| A Streptomyces sp.ⅡR21 strain with resistance to crop pathogenic fungi was isolated from the soil.Its antifungal activity,fermentation conditions,isolation and purification of antifungal compounds,attB site and its whole genome were studied.The aims were to lay the foundation of exploitation of new agricultural antibiotics.Antifungal activity of the secondary metabolite of Streptomyces sp.ⅡR21 strain was detected by the agar block method and cylinder plate method with seven crop pathogenic fungi as indicators.It had strong antifungal activity against Magnaporthe grisea 168,Magnaporthe grisea NO-1,Rhizoctonia solani with the diameter of inhibition zone beyond 40 mm.Streptomyces sp.Ⅱ R21 strain had well degree antifungal activity against Fusarium aminearum,Fusarium culmorumand Botrytis cinerea.The influence of the different cultured conditions on the antifungal activity of the fermentation broth was measured to optimize the conditions of the fermentation broth.The research indicated when strain was cultured in the optimized mycelium fermentation,inhibition activity was better and inhibition zone was 53.31 mm;when strain was cultured for seventh days,inhibition activity was better and inhibition zone was 49.40 mm;when strain was cultured at 30℃,inhibition activity was better and inhibition zone was 52.30 mm;when strain was cultured at rotating speed of 180 r/min,inhibition activity was better and inhibition zone was 50.02 mm.The results of optimize experiment indicated the antifungal activity of Streptomyces sp.ⅡR21 strain was much better which was fermented in shaking flash at 30℃ and 180r/min for 7 days in optimized mycelium fermentation medium.Streptomyces sp.ⅡR21 strain fermentation liquid was extracted,using petroleum ether,ethyl acetate and butanol as the extraction agent.The main antimicrobial ingredients of Streptomyces sp.ⅡR21 strain fermentation broth consisted in the ethyl acetate fraction.Streptomyces sp.ⅡR21 strain ethyl acetate extract was further separated by thin layer chromatography and silica gel column chromatography.Eventually three activated components,F1,F2 and F3 were acquired after separation.Their inhibition zone was respectively 21.26 mm,47.28 mm,30.78 mm.Activated components were purified by preparative HPLC,the antifungal compound of purity over 90% purified from the F1 component.The attB site sequence of Streptomyces sp.ⅡR21 strain was cloned by PCR.The length of attB site sequence was 269 bp.Sequence from Streptomyces sp.II R21 strain was the most similar with attB site core region in Streptomyces.natalensis,reaching 92%,where the cross-over occured in the core region 5’-TT.The integrative plasmid pSET152 including ΦC31 att P site recombined specificly between the attP site in pSET152 and the attB site in the chromosome of Streptomyces sp.II R21 strain.After analysis of secondary metabolites synthesis gene clusters about 580 contigs in Streptomyces sp.II R21 strain genome,61 contigs containing synthetic gene clusters was predicted.Six productions with antifungal activity was synthesized by eights of these synthetic gene clusters.Analysis of synthetic gene cluster in contig51 indicated that Streptomyces sp.II R21 strain had the ability to synthesize clavams metabolites. |
