Construction The Protoplast Transformation System Of Potato Common Scab Pathogen Streptomyces Galilaeus

A new potato common scab pathogen strain CPS-2 was identified to be Streptomyces galilaeus species during classification the scab-related strains of China in our laboratory. Further researches on the genetic features of the strain CPS-2 will help to understand the reasons of genetic diversity and the pathogenic mechanism of potato scab pathogens. In this work, the transform system of S.galilaeus CPS-2 was constructed, which will lay the foundation for molecular genetic studies of the plant pathogenic S.galilaeus species .The factors which affect the protoplast formation and regeneration of S.galilaeus CPS-2 were studied in detail. The results showed that using secondary media culture was suitable for the protoplast formation. The modified YEME (contains 10% glucose) appeared to be better for the protoplast formation than originality medium CP, GPYP, SGGP and R2YE. The strains were shake-cultured at 28℃for 24 h in medium TSB, then 10% of the cultures were transferred to fresh modified YEME supplemented with 1.0% glycine, and shaking at 28℃for 60 h. The mycelium growth was in the middle of logarithmic growth phase when the bioactivity is highest. The number of protoplasts could up to 4.4×109 /mL after the washed mycelium incubated in P buffer containing 3 mg/mL lysozyme at 37℃for 90 min. Then the regeneration rate were tested on the RM1, RM2, RM3, RM4 and R2YE regeneration agar plate. The result demonstrated that the regeneration rate was higher on R2YE than others. The osmotic pressure of R2YE was tested in order to improve the the conidium-producing ability of protoplast. The R2YE containing 0.2 mol/L glucose was efficient and the regeneration rate was higher than 22% when the protoplast was plated on dry plates by spreading with a glass spreader.The Streptomyces plasmid vectors pIJ702 was used to evaluate the transform system of the strain CPS-2. The effect factors of transformation were analyzed through the transfer buffer, the PEG choice, adding time of the antibiotics, etc. The optimum conditions were selected as follows: using T buffer containing 25% PEG 1000, covering SNA(contains 50μg/mL tsr) after 24 hours on transfered plates and the transformants can be obtained after four days. The transformation rate was 102~103 perμg plasmid DNA. The plasmids of the transformants were extracted after cultured in TSB for 48 hours. The electrophoresis detected the plasmid after enzyme digestion by SacI showed that the pIJ702 transferd into protoplasts of CPS-2 successfully. The development of protoplast transformation system of S. galilaeus CPS-2 would contribute to cloning and expression of pathogenic-related genes, which should be able to benefit to the research on the pathogenicity mechanism and genetic diversity.