Natural Occurrence of the Entomopathogenic Fungus Nomuraea rileyi in the Soybean Green Semilooper, Chrysodeixis acuta, in India

In this study, proteins were extracted through ammonium sulfate salting-out from the entomopathogenic fungus, Nomuraea rileyi, cultured on PDB+Y liquid medium at 12 days after incubation. Four proteins with molecular weights of >100, 50-100, 30-50, and 10-30 kDa were isolated and purified from desalting and centrifugal concentrators. These proteins were quantitated using BCA (bicinchoninic acid) protein assay kit, injected into Spodoptera litura 5th instar larvae, and added into culture media for Sf-21 cell line. The protein contents of 50 kDa or above in salting-out precipitants were 45 times more than those of 10-50 kDa proteins. The larval mortality caused by 50-100 kDa was the highest among all proteins, the LC50 being ca. 348.77 μg/ml. In addition, this protein could lower the larval activity, reduce ingestion of foods and larval weight, fail in molting, and prolong instar duration. However, the proteins with 10-30 kDa could induce apoptosis in Sf-21 cells. Our results proposed that N. rileyi could secrete toxic proteins into cultured fluids to exert at least two toxin strategies, causing hypoplasia and apoptosis against the growth of its insect hosts.本文藉由硫酸銨沉澱法鹽析出蟲生真菌綠殭菌(Nomuraea rileyi)在PDB+Y培養液培養12天所含的蛋白質再經由離心濃縮並依照分子量大小進行初步的分離。分析結果分別得到>100、50-100、30-50及10-30 kDa等四種不同分子量的蛋白並經去鹽處理可提高溶液中蛋白質的純度。再通過BCA (bicinchoninic acid)蛋白質定量分析組將各種蛋白質進行定量後注射斜紋夜蛾(Spodoptera litura) 5齡幼蟲與添入Sf-21細胞培養液中培養。結果觀察到鹽析沉澱物中分子量>50 kDa的蛋白質比大小介於10-50 kDa之間的蛋白質含量高出逾45倍且各種蛋白質皆會引起幼蟲死亡但以50-100 kDa的蛋白質造成的死亡率最高半致死濃度(LC50)約為348.77 μg/ml同時併發活動力下降、取食量減少、體重減輕、蛻皮失敗與齡期延滯等不良之影響。而10-30 kDa的蛋白質則能引起Sf-21細胞凋亡。此顯示綠殭菌的生長過程與致病策略中可分泌多種不同的有毒蛋白質且至少包含有造成發育不良與細胞凋亡兩種影響寄主昆蟲生長的毒素策略之應用。誌謝 I摘要 IIIAbstract V前言 1前人研究 3材料與方法 16結果 28討論 34參考文獻 39圖表 51附錄 6