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采用正交试验与单因素设计相结合的方法,对四数獐牙菜ISSR-PCR反应体系中的4种主要因素(Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化筛选,PCR结果用统计软件SPSS16.0分析。结果显示,Mg2+、TaqDNA聚合酶、dNTP这3因素的不同水平对PCR反应结果都有显著影响,其中Mg2+的浓度影响最大。筛选出各反应因素的最佳水平,建立四数獐牙菜ISSR-PCR反应的最佳体系(25μL)为3.0 mmol/L Mg2+,250μmol/L dNTP,0.6μmol/L引物,1UTaqDNA聚合酶,40 ngDNA,2.5μL10×buffer。这一体系的建立为今后利用ISSR技术进行四数獐牙菜遗传多样性分析以及物种保护奠定了技术基础。
Four factors (Mg2 +, TaqDNA polymerase, dNTP and primers) in ISSR-PCR reaction system were screened by orthogonal experiment and single factor design. The PCR results were analyzed by using statistical software SPSS16.0 analysis. The results showed that different levels of Mg2 +, Taq DNA polymerase and dNTP had significant effects on the PCR results, and the effect of Mg2 + concentration was the most significant. The optimum conditions for the ISSR-PCR reaction of S. sclater were screened out. The optimum conditions (25μL) for the ISSR-PCR reaction were 3.0 mmol / L Mg2 +, 250μmol / L dNTP, 0.6μmol / L primer and 1UTaq DNA polymerase ng DNA, 2.5 μL 10 × buffer. The establishment of this system laid the technical foundation for the future use of ISSR technology to analyze the genetic diversity of four species of Swertia and species conservation.