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標題: Enzyme activity, cDNA cloning and expression of acetylcholinesterase in Bactrocera dorsalis
作者: 黃泰鈞
Tai-Chung Huang
關鍵字: 東方果實蠅;乙醯膽鹼酯酶;Bactrocera dorsalis;Acetylcholinesterase
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摘 要

有機磷和氨基甲酸鹽神經毒類殺蟲劑是藉由與昆蟲體內的乙醯膽鹼酯酶(acetylcholinesterase)結合,抑制其酵素活性,干擾其神經傳導,進而達到殺蟲效果。近年來食品安全意識高漲,對各類農產品的農藥殘留程度均嚴格把關,而此類殺蟲劑與酵素分子結合之特性,使這類酵素能成為具潛力之生物檢驗工具,可用以檢測農作物之殺蟲劑殘留。為了建立東方果實蠅(Bactrocera dorsalis (Hendel))此酯酶活性檢測方法,本研究以其頭部均質液和碘化乙醯硫膽素(acetylthiocholine iodide;類乙醯膽鹼化合物)混合,經過數次吸光值變化試驗之後,找出觀察上升段變化的合適時間為前6分鐘。另外,以分子生物技術選殖乙醯膽鹼酯酶基因,以RT-PCR方式分別增幅出全長及成熟蛋白兩cDNA片段,並將之分別重組於pET 29a表現載體,再轉染入表現宿主E. coli (BL21);最後,檢測乙醯膽鹼酯酶蛋白在系統中之表現情況與活性。目前試驗之結果顯示,建構之兩基因片段皆無法被表達出蛋白,推論可能是此真核生物基因內含有一些此原核表現宿主無法表達的稀有密碼子所致,詳細情況尚有待進一步深入探討。


The insecticidal effect of organophosphate and carbamate neurotoxic insecticides is mainly because of the inhibition of acetylcholinesterase (AChE) activity by chemicals, and thus disrupts the normal nerve conduction in insects. In recent years, food safety becomes the most concerned issue; and the pesticide residues on various agricultural products are strictly confined under safe ranges. Characteristics of the interaction of AChE and pesticides are considered that it could be used as potential biological tool for detecting pesticide residues on crops. In this study, therefore, an attempt to establish a detection method was conducted by targeting on the AChE of the oriental fruit fly, Bactrocera dorsalis (Hendel). Firstly, various amounts of the head homogenate of B dorsalis were reacted with acetylthiocholine iodide (a synthetic ACh compound) to establish appropriate detection ranges and standard methods. After several replicates of the reaction tests, the first six minutes was determined as an optimal light absorption observing time period. Furthermore, RT-PCR and molecular cloning technology were applied to isolate the cDNA fragments of full length and mature AChE proteins, respectively, to recombine the individual cDNA fragment into the pET 29a expression vector, and to transform the expression host E. coli (BL21). The current results showed that both full length and mature AChE proteins were not able to be expressed in the system. The possible reasons to lead these results may be due to that the AChE gene contains rare eukaryotic codons which are not able to be expressed in the prokaryotic host. However, the details have yet to be further investigated.
其他識別: U0005-2811201416181128
Rights: 同意授權瀏覽/列印電子全文服務,2014-08-31起公開。
Appears in Collections:昆蟲學系

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