臺灣博碩士論文加值系統

嚴苛環境所造成的壓力，病原菌的感染，及倉儲害蟲的傷害一直是造成農作植物產量及品質受損之重要原因。而植物本身為了抵禦這些天然的生存競爭也發展出了各種重要的防衛策略，諸如:在外層形成以脂質為主要成份的角明質層(cutin)以抵禦外來侵犯，或是直接產生對病原菌或害蟲有毒的化合物及蛋白質。 近來，許多研究已經證明將一些在植物防禦中扮演重要角色的蛋白質(如：植物非專一性脂質運輸蛋白，植物防禦素…等等)以蛋白質工程做修飾可以增強其植物相關抵禦能力。因此，瞭解這些參與植物防禦的蛋白質的基本特性與其反應機制是相當重要的研究目標。
植物非專一性脂質運輸蛋白是一個可以在試管內(in vitro)實驗中細胞膜間傳輸脂質分子的熱穩定蛋白質。這個家族的蛋白曾被報導可能參與：植物防禦，授粉，發芽…等重要功能。但其在生物體內所扮演的真實功能及相關機制卻一直未被釐清。在本論文中，我們從綠豆中純化出一個新的非專一性脂質運輸蛋白並測定其氨基酸序列。藉由分析核磁共振方法所解出來的三級蛋白質結構，我們發現此蛋白質的C端與中空疏水腔的結構和稻米中的脂質運輸蛋白相當不同。因為此一中空疏水腔環境被認為在功能及結構上都具有相當重要的影響。因此，我們利用圓二色光譜，螢光光譜及蛋白質嵌合模擬來瞭解並分析他們的熱穩定特性和脂質運輸能力與三級結構的相關性。
植物防禦素也是一種多功能的植物重要蛋白質。他們已被報導的相關功能包括:酵素抑制，無細胞系統中抑制蛋白質合成，及最有名的抵抗植物病原菌的能力。在此論文中我們利用核磁共振解出一個新植物防禦素VrD2的三級結構。有趣的是此蛋白質雖然和之前被報導可抗蟲的VrD1蛋白質結構極為相似，VrD2卻完全沒有抗蟲能力。另外，在��-澱粉酶的抑制實驗中VrD2也和VrD1表現出不同的抑制能力。因為��-澱粉酶在目前被認為與抗蟲能力相關，因此我們進一步利用蛋白質工程方法以VrD2結構為主體去設計出具��-澱粉酶抑制能力的VrD2 chimera (VrD2c)。我們也利用同源模擬法和蛋白質嵌合分析比較VrD1，VrD2及VrD2c三級結構且討論VrD2c和澱粉酶結合模式。我們相信這些結果證明了植物防禦素適合利用於蛋白質工程且將有助於其在農作植物上的應用。

It is well recognized that environmental stresses, pathogenic infections, and insect damage significantly result in agricultural losses. Plants have developed defense mechanisms such as evolving stress-resistant system, developing barriers to entry and producing the plant poisons against these damage factors. Extensively studies have indicated that the modifications of the proteins related to the plant defense by protein engineering improve protein ability of defense functions. Thus, understanding the mechanisms as well as the biochemical properties of the proteins participate in regulating plant defense mechanisms is important.
Plant non-specific lipid transfer proteins (nsLTPs) are thermal stable proteins that are capable of transferring lipid molecules between membranes in vitro. This family of proteins is proposed to be involved in defense, pollination and germination; the in vivo biological function remains, however, elusive. Here we report the purification and sequencing of an nsLTP1 (Vr-nsLTP1) from sprouts of Vigna radiate (Vr). By determining and analyzing the solution structure of Vr-nsLTP1, some notable differences in the C-terminal tails and internal hydrophobic cavities were found between Vr-nsLTP1 and Os-nsLTP1 (purified from oryza sativa). Circular dichroism and fluorescence spectroscopy were used to compare the thermodynamics and lipid transfer properties of Vr-nsLTP1 with that of Os-nsLTP1. Docking of a lipid molecule into the solution structure of Vr-nsLTP1 reveals similar binding cavities and hydrophobic interactions as in Os-nsLTP1 consistent with their comparable lipid transfer properties measured experimentally.
Plant defensins are important constituents of the innate immune systems of plants. They are multi-function proteins reported to inhibit protease, protein synthesis in cell-free system or act against microbe and fungi. Here, the structure of a novel plant defensin isolated from the seeds of the Vigna radiata has been determined by 1H nuclear magnetic resonance spectroscopy. Interestingly, this protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera produced by substitution of the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. The application of protein engineering to this protein family may provide an efficient method for protection against crop losses.

Abstract in Chinese 1
Abstract 3

Chapter I. Introduction
1.1 Introduction 6
1.2 Non-specific Lipid Transfer Proteins 6
1.3 Plant Defensins 9
1.4 Figures and Tables 13

Chapter II. Experimental Procedures
2.1 Sequence Determination and Characterization of NsLTP1s 27
2.2 Structural Analyses of NsLTP1s 29
2.3 Lipid Transfer Assay of NsLTPs 30
2.4 NMR Experiments and Structure Calculations of Plant Defensin VrD2 30
2.5 Construction of VrD2 Derivative Expression Plasmids 32
2.6 Overproduction and Purification of Recombinant VrD2 and Derivatives 33
2.7 Plant Defensins Identification and Characterization 34
2.8 Purification and Activity Assay of Tenebrio Molitor α-Amylase 35
2.9 Model Building and Docking Study of Plant Defensin 35
2.10 Figures 36

Chapter III. Characterization and Structural Analyses of nonspecific Lipid Transfer Protein 1 from Mung Bean
3.1 Introduction 42
3.2 Result and Discussion 43
3.2.1 Purification and Sequencing of Vr-nsLTP1 43
3.2.2 Secondary Structure of Vr-nsLTP1 44
3.2.3 Comparison with Homologous NsLTP1s 44
3.2.4 Lipid Transfer Activity and Docking of a Lipid Molecule into Vr-nsLTP1 46
3.2.5 Comparisons of the Thermodynamic Properties of Vr- and Os-nsLTP1 48
3.2.6 Probable Role of NsLTP1 as Plant Defensin 48
3.3 Conclusion 49
3.4 Figures and Tables 51

Chapter IV. Structure-Based Protein Engineering for Alpha-amylase Inhibitory Activity of Plant Defensin
4.1 Introduction 69
4.2 Result and Discussion 70
4.2.1 Resonance Assignment and Secondary Structure of VrD2 70
4.2.2 Structure Calculation and Refinement 71
4.2.3 VrD2 Structure 72
4.2.4 Structural comparison of VrD2 with VrD1 73
4.2.5 VrD2 chimera 74
4.2.6 Insect alpha-Amylase Inhibitory activity 74
4.3 Discussion 75
4.4 Figures and Tables 79
Research Publications 94
References 95

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