CAJ | 학술논문

为建立粗皮桉 ISSR-PCR 优化反应体系，先通过单因素试验确定影响粗皮桉 ISSR-PCR 5个因素(Mg2+、dNTP、Taq DNA 聚合酶、DNA 模板、引物)的较适宜浓度范围，在此基础上进一步通过正交试验对5个因素4个水平进行优化，并用 DPS 软件分析试验结果。结果表明粗皮桉 ISSR-PCR 的优反应体系为：在25μL 反应体系中，10×PCR buffer 2.5μL、MgCl 2.0 mmol·L-1、dNTPs 0.3 mmol·L-1、Taq DNA 聚合酶1.25 U、DNA 模板60 ng、引物0.8μmol·L-1。通过梯度试验确定的扩增程序为：94℃预变性5 min，然后按94℃变性1 min，51℃退火3 min，72℃延伸2 min，进行35个循环，最后72℃延伸5 min，4℃保存。
위건립조피안 ISSR-PCR 우화반응체계，선통과단인소시험학정영향조피안 ISSR-PCR 5개인소(Mg2+、dNTP、Taq DNA 취합매、DNA 모판、인물)적교괄의농도범위，재차기출상진일보통과정교시험대5개인소4개수평진행우화，병용 DPS 연건분석시험결과。결과표명조피안 ISSR-PCR 적우반응체계위：재25μL 반응체계중，10×PCR buffer 2.5μL、MgCl 2.0 mmol·L-1、dNTPs 0.3 mmol·L-1、Taq DNA 취합매1.25 U、DNA 모판60 ng、인물0.8μmol·L-1。통과제도시험학정적확증정서위：94℃예변성5 min，연후안94℃변성1 min，51℃퇴화3 min，72℃연신2 min，진행35개순배，최후72℃연신5 min，4℃보존。
In order to develop an optimized ISSR-PCR analytical system for Eucalyptus pellita, single factor experiments were conducted to determine suitable concentrations of different factors (Mg2+, dNTP, Taq DNA polymerase, DNA template, primer) that influence ISSR-PCR analyses. Based on the optimal concentrations identified, an orthogonal designed trial was carried out in order to optimize 5 factors at 4 levels. The results showed that a suitable ISSR-PCR reaction system involved: 25 μL reaction system containing 10×PCR buffer 2.5 μL, MgCl2 2.0 mmol·L-1, dNTPs 0.3 mmol·L-1, Taq DNA polymerase 1.25 U, DNA template 60 ng and primer at 0.8 μmol·L-1. Through gradient testing, the optimized PCR amplification program was determined to involve pre-denaturing at 94℃ for 5 min, then denaturing at 94℃ for 1 min, annealing at 51℃ for 3 min, extension at 72℃ for 2 min over 35 cycles, and finally extension at 72℃ for 5 min. The PCR amplification products were stored at 4℃.