Clone And Analysis Of Sequence Involved In Site-specific Recombination In Micromonospora Sp. 40027 Strain

The bacteria of the genus Micromonospora belong to rare actinomycetes, which produce wide varieties and diverse structure types of antibiotics. In recent years new antibiotics that have been found from the genus Micromonospora is more than Streptomyces. However, the research of the genus Micromonospora on genetics, molecular biology especially on system of gene cloning is seldom. pJTU112, Micromonospora phage, and pSET152 (Streptomyces phageΦC31 derivative plasmid) can integrate into the chromosome of Micromonospora sp. 40027 strain by site-specific recombination, which provides an important foundation for developing mechanism of site-specific recombination in the genus Micromonospora. The main purpose of this study is to clone and analyze the sequence involved in site-specific recombination in Micromonospora sp. 40027 strain.pJTU112 (ca. 14.1 kb), a low-copy-number, self-transmissible covalently closed circular plasmid, is present in the Micromonospora sp. 40027 strain in both integrated and autonomous states. The integration site attP was localized on the 0.4kb PstⅠfragment of pJTU112, and pJTU113 which derivatives from pJTU112 also contained this fragment. For sequencing and analysis the 0.4kb PstⅠfragment, pSCU200 was constructed by this fragment and vector pUC19.The effect of DMSO concentration on the PCR amplification of GC rich Micromonospora genome DNA was investigated. The results showed that PCR amplification was significantly improved at concentration of 2.5% or 4.0% DMSO.By using conjugal transfer method, pSET152 got into Micromonospora sp. 40027 strain and integrated into the chromosome by the attP site of Streptomyces phageΦC31. It is important to optimize the conditions of conjugative transfer for improving the efficiency of conjugal transfer. The results showed that high frequency exconjugants was obtained under the condition of 400μL mycelium of Micromonospora sp. 40027 strain mixed with 40μL Escherichia coli ET12567/pUZ8002.Cloned the left and the right attB of site-specific recombination between pSET152 and Micromonospora sp. 40027 strain by different strategies. Using BamHⅠto digest the DNA of Micromonospora sp. 40027 strain containing pSET152, recovered the 6.3kb fragment which contains apramycin resistance gene acc(3)Ⅳand E.coli replication origin ori. Then self-linked and transformed it into E. coli for sequencing and analyzing (named pSCU202). Sequence analysis certificated that pSCU202 contains the right sequence of attB site. According to the sequence of pSET152, three specific nested premiers PF1, PF2 and PF3 were designed. Taking the DNA of Micromonospora sp. 40027 strain contained pSET152 as a template, the left attB of site-specific recombination between pSET152 and Micromonospora sp. 40027 strain was amplified by TAIL-PCR. The third round product of TAIL-PCR (ca. 250 bp) was cloned and sequenced (named pSCU203). The results of sequence analysis showed that pSCU203 contains the left sequence of attB site.