木荷ISSR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction System of Schima superba

在利用ISSR技术对木荷的遗传多样性进行研究的试验过程中,对影响PCR扩增效果的一些因素诸如模板DNA用量、引物用量、牛血清白蛋白浓度T、aq DNA聚合酶用量、4×dNTP浓度、Mg2+浓度以及退火温度等指标进行筛选和优化,确立了可用于木荷ISSR-PCR分析最适宜的PCR反应条件:10lμPCR反应体积中,1×Taq酶配套缓冲液(10 mmol/L Tris-HCl,pH值9.0,50 mmol/L KCl,0.1%TritonX-100),0.75 U Taq DNA聚合酶,1.5 mmol/L MgCl2,0.2 mol/L 4×dNTP,6 pmol引物,2 mg/ml牛血清白蛋白,10 ng模板DNA;适宜木荷ISSR扩增的退火温度为56.3℃。

In order to study the genetic diversity of Schima superba with ISSR technique, the factors which affected the ISSR amplification such as template DNA dosage, primers dosage, bovine serum albumin concentration, unit ofTaq DNA polymerase, dNTP concentration, Mg^2＋ concentration and annealing tempemture were selected and optimized. The results showed that the conditions being suitable for ISSR-PCR of Schima superba were as follows： 1×Taq buffer（ 10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KCl and 0.1% Triton X-100）, 0.75 U Taq DNA polymerase, 1.5 mmol/L MgCl2, 0.2 mol/L 4× dNTP, 6 pmol primers, 2 mg/ml BSA and 10 ng template DNA. The suitable annealing temperature of Schima superba in the ISSR-PCR reaction system was 56.3 ℃.