臺灣博碩士論文加值系統

綠豆防禦素第一型 (VrD1) 是第一個被報導具有抵抗豆象的植物防禦素。我們實驗室已經利用核磁共振光譜儀解出綠豆防禦素第一型之三度空間立體結構。然而，其殺蟲機制仍不清楚。根據先前的研究結果顯示，綠豆防禦素第一型具有抑制麵包蟲α-澱粉水解酶的能力，推測此抑制功能極可能與其抗蟲機制有關。同時以電腦模擬計算結果顯示，在綠豆防禦素第一型與麵包蟲α-澱粉水解酶的複合結構中，綠豆防禦素第一型的第三環型結構 (loop 3)，推測與其抑制能力息息相關。在所有已知植物防禦素的結構中，綠豆防禦素第一型是第一個具有310 螺旋結構的例子。根據蛋白質序列比對分析，不同於其他植物防禦素，在第26個胺基酸位置，綠豆防禦素第一型是由精胺酸取代麩胺酸，而此結果可能造成第10個胺基酸位置色胺酸其支鏈的方向改變，因而誘發310 螺旋結構形成，進而促進綠豆防禦素第一型整體結構的穩定。在此論文中，我們主要針對第三環型結構與310 螺旋結構之形成對於��-澱粉水解酶抑制能力之影響。利用定點突變分析方法來研究綠豆防禦素第一型中對於抑制麵包蟲��-澱粉水解酶有重要影響的胺基酸。並且進一步地利用圓二光偏極光譜儀來分析純化之突變綠豆防禦素第一型的二級結構。實驗結果顯示數個突變綠豆防禦素第一型抑制麵包蟲α-澱粉水解酶的能力有明顯降低，證明第三環型結構與310 螺旋結構在其抑制功能上，扮演重要角色。

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity against Callosobruchus chinensis (bruchid). Three dimensional structure of VrD1 has been determined by nuclear magnetic resonance spectroscopy in our laboratory. Nevertheless, the mechanism of insecticidal activity is unclear. According to our previous work, VrD1 has shown to inhibit Tenebrio molitor α-amylase (TMA) in vitro which may trigger insecticidal activity. Computational docking model of VrD1-TMA complex also implied that loop L3 of VrD1 is important for this inhibition. Among plant defensins of known structure, VrD1 is the first case containing a 310 helix. Based on protein sequence alignments, VrD1 is different from other defensins and contains an arginine in place of glutamate at the residue 26. We propose that this residue may induce a shift in the orientation of Trp10, thereby facilitating the 310 helix formation and then contributing to the stability of global structure. Therefore, this study would focuse on the influence of loop L3 and 310 helix of VrD1 on its inhibitory function. Site-directed mutagenesis was carried out to study critical residues of VrD1 involving in TMA inhibitory activity. The secondary structures of purified proteins were examined by circular dichroism (CD). Our results showed that several mutants significantly decreased in TMA inhibition, indicating that the loop L3 and 310 helix indeed play important roles in VrD1 insecticidal function.

Contents 1
Abstract 3
中文摘要 4
Abbreviations 5
Chapter 1. Introduction 6
1.1 Plant Defensins 6
1.2 Vigna radiata plant defensin 1(VrD1) 7
1.3 Tenebrio molitor α-amylase (TMA) 8
1.4 The theme of this thesis 9
Chapter 2. Materials and Methods 11
2.1 Materials 11
2.2 Construction of expression plasmids of recombinant VrD1 11
2.3 Expression and purification of rVrD1 12
2.4 Purification and activity assay of α-amylase from T. molitor larvae 13
2.5 Mass spectrometry 15
2.6 Assay of inhibitory function of VrD1 against TMA 15
2.7 Circular dichroism (CD) spectra 16
2.8 Fluorescence spectroscopy 17
2.9 Protein concentration 18
Chapter 3. Results and Discussion 19
3.1 Purification of rVrD1 19
3.2 Purification of TMA 19
3.3 Target selection for site-directed mutagenesis 20
3.4 Characterization of VrD1 variants 21
3.5 Effects of VrD1 mutants on TMA activities 22
3.6 Fluorescence of intrinsic tryptophan of VrD1 23
Chapter 4. Conclusion 25








Contents of Tables and Figures

Table 1. Molecular weight of VrD1 mutants 27
Table2. Summary of biophysical properties 28
Table 3. Sequence of oligonucleotides used for PCR and site-directed mutagenesis. 29
Figure 1: Solution structure of VrD1 (PDB code: 1TI5). 30
Figure 2: 3-D structure of TMA (PDB code:1JAE). 31
Figure 3: Sequence alignment of various plant defensin. 32
Figure 4: The mutated residues in the structural element of VrD1. 33
Figure 5: Construction of the expression system. 34
Figure 6: Protein purification was analyzed by SDS-PAGE. 35
Figure 7: RP-HPLC profile of rVrD1. 36
Figure 8: VrD1 mutants were analyzed by Tricine-SDS-PAGE. 37
Figure 9: Identification by ESI-MS spectroscopy. 38
Figure 10: Flowchart for TMA purification. 39
Figure 11: Purification of TMA on FPLC system. 40
Figure 12: TMA purification was analyzed by SDS-PAGE. 41
Figure 13: Determination of activity unit of purified TMA 42
Figure 14: The titration curve of the VrD1 mutants. 43
Figure 15: The inhibitory activity of the VrD1 variants. 44
Figure 16: Far-UV CD spectra of wild-type and mutant proteins. 45
Figure 17: The thermal stability of wild-type and mutant proteins. 46
Figure 18: The procedure of insect α-amylase inhibitory assay. 47
Figure 19: Computational docking analysis of TMA-VrD1 complex. 48
Figure 20: Fluorescence of intrinsic tryptophan of VrD1 variants involving 310 helix formation. 49
Figure 21: Intramolecular interactions between Trp10 and other residues in VrD1 50
References 51

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