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標題: The establishment of Chilo suppressalis acetylcholinesterase gene expression systems
作者: 錢宣仁
Chien, Hsuan-Jen
出版社: 昆蟲學系所
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摘要: Thousand-fold carbofuran resistance differences have been observed among Chilo suppressalis populations in Taiwan. Since the major target of carbamate insecticides is acetylcholinesterase (AChE), it prompts us to examine the possible genetic changes on acetylcholinesterase gene (ace1) of these local insect pests. Comparing to the sequence of the acetylcholinesterase gene (ace1) of relatively sensitive Hsinchu strain, there is a point mutation located close to 3' end of the ace1 in Changhua and Chiayi strains respectively. The point mutations lead to a H668P mutation (CAC to CCC) in Chilo suppressalis ace1 in Changhua strain, and a R667Q mutation (CGA to CAA) in Chiayi strain. In this study, we cloned ace1 genes of C. suppressalis from Hsinchu strain, Changhua, Chiayi strains first, and then express them using both prokaryotic (Escherichia coli) expression system and eukaryotic (insect cells) expression system. In prokaryotic expression system, the pET-30a vector harboring cloned ace1 was expressed in the host cell BL21(DE3). After IPTG induction, the products of ace1 had been found in lysate as inclusion body. Several bands with unexpected size on sodium dodecyl sulfate polyacrylamide gel. They were proved to be degraded products of AChE by Mass Spectrometer analysis. We tried to dissolve inclusion body in 6 M urea, refold, and purification; however, the AChE activity still can be restored. Although the AChE products from the prokaryotic expression system without detectible activity, it can be used to raise polyclonal antibody. In addition, the pIZT-V5/His vector was used in eukaryotic expression system. After subcloning of ace1 and transfecting expression constructs into Sf9 cells, the stable cell lines were established. These stable lines could express exogenous ace1 genes; the transcribed ace1 mRNAs were verified by RT-PCR assay. Whereas, there was no significant protein expression found on SDS-PAGE, and the AChE activity was undetectable. Therefore, we suspect that the protein expression of ace1 is low and results in undetectable AChE activity.
由於臺灣部分地區的二化螟族群對乙醯膽鹼酯酶抑制劑之一的加保扶出現高達千倍的抗藥性,並造成乙醯膽鹼酯酶親和力與反應速率改變,因此針對二化螟乙醯膽鹼酯酶基因序列進行分析。經比較感性新竹品系與彰化及嘉義品系的二化螟乙醯膽鹼酯酶基因,在靠近3’端處,分別發現一個核苷酸點突變。在彰化品系中,乙醯膽鹼酯酶基因序列由原本的CAC改為CCC,造成胺基酸由原本的組胺酸 (Histidine) 轉變為脯氨酸 (Proline);而於嘉義品系中的點突變使得乙醯膽鹼酯酶基因則由原本CGA變為CAA,胺基酸則由晶胺酸 (Arginine) 變為麩醯胺酸 (Glutamine)。本研究第一部分是先取得相對感性的新竹品系和含有突變點的彰化及嘉義品系二化螟乙醯膽鹼酯酶基因,接著建立並利用原核的大腸桿菌表現系統及真核的昆蟲細胞表現系統表現此三個不同品系的二化螟乙醯膽鹼酯酶基因,擬經由比較酵素活性上的變化,探討這些突變與產生千倍加保扶抗性間的關係。本研究原核表現系統使用表現載體為pET-30a,在宿主為 BL21 (DE3)中,可利用 IPTG 於37°C 誘導外源蛋白表現4小時。此原核表現系統表現出的二化螟乙醯膽鹼酯酶易形成包涵體 (Inclusion body),可在裂解物 (lysate) 的沉澱部分被發現。所表現的乙醯膽鹼酯酶在對鈉十二烷基硫酸鹽聚丙烯醯胺凝膠電泳 (SDS-PAGE) 上出現許多非預期之小片段,經質譜分析証實是降解現象所造成。雖使用6 M 尿素溶解包涵體,再經過重新折疊 (refolding) 及純化過程,仍未能使菌體表現的乙醯膽鹼酯酶恢復活性。此不具有活性的乙醯膽鹼酯酶,可使用於抗體製作。本實驗真核表現系統使用pIZT-V5/His 載體,經加入外源基因並轉染宿主 Sf9 細胞株後,建構出穩定表現細胞株。但在這些細胞株中未如預期偵測出二化螟乙醯膽鹼酯酶的活性,於SDS-PAGE 亦未出 現明顯的蛋白表現。可是經RT-PCR檢測發現,在這些含有外源基因的穩定細胞株中,確實有二化螟乙醯膽鹼酯酶基因的轉錄。推測因植入基因的蛋白表現量過低,以致未能測出乙醯膽鹼酯酶活性。
其他識別: U0005-2208201001343200
Appears in Collections:昆蟲學系



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