不同聚合形式之甘藷凝集素其抗菌和抗蟲機制之研究

Molecular function of Ipomoelin in different oligomerizations for antibacterial and insecticidal ability

黃詠琪(Yung-Chi Huang)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10471

摘要

甘藷凝集素Ipomoelin(IPO)為甘藷受到傷害時，在葉子誘導表現的植物性防禦蛋白質。根據文獻結果，植物凝集素(lectins)可用來抵抗病毒、細菌、真菌以及昆蟲。IPO是屬於mannose-specific jacalin-related lectin(mJRL)次家族的醣結合蛋白，但亦能與半乳糖(galactose)結合，顯示其具有多種醣類結合的能力。IPO晶體結構呈現四聚體形式，每個單體N端具兩個短鏈的β摺板，約10個胺基酸，具有改變對醣類的結合能力，並且和IPO形成不同的多聚體相關聯。本研究進一步對於IPO的N端序列及其抗蟲抗病能力進行探討，首先構築了N端去掉前6個胺基酸的IPO(ND6)，相當去掉N端一個β摺板，晶體結構及膠體過濾色層分析法(gel filtration chromatography)結果顯示ND6為二聚體。進一步比較wtIPO(四聚體)和ND10(單體)，利用等溫滴定微熱量法(isothermal titration calorimetry, ITC)得知ND6對甲基化甘露醣(methyl-α-D-mannopyranoside, Me-Man)的結合能力介於wtIPO和ND10之間，呈現過渡態的形式。IPO和不同物種同屬JRL家族的凝集素序列比對的結果，將醣結合區域(carbohydrate binding pocket )之Trp-142位置定位點突變(site-directed mutagenesis)成其他物種之胺基酸，利用ITC測試其醣結合能力是否改變，以確定Trp-142是穩定甲基化單醣結合的重要胺基酸。在進行家蠶餵食實驗結果顯示，抑蟲能力為ND10 > ND6 > wtIPO；而在抑制大腸桿菌(Inhibition assay)的實驗中，抑菌能力為wtIPO > ND6 > ND10。利用差異性掃描螢光法(Differential scanning fluorimetry, DSF)測定變性溫度(melting temperature, Tm) 得知IPO不同蛋白質之熱穩定性，而wtIPO 在不同的酸鹼值之下會比ND6 及ND10來得相對穩定；利用次細胞定位(subcellular localization)得知IPO不只會存在在細胞質中，也會出現於細胞核中，代表其有可能具有訊息傳遞的功能。

關鍵字

甘藷凝集素 ； 凝集素 ； 多聚體 ； 膠體過濾色層分析 ； 等溫滴定微量熱法

並列摘要

Ipomoelin (IPO), a plant defense protein, is induced by mechanical wounding or herbivorous attacking. It was isolated from sweet potato (Ipomoea batatas cv. Tainung 57) and belongs to mannose-specific jacalin-related lectin (mJRL) family. The quaternary structure of IPO reveals a tetrameric association. However, previous results indicated that IPO could also bind to galactose and showed more diverse carbohydrate binding ability than that of mJRL. The first 10 amino acids of N-terminus of IPO are important to carbohydrate binding and might be involved in different oligomerization of IPO. Several experiments were used to identify the roles of N-terminus of IPO. A mutant protein with truncated N-terminal 6 residues of IPO (ND6) was constructed. The dimeric form of ND6 was identified by X-ray crystallography and size exclusion chromatography (SEC). In comparison with wtIPO (tetramer form) and ND10 (monomer form), the binding affinity of ND6 to methyl-α-D-mannopyranoside (Me-Man) showed its transition manner in the middle of wtIPO and ND10 by isothermal titration calorimetry. According to the sequence alignment between IPO and other JRL family lectins, we use site-directed mutagenesis change the Trp-142 of IPO to other lectins residue. Use the ITC studies to confirm the residue Trp-142 as a key residue for stabilizing the binding of methylated carbohydrates. In the silkworm feeding experiment, the retarded growth ability is ND10 > ND6 > wtIPO. For the antibacterial assay, the inhibition growth of Escherichia coli is wtIPO > ND6 > ND10. The melting temperature determined by differential scanning fluorimetry (DSF) showed that wtIPO was more stable in wide range of pH than those of ND6 and ND10. Furthermore, wtIPO would exist in both cytoplasm and nucleus by subcellular localization experiment.

並列關鍵字

lectin ； jacalin ； oligomerization ； ipomoelin ； gel filtration chromatography ； isothermal titration calorimetry

參考文獻

邱宜芳 (2003) 甘藷傷害誘導之蛋白質Ipomoelin的功能測定。國立台灣大學植物學研究所碩士論文。

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