博碩士論文 952204003 詳細資訊




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姓名 王綉華(Siou-hua Wang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Galectin-1 蛋白對小菜蛾的殺蟲活性及作用機制之研究
(Insecticidal action of a mammalian Galectin-1 toward Plutella xylostella)
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摘要(中) Galectins 是ㄧ類哺乳動物之醣類結合蛋白,可以專一性結合半乳糖。先前研究顯示,純化之重組 galectin-1 (GAL1) 蛋白可以干擾小菜蛾(Plutella xylostella) 正常生長發育;為了進一步探討 GAL1 發展成生物殺蟲劑之潛力,本研究深入研究 GAL1 蛋白殺蟲活性及其毒性作用機制。我們首先利用原二色光譜儀 (Circular Dichrosim) 研究 GAL1 在不同 pH及溫度的穩定性,結果發現 GAL1 在 pH 8~9 時較為穩定,而這相當接近於昆蟲腸道的鹼性環境;實驗結果也顯示 GAL1 在 50 0C下結構相對穩定。幾丁質 (Chitin) 及圍食膜 (Peritrophic membrane) 為目前生物殺蟲劑新標的,本實驗結果也證實 GAL1 可和幾丁質以及圍食膜結合。為了瞭解 GAL1 的應用潛力,本實驗成功篩選了 GAL1 的阿拉伯芥轉殖株 (包括全株表現 GAL1-Arabidopsis 及維管束專一性表現 GAL1-Arabidopsis-vas 轉殖株),於全株表現的轉殖株中,GAL1 表現量約占 0.5 % ~ 1 %;餵食試驗發現 GAL1-Arabidopsis 轉殖株對小菜蛾有明顯的殺蟲活性,包括存活率、平均體重及攝食量都有隨著時間而下降。而組織化學及免疫染色切片都顯示,小菜蛾的腸道都觀察到 GAL1 蛋白的存在 ; 而超微結構顯示,餵食 GAL1-Arabidopsis 轉殖株之小菜蛾腸道表皮細胞和微絨毛都呈現不正常的形態。掃描式電子顯微鏡結果顯示餵食 GAL1 的小菜蛾,其圍食膜都呈現被破壞的現象。此一實驗顯示 GAL1 對小菜蛾之毒性機制可能與幾丁質產生結合作用,而影響昆蟲圍食膜的結構,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。
摘要(英) Galectins (GALs) are a family of mammalian sugar-binding proteins specific for β-galactosides. Previous studies have shown that the larval development of Plutella xylostella are significantly disturbed when they are fed them with E. coli-expressed recombinant galectin-1 (GAL1). To explore the applicability of GAL1 as a bio-insecticide, the insecticidal activity and mechanism of GAL1 were further investigated. The circular dichrosim (CD) spectrum showed that the secondary structure of GAL1 was not affected under pH 8-9 or below 50 oC. These results indicated that GAL1 was workable in the midgut’s microenvironments of most insects (pH 8-9) and also thermo-stable below 50 oC. Besides, GAL1 could interact in vitro with chitin and peritrophic membrane (PM) that are the potential targets for new bio-insecticides development. Two GAL1 over-expressed Arabidopsis 【GAL1-Arabidopsis (whole plant) and GAL1-Arabidopsis-vas (vascular bundle-specific)】.These studies also screen for insecticidal activity and mechanism studies. The results showed that expression level of GAL1 in GAL1-Arabidopsis transformants ranges from 0.5 % ~ 1 % of total leaf soluble protein. The survival rate, body weight and food consumption were significantly decreased in a time-dependent manner in P. xylostella fed on GAL1-Arabidopsis. The results of histochemical structure and immunostaining suggested that GAL1 dose- and time-dependently bound to the midgut epithelium of P. xylostella fed on GAL1-containing diet or GAL1-Arabidopsis. The ultrastructural studies further showed the disruption of the microvilli and abnormalities in epithelial cells. The scanning electron micrographs showed no PM present in P. xylostella fed on GAL1-containing diet or GAL1-Arabidopsis. Inferred from these results, the insecticidal mechanism of GAL1 involves direct binding with chitin to interfere with the structure of the PM, and GAL1 could be a potential candidate for bio-insecticides development.
關鍵字(中) ★ Galectin-1 蛋白
★ 幾丁質
★ 圍食膜
★ 小菜蛾
★ 生物殺蟲劑
關鍵字(英) ★ Plutella xylostella bio-insecticide
★ chitin
★ Galectin-1
★ peritrophic membrane
論文目次 Contents..............................................Ⅰ
Figure Contents.......................................Ⅲ
Abbreviations.........................................Ⅳ
Introduction
1. Bio-insecticides....................................1
2. Plant lectins.......................................3
3. Peritrophic membrane................................6
4. Galectins...........................................9
5. Preliminary study and specific aims................11
Materials and Methods
1. Insect culture.....................................12
2. Over-expression and purification of GAL1 from E. coli…................................................12
3. Screening of GAL1-Arabidopsis transformants........13
4. Insect feeding trials..............................14
4.1 Insect bioassay on E. coli-expressed GAL1.........14
4.2 Insect bioassay on transgenic Arabidopsis.........14
5. Circular dichrosim spectrum of GAL1................15
6. Pull-down assay of GAL1 binding with chitin........16
7. Interaction of E. coli-expressed GAL1 with PM of P.
xylostella in vitro................................16
8. Preparation and sectioning of larvae for light
microscopy.........................................17
9. Histochemical staining of midgut section for
carbohydrates......................................18
10. Immunocytochemistry for confocal laser scanning
microscopy........................................18
11. Effect of GAL1-Arabidopsis on the ultrastructure
of PM.............................................19
12. Cryosection and immunohistochemistry of GAL1-
Arabidopsis leaves................................19
13. Electrophoresis and Western blotting..............20
14. Statistical analysis..............................21
Results
1. Structural stability of GAL1.......................22
2. Interaction of GAL1 with chitin in vitro...........22
3. Interaction of GAL1 with PM of P. xylostella in
vitro..............................................23
4. Effect of GAL1 on the midgut ultrastructure of P.
xylostella.........................................23
5. Effect of GAL1 on carbohydrate metabolism in P.
xylostella.........................................24
6. Expression of GAL1 in transgenic GAL1-Arabidopsis
and GAL1-Arabidopsis-vas...........................25
7. Effect of GAL1-Arabidopsis on survival, growth and
food consumption of P. xylostella..................25
8. Effect of GAL1-Arabidopsis on midgut ultrastructure
of larval P. xylostella............................27
9. Effect of GAL1-Arabidopsis on isolated PM of P.
xylostella.........................................27
Discussion............................................29
Figures...............................................35
References............................................49
Appendix..............................................60
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指導教授 黃榮南(Rong-nan Huang) 審核日期 2008-7-12
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