Establishment Of Metarhizium Anisopliae Transformation System And Characteristic Of Transformations

Metarhizium anisopliae Sorokin has been widely applied in biological control of the agricultural and forestry pests, which is also the main focus of the microbial pesticide at present. M. anisopliae has a lot of advantages over the conventional chemical pesticides: broad host range, high pathogenicity, and no toxicity to human being, livestock, and crops. So it can be used widely for killing the pest in future. But the natural isolate has low virulence or adapt the environment poorly, so it can only be used for biological control after improvement.The present study investigated the protoplast preparation and the establishment of transformation system of M. anisopliae IMI330189. The biological characteristics of stable transformants were also investigated. The present study provided some references for the research of genetic transformation of M. anisopliae. The main results of present study are as follows:Mycelium was collected by two-steps cultures, and then it was processed for 5h with protoplast buffer at 30℃, 0.7 mol/L NaCl solution was used as osmosis stabilizer, 5～6×10⁷～10⁸sp protoplast/g wet mycelium was formed and regeneration rate was 15ï¼….The genetic transformation system of M. anisopliae was constructed by PEG-mediated protoplast and G418 was used as selection marker. M. anisopliae growth was inhibited completely when the medium contained 300μg/ml G418.The results of sensitivity examinations showed that the efficiency of transformation was 15～23 transformants/μg vector DNA when the concentration of G418 was 300 g/mL, and transformants existed stably. The construction of M. anisopliae transformation system of PEG-mediated laid a good foundation for researching the M. anisopliae from the molecular level and gaining stable, and effective re, combinant strain.This research mainly focus on the optimal medium, growth speed, spore production, and pathogenecity of stable transformants. The result manifested as follows: 1. PDAY was the best medium for the optimal medium after selection research. 2. The daily rate of growth of transformants which were lower than CK, varied from 3.30 to 3.79mm, compared to CK (3.85mm). Faster growth presented in transformants TM1 and TM3 in contrast to TM6 of 3.85±0.13mm, the number was 3.78±0.02mm and 3.81±0.07mm respectively. 3. As for the spore production, the transformants produced 0.68～1.72×10⁸sp/ml. Compared to the control, the production of TM2 was higher than the control, but did not show the remarkable difference. 4. The result on virulence of transformants manifested that TM19 and TM20 showed remarkable difference in LT₅₀ being of 8.4340d and 8.3067d respectively, compared to other transformantants. The Lethal center showed longer than other strain. Therefore, the pathogenic efficacity of two strains is weaker to L. migratoria.PEG-mediated transformation system constructed in the present study would be helpful for the study of genetic transformation and the clone of pathogenic genes of M. anisopliae.