綠豆防禦素第一型是個由四十六個胺基酸所組成的鹼性蛋白質，其中又以胱胺酸最為豐富。在前人的研究中發現這個蛋白質具有殺蟲的效果，立體結構也已經被用核磁共振光譜所解出，然而對於殺蟲的機制仍然是不清楚。此外在我們先前的研究中發現綠豆防禦素第一型除了殺蟲以外也具有抑制麵包蟲(α-)澱粉水解□的能力，抑制澱粉水解□是否對於殺蟲的效果有何影響是我們感興趣的，但是綠豆防禦素第一型在結構上是如何與澱粉水解□發生作用？哪些位點上的胺基酸對於抑制功能是重要的？對於這些疑問我們需要大量的蛋白質來實驗，所以我們建構綠豆防禦素第一型在能夠在大腸桿菌上表現的載體，利用定點突變的技術來觀察胺基酸的性質與位置對於功能上的影響。根據一級結構的分析與三級結構的比對，我們預測十一個胺基酸會是重要的，十三個突變經過抑制澱粉水解□的檢驗下，我們發現了三個帶正電的胺基酸(Lys6、Arg26和Arg38)對於功能有很大的影響。此外我們發現二級結構在點突變下改變並不大，在結構沒改變下卻影響到了功能，所以我們認為在這三個位點與澱粉水解□的作用對於抑制功能是重要的。

Vigna radiata defensin 1 (VrD1) is a small, basic and cysteine-rich peptide of 46 amino acids. In former study, VrD1 was reported to exhibit insecticidal activity, and three dimensional structure of VrD1 have been determined by nuclear magnetic resonance (NMR) spectroscopy. However, the insecticidal mechanism of VrD1 is still indistinct. Our preliminary data showed that VrD1, which was purified from mung bean, inhibited Tenebrio molitor α-amylase. To elucidate the α-amylase inhibition mechanism of VrD1, recombinant VrD1 was constructed, expressed and purified from Escherichia coli. According to amino acid sequence analysis and protein structure comparison, specific residues involved in α-amylase inhibition were identified by site-directed mutagenesis. Eleven mutants were totally obtained and analyzed by circular dichroism (CD) for secondary structure and α-amylase activity assay for the inhibition function. The CD spectra showed that all recombinant VrD1 proteins have similar secondary structures. The results of α-amylase inhibition assay show that three mutants, K6A, R26E and R38A, significantly decrease in □-amylase inhibition. These three residues may play important roles in inhibitory function in VrD1.

Abstract 3
摘要 4
Abbreviations 5
Chapter 1. Introduction 6
1.1 Plant defensins 6
1.2 Vigna radiata plant defensin 1 (VrD1) 6
1.3 Tenebrio molitor α-amylase (TMA) 7
1.4 The theme of this thesis 8
Chapter 2. Materials & Methods 10
2.1 Materials 10
2.2 Construction of recombinant VrD1 (rVrD1) 10
2.3 VrD1 variants construction 11
2.4 Expression and purification of rVrD1 11
2.5 Purification of α-amylase from T. molitor larvae 12
2.6 Mass spectrometry 13
2.7 Protein concentration 15
2.8 Assay of inhibitory function against TMA 14
2.9 Circular dichroism (CD) spectra 15
Chapter 3. Result & Discussion 16
3.1 Cloning of the recombinant VrD1 16
3.2 Purification of rVrD1 16
3.3 Characterization of rVrD1 17
3.4 Purification of TMA 18
3.5 Targets for site-directed mutagenesis 18
3.6 Expression and purification of VrD1 variants 19
3.5 Titration of VrD1 mutants 19
3.7 K6A and K7A 20
3.8 R26E and R26K 20
3.9 K12A and R38A 21
3.10 Modification of the loop L2 (T37D and 39insA) 21
Chapter 4. Conclusion 23

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