| Stremptomyces roseoflavus Men-myco-93-63 was a biological agent isolated frompotato scab disease declined soil of Minnesota, which was discovered by professor LiuDa-qun in 1992. Men-myco-93-63 was identified as Stremptomyces roseoflavus accordingto its morphological characteristics, physiological and biochemical characteristics, and16SrDNA homology analysis. It was found that both of the strain and its fermentationbroth showed great inhibition activities on many important plant pathogens, such. asVerticillium dahlia, Cucumber powdery mildew, Botrytis cinerea, and so on. The purposeof my research is to develop the transform system of Men-myco-93-63 includingPEG-mediated protoplasts transformation, conjugal transformation, and electroporation.Parameters which affect the protoplast formation and regeneration of Men-myco-93-63were firstly studied in derail. Medium SGGP appeared to be more suitable for theprotoplast formation and regeneration than originality medium, R2YE, YEME, GPYP, R3,SGGP and CRM. A procedure of effective formation and regeneration of the protoplastswas developed. The strains were shake-cultured at 28℃for 24 h in medium TSB, then thecultures were transferred to fresh medium SGGP supplemented with 2% glycine, andshake-cultured at 28℃for 48h. The washed mycelia were incubated in buffer P containing2mg/ml lysozyme at 37℃for 1h. Estimating by microscopic counts, the amount of theprepared protoplasts was 107～108/mL. The protoplasts were plated on the R2 YEregeneration agar plate. The concentration of MgCl2-6H2O and CaCl2·2H2O were 50mmol/L and 100 mmol/L respectively. It's different with norm R2YE. The plates wereincubated at 28℃for 5d, and the regeneration number was 4.0×106/mL. The regenerationfrequency was about 15%. According the transform method of protoplasts of S. lividans,many attempts to transform protoplasts of Men-myco-93-63 were unsuccessful using theStreptomyces plasmid vectors pIJ702. The effect factors of infection transformationincluding the condition of protoplast formation and regeneration, the number of protoplastduring the transformation, the source and concentration of plasmid, the desiccation degreeof regeneration plate, the source and concentration of PEG, the choice time of antibiotics,and so on. PEG transform was succeeded using PEG-4000. However, the transform ratewas so low, which is only 1.5×10-3%.If there were restriction-modified system in Men-myco-93-63, the transform frequencywould be low. Restriction-modified system in some Streptomyces spp. could be circumvented by the conjugation. A recombinant E. coli ET12567(pUZ8002, pKC1139)was obtained by transforming E. coli ET12567(pUZ8002) with pKC1139, anE. coli-Streptomyces shuttle plasmid incorporating oriT. In mating experiments, E. coliET12567(PUZ8002, pKC1139) was the donor, and the recipient was spores ofMen-myco-93-63 after pregerminating by heat shock. The donor and the recipient weremixed and spead on MS agar plate. After selecting by nalidixic acid and apramycin, severalhundreds of conjugants per plate were achieved. The transform frequency was103～104No./μg pKC1139. Electrotransformation was investigated in cells of plants,animals, and bacteria successfully. Protoplasts, spores of prebourgeon, mycelium dealed bylysozyme of Men-myco-93-63 were used for the elctroporation. All the results showed thatno transformant was achieved.The development of gene transfer system of Men-myco-93-63 by PEG-mediatedprotoplast transformation and conjugal transfermation would contribute to the study ofcloning and reforming of the antibiotic biosynthesis genes of Stremptomyces roseoflavus.
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