石蒜属植物种质资源ISSR-PCR反应体系的建立

An inter simple sequence repeats(ISSR) reaction system for Lycoris(Amaryllidaceae)

为进一步开展石蒜属Lycoris植物种质资源遗传多样性研究,利用正交试验设计的方法,从Taq酶、Mg2+、模板DNA、dNTP和引物等5因素4水平,对石蒜属植物ISSR(intersimple sequence repeats)反应体系进行优化,确立了石蒜属植物ISSR的最佳反应体系:在20μL反应体系中,含1×Buffer,模板DNA 50 ng,2.5 mmol.L-1氯化镁(MgCl2),0.15 mmol.L-1dNTP,25.05 nkatTaq聚合酶,0.5μmol.L-1引物。进一步进行梯度退火试验,确定最适宜退火温度为57℃。

To study the genetic diversity of Lycoris Herb. （Amaryllidaceae）, an orthogonal design was used to optimize an inter simple sequence repeats （ISSR） amplification system on Lycoris using five factors （Mg^2＋, deoxyribonucleotide triphosphate （dNTP）, primers, DNA template, and Taq polymerase ） at four levels of concentrations respectively. A suitable ISSR reaction system was constructed with the 20 μL reaction system containing 1 x polymerase chain reaction （PCR） buffer, 2.5 mmol· L^- 1 MgC12, 150 μmol· L^- 1 dNTP, 0.5 μmol· L^-1 primer, 50 ng DNA template, and 25.05 nkat Taq DNA polymerase. According to the gradient PCR, the optimal annealing temperature for the ISSR reaction was 57 ℃. [Ch, 7 fig. 2 tab. 23 ref. ]