利用氯化苄提取真菌基因组DNA及其分子生物学分析

Isolation and molecular biological analyses of genomic DNAs from fungi using benzyl chloride

研究了利用氯化苄提取丝状真菌的基因组DNA。在 pH9.0条件下 ,使氯化苄与菌体细胞充分混合后于 50℃保温 1h ,可以从中华根霉 (Rhizopuschinensis) Rc0 1和少根根霉 (Rhizopusarrhizus)Ra0 1的完整细胞及经液氮研磨的无花果曲霉 (Aspergillusficuum ) AS3.32 4和无花果丝孢酵母 (Trichosporonficuum) Tf0 1的菌粉中提取基因组DNA。所提DNA收获量大 ,蛋白质污染少 ,分子量均在 2 3kb以上 ,一级结构完整。以AS3.32 4基因组DNA为模板在特定引物下 ,可通过PCR扩增出 1 .4kb的植酸酶基因 ( phyA) 。

Benzyl chloride were used for extrcting fungal DNA.Under the condition of pH9.0 and incubated at 50?℃ for an hour after benzyl chloride was mixed enough with fungal cells, Genomic DNAs of Rhizopus chinensis Rc1 and Rhizopus arrhizus Ra1 were extracted and isolated from their integrated cells and from the mycelial powder of Aspergillus ficuum AS3.324 and Trichosporon ficuum Tf1 grinded in liquid nitrogen with high yield and little contamination of protein or other organic components.Molicular sizes of all the isolated DNAs were higher than 23kb.After PCR was operated by using the isolated genomic DNAs of AS3.324 as template and using a double of specifical primers, a 1.4 kb segment of phyA was obtained.