为了探索提取贵阳腐霉Pythium guiyangense菌株总RNA简便而有效的方法,我们分别采用石英沙研磨、超声波破碎、液氮研磨、液氮加石英沙研磨和溶菌酶消化5种方法破碎真菌细胞壁,再用市售RNAsimple total RNA kit试剂盒提取总RNA,最后通过对提取物进行琼脂糖凝胶电泳定性、紫外比色定量及PCR检测来评价各种破壁方法的优弊.结果显示液氮加石英沙研磨破壁法提取的真菌总RNA在5种方法中效率最高,OD260/OD280=2.093±0.264,RNA浓度为(0.134±0.021)μg/μL,RNA制备效率为(26.76±4.10) μg/g菌丝体,并且该方法操作简便,重复性最好,是制备P.guiyangense菌株总RNA的首选破壁方法.%To find a simple and effective wall-breaking method for total RNA extraction from filamentous fungus ( Pythium guiyangense Su) , five kinds of wall-breaking methods were conducted including grinding with silica sand, with liquid nitrogen, with silica sand plus liquid nitrogen, sonication and enzyme digestion. The total RNA in each lysate was purified with RNA simple total RNA kit and the efficiency of each method was evaluated by qualitative and quantitative analysis and PCR. The results showed that the method of grinding with silica sand plus liquid nitrogen was more effective, of which OD260/OD280 = 2. 093 ± 0. 264, the concentration of total RNA was (0. 134 ± 0. 021 ) u,g/u,L and the purified efficiency was (26. 76 ±4. 10) u,g/g mycelium. It was the best means for breaking cell wall during total RNA extraction from this fungus since its simplicity, reliability and efficiency.
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