Galectin-1 蛋白對小菜蛾毒性機制之研究｜國立中央大學博碩士論文系統

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研究生：	陳念廷 Nien-Ting Chen
論文名稱：	Galectin-1 蛋白對小菜蛾毒性機制之研究 Characterization of the insecticidal activity of galectin-1 on Plutella xylostella
指導教授：	黃榮南 Rong-Nan Huang
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
畢業學年度：	93
語文別：	英文
論文頁數：	57
中文關鍵詞：	小菜蛾、幾丁質、蛋白質
外文關鍵詞：	protein(galectin-1), Plutella xylostella, chitin
相關次數：	點閱：17 下載：0
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摘要
GAL1是ㄧ種動物性半乳糖結合蛋白( Glaectin)，具有多重功能，包括細胞貼附、增生、分化及免疫調節之功能。GAL1 蛋白於細胞質合成後，分泌至細胞外，再與細胞膜上含 β-galactosyl 之蛋白進行結合，進而引發細胞內之訊息傳遞。實驗室先前的研究已證實GAL1蛋白可吸附在幾丁質聚醣膜上，而造成3T3細胞之增生。此外實驗也證實GAL1蛋白能與幾丁聚醣做結合。幾丁質是自然界中最重要的天然聚合物之一，也是昆蟲外胚層衍生構造之胞外基質, 如：外骨骼及腸道外基質(matrix)主要成分。因此本研究擬探討GAL1蛋白對昆蟲生長發育之影響及其毒性作用之機制。結果顯示以GAL1蛋白萃取液或純化之GAL1蛋白餵食三到四齡小菜蛾，可以干擾其正常生長發育；組織切片結果顯示，隨著GAL1劑量增加或處理時間延長，小菜蛾之腸道結構破壞越深。此一結果顯示，GAL1蛋白對小菜蛾之毒性作用可能與腸道組織之破壞有關。實驗結果也顯示幾丁質分解酵素(chitinase)之活性及幾丁質合成酵素(chitin synthase)之表現可被GAL1蛋白所抑制。GAL1與哺乳動物細胞膜蛋白結合的 motif 主要具有Galβ1-4GlcNAc 結構，與幾丁質(β-1,4 N-acetyl-D-glucosamine) 結構相似。因此推測，GAL1對小菜蛾之毒性機制可能與幾丁質產生結合作用，而造成昆蟲幾丁質代謝干擾，進而影響其正常發育。由本實驗結果顯示，GAL1蛋白可以抑制小菜蛾幼蟲正常發育，並具有發展成為生物性殺蟲劑之潛力。

Abstract
Galectin-1 (GAL1), a β-galactosyl lectins, has been shown to modulate cell adhesion, cell-matrix interaction, cell proliferation and some immune functions. GAL1 is an extracellular protein acted to cross-linking cell surface and substrate glycoconjugate. Previous work in this laboratory have showed that GAL1-coated chitosan membrane support 3T3 cell proliferation and GAL1 could dose-dependently bind with chitosan (a partial de-acetylated chitin) in vitro. These results suggested that chitosan and chitin could be a natural ligand of GAL1.
Chitin is one of the most important biopolymer in nature. In insect, it functions as scaffold materials, supporting the cuticles of the epidermis and trachea as well as peritrophic matrices lining the gut epithelium. In this study, we investigated the effect of GAL1 on insect larval development and the mechanism of its insecticidal activity. The results showed that GAL1 is toxic to the Lepidoptera of Plutella xylostella. The development of third to fourth instar of P. xylostella were significantly disturbed after feeding with GAL1 extract and/or recombinant proteins purified from E.coli-over-expressed GAL1. Histological study showed that midgut of P. xylostella larva was dose-, and time-dependently destroyed after feeding with GAL1. Moreover, the expressions of chitinase activity and PxCS (P. xylostella chitin synthase) were down-regulated by the treatment of GAL1. These results suggested that insecticidal activity of GAL1 toward P. xylostella results from the disruption of midgut integrity. In view of GAL1 having a single carbohydrate-recognition domain recognized the structural motif Galβ1-4GlcNAc that is similar to that of chitosan membrane (?-1,4 N-acetyl-D-glucosamine). We proposed that insecticidal mechanism of GAL1 involves direct binding with chitin. The present study also indicated that GAL1 could be a candidate for bio-insecticide due to its high insecticidal activity.

