Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30766
標題: The establishment of Chilo suppressalis acetylcholinesterase gene expression systems
建構二化螟乙醯膽鹼酯酶基因表現系統
作者: 錢宣仁
Chien, Hsuan-Jen
關鍵字: http://etds.lib.nchu.edu.tw/etdservice/view_metadata?etdun=U0005-2208201001343200
二化螟
加保扶
乙醯膽鹼酯酶原核表現系統
真核表現系統
出版社: 昆蟲學系所
引用: 田學志、高保宗、石家勝、尤子平、譚福杰。1991。安慶地區水稻二化螟抗藥性研究。安徽農業科學 1:61-66。 何火樹、劉達修。1970。臺中地區水稻二化螟蟲之生態研究。台灣農業季刊 6:7-14。 何火樹、劉達修。1971。水稻二化螟蟲發蛾盛期之推定。臺灣農業季刊 7:77-84。 陳啟吉、劉達修。1994。水稻莖桿特性與其對螟蟲之感受性關係研究。臺中區農業改良場研究彙報 43:1-6。 黃秋蘭、丁文彥、江瑞拱。2008。臺東良質米之栽培技術。行政院農業委員會臺東區農業改良場 pp. 7-8。 劉達修。1977。二化螟蟲對水稻之危害觀察。科學發展月刊 5:185-188。 劉達修、王文哲、王玉沙。1991。臺中地區二化螟蟲多發生地區猖獗因子之研究。中華昆蟲 11:300-309。 鄭清煥。1979。水稻褐飛蝨的經濟危害水平之研究 II. 褐飛蝨棲群密度與水稻產量損失之關係。科學發展月刊 7:1103-1114。 鄭清煥、朱耀沂。1999。台灣光復後水稻害蟲之發生演變及防治研究之回顧。植物保護學會會刊 41:9-34。 鄭清煥。2000。應用性費洛蒙於水稻二化螟蛾族群之發生偵測與預測。植物保護學會會刊 42:201-212。 鄭軒。2008。臺灣二化螟對加保扶抗藥性之研究。國立中興大學昆蟲學系碩士論文 pp. 20-29。 臺灣省政府農林廳。1990。水稻蟲害。植物保護手冊 pp. 31-37。 Aldridge, W. N., and E. Reiner. 1975. Kinetics of reaction of B-esterases with carbamates. Enzyme Inhibitors as Substrates. pp. 123-145. Andersson, D. I., K. Boham, L. A. Isaksson, and C. G. Kurland. 1982. Translation rates and misreading characteristics of rpsD mutants in Escherichia coli. Mol. Gen. Genet. 187: 467-472. Baneyx, F. and M. Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 22: 1399-1408. Bourguet, D., M. Raymond, D. Fournier, C. A. Malcolm, J. P. Toutant, and M. Arpagaus. 1996. Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera:Culicidae). J. Neurochem. 67: 2115-2123. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Brondyk, W. H. 2009. Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol. 463: 131-147. Carbonell, L. F., M. J. Klowden, and L. K. Miller. 1985. Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J. Virol. 56: 153-160. Cheng, X., C. Chang, and S. M. Dai. 2010. Responses of striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), from Taiwan to a range of insecticides. Pest Manag. Sci. 66: 762-766. Ellman, G. L., K. D. Courtney, V. Andres, and R. M. Featherstone. 1961. A new and rapid colorimeric determination of acetylcholinesterase activity. Biochem. Pharmac. 7: 95-99. Goustin, A. S., and F. H. Wilt. 1982. Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos. Biochim. Biophys. Acta. 699: 22-27. Gunning, R. V., I. G. Ferris, and C. S. Easton. 1994. Toxicity, penetration, tissue distribution, and metabolism of methyl parathion in Helicoverpa armigera and H. punctigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 87: 1180-1184. Hao, C. j., B. F. Chai, W. Wang, Y. Sun, and A. H. Liang. 2005. Polyclonal antibody against Manduca sexta chitinase and detection of chitinase expressed in transgenic cotton. Biotechnol. Lett. 27: 97-102. Hall, L. M. C., and C. A. Malcolm. 1991. The acetylcholinesterase gene of Anopheles stephensi. Cell. Mol. Neurobiol. 11: 131-141. Hall, L. M. C., and P. Spierer. 1986. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5’ leader. EMBO J. 5: 2949-2954. Heim, J., C. Schmidt-Dannert, H. Atomi, and R. D. Schmid. 1998. Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli. Biochim Biophys Acta. 1396: 306-319. Jana, S., and J. K. Deb. 2005. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67: 289-298. Jarvis D. L., J. G. W. Flemina, G. R. Kovacs, M. D. Summers, and L. A. Guarino. 1990. Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably-transformed lepidopteran insect cells. Bio-Technology 8: 50-55. Jiang, X., M. Qu, I. Denholm, J. Fang, W. Jiang, and Z. Han. 2009. Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae). Biochem. Biophys. Res. Commun. 378: 269-272. Kato T., K. Yoshizuka, and E. Y. Park. 2010. New strategy for rapid isolation of stable cell lines from DNA-transformed insect cells using fluorescence activated cell-sorting. J. Biotechnol. 147: 102-107. Liang, D., S. P. Carvalho, S. Bon, and J. Massoulie. 2009. Unusual transfer of CutA into the secretory pathway, evidenced by fusion proteins with acetylcholinesterase. FEBS J. 276: 4473-82. Looman, A. C., J. Bodlaender, M. deGruyter, A. Vogelaar, and P. H. van Knippenberg. 