无距虾脊兰ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Calanthe tsoongiana

利用正交实验设计的方法,对珍稀植物无距虾脊兰ISSR-PCR反应的5个因素(Mg2+、dNTPs、引物、模板DNA和Taq DNA聚合酶)4个水平进行试验,并通过梯度PCR实验确定引物的最佳退火温度和循环次数,最终确定无距虾脊兰的最佳反应体系为:25μL的体系中含3.0 mmol L-1Mg2+、0.3 mmol·L-1dNTP、0.4μmol·L-1引物、2.5 ng·μL-1模板DNA、0.08 U·μL-1Taq DNA聚合酶以及1×PCR buffer;扩增程序为94℃预变性5 min;94℃变性1 min,退火1 min(根据不同引物选择不同的退火温度),72℃延伸1 min,循环40次;72℃延伸10 min;12℃终止反应。该ISSR-PCR体系的建立,为今后利用ISSR技术对无距虾脊兰及其近缘种的分子系统学以及遗传多样性的研究奠定基础。

Orthogonal design was used to optimize ISSR-PCR amplification system of Calanthe tsoongiana in five factors （Mg2., dNTP, primer, DNA template and Taq DNA polymerase ） at four levels, respectively. The results showed that the suitable ISSR-PCR system was performed in a 25 ~xL volume containing 3.0 mmol ~ L- Mg2 ＋, 0.3 mmol ~ L - 1 dNTP, 0.4 p^mol ~ L- 1 primer, 2.5 ng~ ~L- 1 DNA template, 0.08 U ~ ~L - ~ Taq DNA polymerase and 1 ~ PCR buffer. The optimized augmentation procedure was pre-denaturation at 94~C for 5 rain, followed by 40 cycles of 94~C for 1 min, annealing at suitable temperature for different primers for 1 min, 72~C for 1 min, extension at 72~（2 for 10 rain, and preservation at 12~C. This optimized ISSR-PCR system would play an important role in further research of molecular systematics and ~enetic diversity in C. tsoongiana.