The Establishing Of Nylon Membrane Hybridization Method For Detecting Actinomycete Genome DNA Relatedness

In this paper, strain 061324 isolated from mangrove environment, with high antitumor activity, which was identified to Streptomycete genus by formerly test in our lab, was further studied for genome DNA relatedness via nylon membrane DNA-DNA hybridization method, and confirmed as a new species.According to the on line blastn result at GenBank for strain 061324, the most related type strain was Streptomyces cheonanensis NBRC 100940T; which was selected for the DNA-DNA hybridization in this experiment. And a genome sequence available type strain Streptomyces coelicolor was selected as DNA-DNA hybridization reference. The L-dried culture of type strain S. cheonanensis was baught from NITE Biological Resource Center. It was reactivated on ISP.2 medium. Strains were cultured in YE liquid medium. Genome DNA was isolated via Lysozyme-SDS method. The DNA hybridization probe was prepared by linker-adaptor PCR method. The cohesive ends of genome DNA were digested by Sau3AⅠ(or MboⅠ) , ligated with S and P linker, respectively by T4 ligase. S ligated DNA was amplified by S1 primer and immobilized on nylon membrane via UV irradiating for 1 min; P ligated DNA was amplified by P1 primer for DIG labeling PCR amplifying probe. The hybridization was carried in'High Effective Hybrid Solution'(Innogent Company, Shenzhen) for 20 h at 66.5℃. The nylon membrane was incubated in anti-DIG AP solution for 30 min after being washed strictly. Using NBT/BCIP as substrate, the hybridization signals were detected in the dark and the membrane was photographed after being air-dried. DNA-DNA hybridization value was calculated as 49.7% by tanon dots analysis system (Shanghai Tanon Science & Technology Co., Ltd.). It was concluded that strain 061324 is a new species and very similar with S. cheonanensis.The other taxonomic data of strain 061324 studied by Liu Lihua et al. has great similarity with S. cheonanensis NBRC 100940T. It was showed from this research that DNA-DNA hybridization is very important for bacterial taxonomy, especially for actinomycete.