金龟子绿僵菌遗传转化体系的建立及转化子特性分析-农业微生物学专业论文.docxVIP

AbstractMetarhizium Abstract Metarhizium anisopliae SoTOHn has been widely applied in biological control of the agricultural and forestry pests．which is also the main focus ofthe microbial pesticide at presenL肛anisopl／ae has 衄of advantages over the conventional chemical pesticides：broad host range，high pathogenicity，and 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 Cen only be used for biological control after improvemenL The present study investigated the protoplast preparation and the establislLment of transformation system of jlf．an／aopl／ae【M1330189．The biological characteristics of stable transformants weTe also investigated．The present study provided SOIIt referances for the research of genetic transformation of 肛anisopliae．The main results ofpresent study are私follows： Mycelium Was collected by two·steps celmres，and then it was processed for 5h with protoplast buffer at 30C，0．7 mogL NaCl solution was used as osmosis stabilizer,5--6×lO。7-lOSsp protoplast怛 wet mycelium wag formed and regeneration rate WaS 15％． The genetic transformation system of肛anisopliae Was constructed by PEG-mediated protoplast and G418 WaS used as selection nlarl【eL肛an／sopl／ae growth was inhibitexl completely when the medium contained 300“g／ml G418．The results of sensitivi￡y examinations showed that the efficiency of transformation Was 15-23 transformants，烬vector DNA when the concentration of G418 Was 300p g／mL．and transformants e】【isted stably．砧e construction of M anisopliae transformation system of PEG-mediated laid a good foundation for researching the膨an／sopl／ae from the molecular level and gaining stable,and effective recombinant strain． This research mainly focus 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 mexiium after selection r