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

王綉華(Siou-hua Wang)

指導教授 ： 黃榮南

國立中央大學/理學院/生命科學系/碩士(2008年)

摘要

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。

關鍵字

Galectin-1 蛋白 ； 幾丁質 ； 圍食膜 ； 小菜蛾 ； 生物殺蟲劑

並列摘要

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.

並列關鍵字

Plutella xylostella bio-insecticide ； chitin ； Galectin-1 ； peritrophic membrane

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