关键词: 蘑菇, RAPD-PCR, 体系优化

Abstract: Six key effect factors to RAPD-PCR including Taq enzyme dosage,template DNA dosage,magnesiumion concentration,dNTPs concentration,arbitrary primer concentration and annealing temperature were optimized one by one,in order to establish the optimal RAPD-PCR system for RAPD diversity analysis among some varieties of Agaricus bisporus. The gradient treatments of the six factors were established as follows:(1) 7 gradient annealing temperatures 33.5,36.5,38.3,40.1,43.7,45.4 and 48.1℃; (2) 6 gradient concentrations of magnesiumion 0.5,1.0,1.5,2.0,2.5 and 3 mmol/L; (3)5 gradient dosage of Taq enzyme 0.5,1.0,1.5,2.0 and 2.5 U; (4) 5 gradient concentration of dNTPs 0.10,0.15,0.20,0.25 and 0.30 mmol/L; (5) 5 gradient concentration of primers 0.1,0.2,0.3,0.4 and 0.5μmol/L; (6) 7 gradient dosage of template DNA with 100,20,4,0.8,0.16,0.032 and 0 ng.The results showed that 20μl volume of the reaction system consists of 2.0μl 10×buffer,1.5 unit Taq DNA enzyme,80 ng template DNA,2.0 mmol/L MgCl2,0.2 mmol/L is dNTPs,0.3μmol/L arbitrary primer,adding ddH2O to 20μl final volume. The thermal programmer of RAPD-PCR is 5 min denaturation at 94 ℃,then 1 min denaturation at 94 ℃,1 min annealing at 44 ℃,1 min 50 s polymerization at 72 ℃,for 40 cycles,and final polymerization at 72 ℃ for 7 min.

Key words: Agaricus bisporus, RAPD-PCR, system optimization