铁皮石斛SCoT-PCR反应体系构建及优化

采用正交设计和单因素试验相结合的方法,对铁皮石斛的SCoT-PCR反应中5个因素(DNA 模板、Mg2+、dNTPs、Taq 酶和引物)进行优化试验。建立了适合铁皮石斛既稳定且多态性高的SCoT-PCR的最佳反应体:20μl PCR反应体系中含有1×Buffer、30ng DNA 模板、2.5mmol·L-1Mg2+、0.15mmol·L-1 dNTPs、1.5U Taq DNA聚合酶和0.4μmol·L-1引物。对铁皮石斛的SCoT-PCR最佳反应体系的退火温度进行梯度试验,发现本试验引物S6的退火温度为54.5℃,且最适退火温度因引物而异。这一优化的SCoT-PCR反应体系在32份铁皮石斛材料的验证中表现出良好的稳定性和重复性。该优化的SCoT-PCR反应体系为进一步进行铁皮石斛种质鉴定、遗传图谱构建、基因定位和遗传多样性的分析奠定了技术基础。

ESTABLISHMENT AND OPTIMIZATION OF SCoT-PCR REACTION SYSTEM FOR Dendrobium officinale

Orthogonal design and single factor experiment design were applied for optimizing five factors (DNA template, Mg2+, dNTPs, Taq DNA polymerase and primer) in the SCoT-PCR amplification system on Dendrobium officinale . The results showed that a satisfactory SCoT reaction system for Dendrobium officinale with desirable repeatability and polymorphic bands was established. In a total volume of 20μl SCoT-PCR system, it contained 1×Buffer, 30ng DNA template, 2.5mmol·L-1Mg2+, 0.15mmol·L-1 dNTPs, 1.5U Taq DNA polymerase and 0.4μmol·L-1 primer. The optimal annealing temperature of every primer for SCoT-PCR reaction was proposed by gradient PCR, and it was found that different primers had different annealing temperature. As far as primer S6 was concerned, the optimal annealing temperature was 54.5℃. The optimal reaction system was further verified in 32 Dendrobium officinale varieties with good stability and repeatability. This optimized SCoT-PCR reaction system would provide the basis for germplasm evaluation of Dendrobium officinale , the establishment of genetic maps, gene localization and analysis of genetic diversity.