Contents
Contents………………………..………..…………………….……...Ⅰ
Figure Contents…………….……………………………………….Ⅲ
Abbreviation……………….…………….…………………………..Ⅳ
Chapter 1. Introduction
1.1   Lectin……………………………..……….……...……………..1
1.2   Galectin-1……………………………………………………….3
1.3   Chitin…………………………..………………………………...5
1.4   Chitosan……………………..…………………………………..8
1.5   Chitin synthase……………..…………………………………..9
1.6   Chitinase……………..…………………………………………11
1.7   Preliminary study and specific aim…………………………12
Chapter 2. Materials and Methods
2.1   GAL1 protein purification..................................................14
2.2	Insect rearing……………………………………………..15
2.3	Insect feeding trials……………………………………….15
2.4   Tissue preparation for microscopy ……………………….16
2.5   Preparation and sectioning of insect tissues……………...17
2.6   Immunocytochemistry for light microscopy……………..17
2.7   Protein Quantification…....................................................18
2.8   SDS-PAGE Analysis..........................................................19
2.9    Western Blot Analysis…………………………………19
2.10   Detection of chitinase activity after SDS-PAGE………19
2.11   Isolation of RNA from larvae using TRIZOL reagent…20
2.12   RT-PCR………………………………………………...21
2.13   Ligation into pGEM-Teasy Vector…………………….22
214   Transformation and Screening……………………….....22
2.15   Mini-preparation of plasmid DNA……………………..23
2.16   DNA Sequencing……………………………………….24
Chapter 3. Results
3.1   Effects of GAL1 on larval surviva………………………25
3.2   Effects of GAL1 on the gut tissue of larval Plutella xylostella……………………………………………………………….25
3.3   Detection of GAL1 by Western blot analysis…………...26
3.4	   Localization of GAL1 in larval Plutella xylostella tissues by light microscopy……………………………………………..27
3.5   Effect of GAL1 on chitinase expression………………...28
3.6	 Effect of GAL1on PxCS expression…………………….28
3.7   Analysis of the sequence of PxCS………………………29
3.8   Homologies with other proteins………………………....30
Chapter 4. Discussion………………………………………….31
Figure…………………………………………………………...36
Reference……………………………………………………….47
Appendix …………...…………..………………..……………..52
Figure Contents
Fig. 1. Chitin structure.…………………………………………………...6
Fig. 2. Chitosan structure.………………………………………………...8
Fig. 3. Chitin synthase structure.………………………………………….9
Fig. 4. Chitinase structure.……………………………………………….11
Fig. 5. Effects of GAL1 on mortality of P. xylostella larvae.……………36
Fig. 6. Micrographic structure of midgut epithelium of P. xylostella……37
Fig. 7. Immunodetection of GAL1 in P. xylostella larvae………………38
Fig. 8. Immunolocalization of GAL1 in the midgut of P. xylostella……39
Fig. 9. Localization of GAL1 in P. xylostella…………………………...40
Fig. 10. Effect of GAL1 on chitinases activity of P. xylostella larva……41
Fig. 11. Inhibitory effect of GAL1 on chitinase activity in P. xylostell larva……………………………………………………………………...42
Fig. 12. Effect of GAL1 on PxCS expression in P. xylostell larva………43
Fig. 13. Homologous search of RT-PCR amplified PxCS in the NCBI database using BLASTN computer program…………………………….44
Fig. 14. Homology of PxCS amino-acid sequences……………………...45
Fig. 15. Alternative models of PxCS chitin synthase function…………...46

                                

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