.Furthermore,the optimized reaction conditions were examined for its repeatability.The optimal conditions of ISSR-PCR was established as follows：10 × PCR Buffer 2 μL,2.0 mmol/L Mg2 ＋ 1.6 μL,0.2 mmol/L dNTPs 2 μL,0.4 mmol/L primer 0.8 μL,0.5U Taq DNA polymerase 0.1 μL,40 ng DNA template 1 μL,1.5% formamide deionized 0.3 μL and ddH2O 12.2 μL in 20 μL reaction system.The augmentation procedure was：pree-denaturation at 94℃ for 5 min,denaturation at 94℃ for 30 s,annealing of 45 s due to denaturing temperature of different primer,extension at 72℃ for 90 s,reaction of 37 cycles and extension at 72℃ for 7 min.The establishment of the ISSR-PCR reaction conditions could settle favorable basis for the further study on the genetic diversity of Machilus pingii by using ISSR molecular marker.