珠子参ISSR-PCR反应体系的建立及优化

目的建立珠子参的ISSR-PCR适宜扩增反应体系和程序,为其深入研究奠定基础。方法采用新型植物基因组DNA提取试剂盒提取珠子参总DNA,并用正交设计实验对其ISSR反应体系条件进行筛选及优化。结果提取的基因组DNA纯度和完整性较好,A260/A280值在1.8².0之间,DNA无降解现象,可满足ISSR-PCR扩增要求。珠子参ISSR分析的最适反应体系为:在20μL PCR反应体积中,含10ng模板DNA、0.20mmol·L-1dNTPs、1.5μmol·L-1引物、3U TaqDNA聚合酶、2μL10×PCR Buffer、3mmol·L-1 Mg2+。扩增程序为:95℃预变性5min,94℃变性30s,58℃退火30min,72℃延伸1min,循环40次,72℃延伸10min。由此可获得带型丰富和清晰可辨的DNA指纹图谱。结论建立的ISSR-PCR反应体系可用于研究珠子参的遗传变异和遗传多样性。

Establishment and optimization of ISSR-PCR reaction system for Panax j aponicus C .A .Mey .var .ma j or

Objective The present study was undertaken to establish an ISSR-PCR amplification reaction system for Panax japoni-cus C .A .Mey .var .major for providing basis to further research .Methods The samples of Panax japonicus C .A .Mey .var . major were used to isolate high-quality genomic DNA by using the plant genomic DNA Preps Kit .The different conditions for IS-SR reaction system were optimized .Results The extracted genomic DNA was of high quality as revealed by 1 .8 to 2 .0 absorbance ratio of wavelengths （260/280） ,did not show any degradation ,and was found suitable for ISSR-PCR reaction .The conditions of the optimal ISSR-PCR amplification system were in a total 20μL reaction volume included 10 ng of template DNA ,0 .20 mmol · L-1 of dNTPs ,1 .5 μmol · L -1 primer ,3 U of Taq DNA polymerase ,2 μL 10 × PCR buffer and 3 mmol · L -1 Mg2＋ .The PCR program was set to 5 min at 95 ℃ for pre-denaturing ,followed by 40 cycles of 30 s at 94 ℃ （denaturation） ,30 s at 58 ℃ （annealing） and 1 min at 72 ℃ （extension） ,and the final extension was set at 72 ℃ for 10 min .The optimized reaction system yielded distinct DNA fingerprinting results .Conclusion The established ISSR-PCR amplification reaction system may be useful to assess the genetic variability and diversity in Panax j aponicus C .A .Mey .var .major .