Exploratory Research On Breeding New And Excellent Strains Based On Genome Shuffling Between S.Avermitilis And S.Spectabilis

Genome shuffling is a new-type technology based on protoplast fusion and recursive protoplast fusion.It has attracted a lot of attentions in the breeding world for its advantages of high efficiency and wide range in recombination.In this dissertation,genome shuffling between S.avermitilis and S.spectabilis has been employed to breed new strains.In order to increase the regeneration ratio,the genome shuffling condition was optimized regarding the choice of regeneration medium,fusion ratio and survival rate after UV treatment.It was found that RM14 medium was the best as regeneration medium;the fusion ratio of S.avermitilis to S.spectabilis was from 10:1 to 1:1;the survival rate of protoplasts after UV treatment was 1%.Employing this combination condition,the genome shuffling between S.avermitilis and S.spectabilis was performed.Single colony was screened based on morphological characteristics in batches,followed by bioassay,flask fermentation and HPLC analysis.The strains with high-yield of avermectin B1a,were chosen as parent strains for the next round of genome shuffling.As a result,total 621 recombinants were isolated with each batch of 211,216 and 194 strains,respectively.Among these target isolates,three different groups were found according to the bioassay and HPLC analysis.Group ? including G2-67,G3-29 and G3-31 strains,showed high antibacterial activities and stability;Group ?including G2-123 and other six strains was with high insecticidal activities and stability;Group ? including G2-145,and G3-4 strains showed high yields of avermectins B1a,which are 13.6 and 11.8 folds as that of original strain,respectively.