| Stremptomyces roseoflavu Men-myco-93-63was isolated from potato scab (S. scabies)decline soil and its fermentation products can inhibit many phytopathogenic fungi diseases.As microbial hormones, A-factor homologues have been found in variousStreptomyces species and are characterized by a2,3-disubstituted γ-butyrolactone skeleton.Researches have revealed that A-factor homologues are essential for morphologicaldifferentiation and/or the production of secondary metabolites antibiotic metabolitesprocess, and regarded as regulators of aerial hyphae growth, sporulation, and/or pigmentsbiosynthesis of Streptomyces species. The afsA-family genes were shown to be keyenzymes for biosynthesis of A-factor homologues. In our previous work, the biosyntheticgene clusters of antibiotics against Verticillium dahliae has been located in plasmid pSR47,which is a linear plasmid in S.roseoflavus Men-myco-93-63strain. Bioinformatic analysisrevealed that pSR47plasmid contains an afsA family gene srpA.In this study, Function of srpA in S. roseoflavu Men-myco-93-63was analyzed byusing gene-knockout technology. The results were as follows:1) A knockout vector pKC1139-srpAF-tsr-srpAR for delection of afsA wasconstructed, transferred into S. roseoflavu Men-myco-93-63by conjugation, andthiostrepton-resistant strains, which were preliminary regarded as afsA-deletionmutant, were obtained.2) Total RNAs were extracted from mycelia harvested after cultivation for48,60,72,and84h in liquid PD media, transcriptional analysis of srpA of the wild-typestrain and srpA-disruptant by RT-PCR. The experiment results showedtranscription of srpA gene in the wild-type strain, but no transcript in thesrpA-disruptant, which further demonstrated srpA disruption.3) The growth curves showed that growth rate and biomass of the srpA-disruptantwere distinctly lower than those of the wild-type strain in mycelial growth stage,which suggests that deletion of srpA limits mycelial growth of S. roseoflavus.4) Observation on phenotypes showed that compared to the wild-type strain, theappearance of substrate mycelium, aerial mycelium and spore of the srpA-disruptant was delayed, and the wild-type strain produced abundant aerialmycelia and light pink spores, the srpA-disruptant produced sparse aerial myceliaand colorless spores; substrate mycelium of the wild-type strain produced lemonyellow pigments, while the pigments prodution of srpA-disruptant did notobserved. These results reveal that the srpA gene positively controlsmorphological differentiation and pigments production of S. roseoflavus.5) The atagonism experiments of S. roseoflavus against Verticillium dahliaedisplayed that the wild type strain can inhibit Verticillium dahliae, while thesrpA-disruptant lost ability to inhibit Verticillium dahliae, indicating that the srpAgene positively controls antibiotics production of S. roseoflavus. |
