【作者】 王晓玲；

【导师】 张泽华；

【作者基本信息】 中国农业科学院 ， 农业微生物学， 2007， 硕士

【摘要】 金龟子绿僵菌（Metarhizium anisopliae Sorokin）在农林害虫生物防治中应用广泛，也是目前微生物农药的研究热点之一。与传统的化学农药相比，绿僵菌寄主范围广，致病力强，具有对人、畜、农作物无毒，无残留、菌剂易生产，持效期长，害虫不产生抗药性等优点，具有广阔的应用前景。但作为一种昆虫病原真菌，绿僵菌同时也存在着杀虫速度慢、效率低、防治效果不稳定等缺点，大大影响了其杀虫效果。另外由于自然分离到的菌株往往毒力不强或对环境的适应力较弱，需改良后才能应用于生物防治，国外对其遗传转化方面已有大量研究，然而国内在这方面尚未进行深入研究从而制约了绿僵菌优良菌株的开发与利用。本研究以金龟子绿僵菌IMI330189为目标菌株，研究其原生质体制备、转化体系的建立并对稳定转化子的生物学特性进行了研究，为我国绿僵菌遗传转化的研究提供参考。主要研究结果如下：采用二级培养法收集菌丝体在采用0.7mol／L的NaCl溶液作为渗透压稳定剂，20ml原生质体Buffer，30℃下处理5h的酶解条件下可形成5～6×10⁷～10⁸个／g湿菌丝原生质体，再生率为15％。用CaCl₂-PEG介导法对绿僵菌原生质体进行了遗传转化，用G418作为筛选标记，构建了绿僵菌的遗传转化体系。通过敏感性试验得出G418的使用浓度为300μg／mL，转化频率达到15～23个／μg质粒，转化子能够稳定遗传。CaCl₂-PEG介导的绿僵菌遗传转化体系的建立，为从基因水平利用分子生物学手段对绿僵菌进行改造，得到稳定高效的杀毒工程菌株奠定了基础。对得到的稳定的转化子进行了培养基筛选、生长速度、产孢量、致病性等方面进行了研究。通过对转化子生长的培养基进行筛选，筛选出PDAY为转化子生长的最适培养基。对生长速度进行测定结果表明，对照菌株的平均日增长量为3.85mm，转化子的日增长量为3.30～3.79mm之间，生长速度均比对照慢；在转化子中TM1与TM3菌株生长速度最快，分别为：3.78±0.02mm，3.81±0.07mm；TM6菌落生长速度最慢，日增长量为3.85±0.13mm。转化子的产孢量介于0.68～1.72×10⁸sp／ml之间，与对照1.58×10⁸sp／ml相比，TM2菌株的产孢量为1.72×10⁸sp／ml，高于对照但不存在显著性差异。对转化子进行毒力测定发现：TM19和TM20两株转化子的LT₅₀值与其它几株转化子之间均存在显著性差异，LT₅₀值分别为8.4340 d和8.3067 d，致死中时长于其它菌株，说明这两株菌株对尔亚飞蝗的致病力比其它菌株减弱。本研究建立的PEG转化体系，并对得到的转化子的生物学特性进行了测定，为进一步深入研究绿僵菌的遗传转化、工程菌的构建、以及产孢量和致病性等相关基因的克隆鉴定奠定了基础。更多还原

【Abstract】 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 15％.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.更多还原