1986. Secondary structure as primary determinant of the efficiency of ribosomal binding sites in Escherichia coli. Nucleic Acids Res. 14: 5481-5497. Mallender, W. D., T. Szegletes, and T. L. Rosenberry. 2000. Acetylthiocholine binds to Asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway. Biochemistry 39: 7753-7763. Maroni, M., C. Colosio, A. Ferioli, and A. Fait. 2000. Biological Monitoring of Pesticide Exposure: a review. Introduction. Toxicology 143: 1-118 Matsumura, F. 1975. Toxicology of Insecticides. Plenum Press. New York and London pp. 503. Murphy C. I., and H. Piwnica-Worms. 2001. Overview of the baculovirus expression system. Curr. Protoc. Protein Sci. Unit 4.18. Nettleship J. E., R. Assenberg, J. M. Diprose, N. Rahman-Huq, and R. J. Owens. 2010. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. Epub ahead of print. Ni, Y., and R. Chen. 2009. Extracellular recombinant protein production from Escherichia coli. Biotechnol. Lett. 31: 1661-1670. Park, S. E., N. D. Kim, and Y. H. Yoo. 2004. Acetylcholinesterase plays a pivotal role in apoptosome formation. Cancer Res. 64, 2652-2655. Sato R, T. Matsumoto, N. Hidaka, Y. Imai, K. Abe, S. Takahashi, R. H. Yamada, and Y. Kera. 2009. Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme. Protein Expr. Purif. 64: 205-512. Semeniuk D. J., R. Boismenu, J. Tam, W. Weissenhofer, and R. A. Murgita. 1995. Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule. Adv. Exp. Med. Biol. 383: 255-269. Silman, I., and J. L. Sussman. 2008. Acetylcholinesterase: How is structure related to function? Chem. Biol. Interact. 175: 3-10. Smith, G. E., M. D. Summers, and M. J. Fraser. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3: 2156–2165. Sondergaard L. 1996. Drosophila cells can be grown to high cell densities in a bioreactor. Biotechnol. Techniques 10: 161-166. Sussman, J. L., M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, and I. Silman. 1991. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253: 872-879. Temeyer, K. B., and A. C. Chen. 2007. Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae). DNA seq. 18: 85-91. Theilmann D. A. and S. Stewart. 1992. Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 187: 84-96. Vaughn, J. L., R. H. Goodwin, G. J. Tompkins, and P. McCawley. 1977. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13: 213-217. Wang, Q. M., H. I. Jiang, J. Z. Chen, K. X. Chen, and R.Y. Ji. 1998. On the possible reaction pathway for the acylation of AChE of AChE-catalyzed hydrolysis of Ach: semiempirical quantum chemical study. Int. J. Quantum Chem. 70: 515-525. Wang, Q. M., H. I. Jiang, K. X. Chen, R.Y. Ji, and Y. J. Ye. 1999. Theoretical studies on the possible reaction pathway for the decylation of the AChE-catalyzed reaction. Int. J. Quantum Chem. 74: 315-325. Wickham, T. J., T. Davis, R. R. Granados, M. L. Shuler, and H. A. Wood. 1992. Screening of insect cell lines for the production of recombinant proteins and infectious virus. Biotechnol. Prog. 8: 391-396. Zhang, X. J., L. Yang, Q. Zhao, J. P. Caen, H. Y. He, Q. H. Jin, L. H. Guo, M. Alemany, L. Y. Zhang, and Y. F. Shi. 2002. Induction of acetylcholinesterase expression during apoptosis in various cell types. Cell Death Differ. 9: 790-800. Zhu, K. Y., and J. M. Clark. 1995. Cloning and sequencing of a cDNA encoding acetylcholinesterase in Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem. Mol. Biol. 25: 1129-1138. Zingde, S., V. Rodrigued, S. M. Joshi, and K. S. Krishnan. 1983. Molecular properties of Drosophila acetylcholinesterase. J. Neurochem. 41: 1243-1252. Zouhar, J., E. Nanak, and B. E. Brzobohaty. 1999. Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific β-glucosidase. Protein Expression and Purification 17: 153-162.
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。
URI: http://hdl.handle.net/11455/30766
其他識別: U0005-2208201001343200
Appears in Collections:昆蟲學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.