烟草靶斑病菌基因组DNA提取及RAPD反应体系的优化

Extraction of Genome DNA and Optimization of RAPD Reaction System of Rhizoctonia solani

采用改良的CTAB法,对16个烟草靶斑病菌菌株进行基因组DNA的提取,所得的DNA样品的OD260/OD280值在1.76～1.89之间,电泳主带清晰,无弥散、拖尾现象,DNA质量较高。通过单因素和正交设计结合的方法对影响RAPD-PCR反应的主要因子Mg2+,dNTP,引物的浓度和模板DNA,Taq酶的用量进行了优化,建立了适宜于病菌的RAPD分子标记的最佳反应体系。优化的反应体系为:模板DNA 10 ng,Mg2+浓度3.25 mmol/L,dNTP浓度0.175 mmol/L,Taq酶1.4 U,引物浓度0.5μmol/L,反应体系总体积为25μL。通过该反应体系可以获得清晰、稳定、特异性的分子标记谱带,扩增结果较好。

The genome DNAs of 16 strains of Rhizoctonia solani isolated from tobacco target spot were extracted by the improved CTAB method. The OD260/OD280 values of the obtained DNAs ranged from 1.76 to 1.89, the eleetrophoretie bands were clear and free of dispersion and tailing, the DNAs were of better quality. The main feators influencing RAPD-PCR reaction, including the concentrations of Mg2＋, dNTP and primer, and the dosages of template DNA and Taq DNA polymerase, were optimized by the combination of single factor and orthogonal experiments and an optimal RAPD reaction system was established, The total volume of the optimal reaction system was 25 μL, including 10 ng template DNA, 3.25 mmol/L Mg2＋, 0.175 mmol/L dNTP, 1.4 U Taq DNA polymerase and 0.5 μmol/L primer. Clear, stable and specific bands and satisfactory amplification results were obtainable by the optimized reaction system.