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標題: 綠殭菌毒素之純化及其對斜紋夜蛾幼蟲生長發育之影響
Purification of toxin produced by Nomuraea rileyi and its effects on growth and development of Spodoptera litura larvae
作者: Chen, Shiao-Han
出版社: 昆蟲學系所
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Cultured filtrate of the entomopathogenic fungus, Nomuraea rileyi SH1, seems contain proteinaceous substances which are inhibitory to larval growth of Spodoptera litura. The cultured filtrate of 12-h-culture were purified with ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. SDS-PADE profile showed that the final toxic fraction consisted of two protein bands, both with a molecular weight of ca. 30 kDa and an isoelectric point of 9.7. Injection of S. litura fifth-instar larvae with the purified proteins caused 95.56% mortality. However, 40% of the filtrate-injected fifth-instar larvae was able to recover from growth retardation and to pupate normally when injected with 10-6 M 20-hydroxyecdysone every 24 h, totaling 7 times. The rescue effect on larval death by injection of 20-hydroxyecdysone was a dose-dependent response. Although 20-hydroxyecdysone at 10-4 M could rescue the filtrate-injected larvae from intoxication, their body weight increased slowly and fifth-instar duration extended 1-2 days. Once the sixth-instar larvae molted from the fluid-injected fifth-instars, they grew normally without any adverse effect and could emerge to adult. Injection of fifth-instar larvae with 20-hydroxyecdysone which had been treated with N. rileyi cultured filtrate (M20E) could not rescue the filtrate-injected larvae, resulting in only 7.5% pupation of these larvae. However, M20E-treated larvae which had been injected with fungal cultured filtrate could be rescued by injecting 10-4 M 20-hydroxyecdysone, resulting in 47.75% pupation. Therefore, it seems that the toxic proteins produced by N. rileyi SH1 interfere with hormonal action in S. litura.

綠殭菌 (Nomuraea rileyi) 菌株 SH1 之培養液中,含有能抑制斜紋夜蛾 (Spodoptera litura) 幼蟲的生長,造成致死之成份。取其 12 天之培養液,經 80% 飽和濃度的硫酸銨沉澱分離後,利用快速液相層析儀,配合 Sephacryl S-200 HR 凝膠過濾層析及 Butyl Sepharose 4 FF 疏水性層析管柱,純化出兩個分子量相近之蛋白帶,分子量約 30 kDa,等電點約為 9.7。將其注入斜紋夜蛾五齡幼蟲造成 95.56% 之死亡率。另將培養液與脫皮激素 (20-hydroxyecdysone) 注入斜紋夜蛾五齡幼蟲後,每隔 24 小時再注入蟲體脫皮激素一次,共七次,對幼蟲生長發育有顯著的影響,幼蟲能克服綠殭菌培養液造成之生長抑制而正常化蛹比例達 40 %,且脫皮激素的濃度與幼蟲生長回復具劑量依變反應,在 10-4 M 之處理下,具較佳的回復效果,化蛹率可提升至 60%。注入毒素之五齡斜紋夜蛾幼蟲,雖在持續提供脫皮激素下能回復正常發育,但與對照組相比較,有體重增加遲緩以及第五齡期延長 1-2 天之現象,若成功脫皮至六齡後,則能正常完成生活週期,其齡期以及羽化率均不受影響。經綠殭菌 SH1 培養液處理後之脫皮激素,能抑制幼蟲之生長發育,僅7.5% 能正常化蛹。若注入蟲體額外之脫皮激素,則能使部分幼蟲克服此生長之抑制,化蛹率可回復至47.75%。因此,本文推測綠殭菌菌液所含之毒性蛋白會干擾蟲體脫皮激素之作用。
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