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研究生：	林彥志 Lin, Yan-Jhih
論文名稱：	探討蝴蝶蘭中Vicilin蛋白質家族的特性 Characterization of Vicilin Family from Phalaenopsis
指導教授：	蔡文杰 Tsai, Wen-Chieh
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	63
中文關鍵詞：	蘭花種子、台灣白花蝴蝶蘭、豌豆球蛋白、抗真菌活性
外文關鍵詞：	orchid seeds, Phalaenopsis aphrodite, vicilin, antifungal activity
相關次數：	點閱：131 下載：1
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在野外的情況下，蘭花種子需要在潮濕的森林中與真菌共生才能順利萌發。在此種環境底下，蘭花種子有極大的可能性受到致病性真菌或是黴菌的入侵。有趣的是，在我們的觀察中，長時間處於濕潤環境下的蝴蝶蘭種子並不會被致病性真菌或是黴菌感染。研究顯示，一群屬於種子儲存蛋白家族的豌豆球蛋白具有抑制酵母菌以及真菌生長的功能。為了研究蝴蝶蘭種子在萌發過程中抵抗真菌入侵的機制，首先於台灣白花蝴蝶蘭原球體的轉錄體中鑑定出豌豆球蛋白家族的同源基因（PaVIC1及PaVIC2），進一步探討這兩個基因在蝴蝶蘭種子萌發過程中扮演的角色。首先在定序結果分析中發現PaVIC2有六個核苷酸的缺失，可進一步分成PaVIC2.1及PaVIC2.2。經由蛋白質分子量預測結果顯示 PaVICs的分子量接近50 kDa。在種子萌發後之原球體發育的過程中，PaVIC1在不同時期中皆呈現持續性大量的表現情形，而PaVIC2則表現量輕微，但有逐步上升，且是在發育過程中受到調控。此外，PaVIC2在萼片、花瓣及唇瓣等有高量表現。SDS-PAGE的分析結果顯示，在分子量大小約40至55 kDa處有大量的蛋白質累積。進一步使用MALDI-TOF 質譜儀分析，發現PaVIC2.2有極大的可能性存在於蝴蝶蘭原球體中。次細胞定位結果顯示，PaVIC1-GFP、PaVIC2.1-GFP及PaVIC2.2-GFP融合蛋白皆在細胞質中呈現顆粒狀構造之螢光訊號。在紙錠擴散實驗（agar disc diffusion assay）中，我們發現蝴蝶蘭原球體的萃取蛋白針對植物致病性真菌具有抑制生長的活性。進一步萃取PaVIC轉植阿拉伯芥的蛋白質進行紙錠擴散實驗，發現轉植株的萃取蛋白相較野生型阿拉伯芥之萃取蛋白，有顯著的抑制真菌生長現象。這些結果證明蝴蝶蘭豌豆球蛋白具有抑制真菌生長的功能。此外，最小抑制濃度（Minimum inhibitory concentration）分析結果亦顯示，蝴蝶蘭原球體萃取蛋白對人類致病酵母菌具有抑制生長的功能。綜合以上，蝴蝶蘭中的豌豆球蛋白除了參與在蝴蝶蘭種子發育過程中，抵抗真菌的入侵，同時也提供了從蝴蝶蘭中發展嶄新抗真菌藥物的可能性。

In the wild, orchid seeds need fungus to in symbiosis under the moist and humid forests. Strikingly, the orchid seeds in humid environment were not invaded by mold nor fungi during germination. Vicilin, a seed storage protein identified in pea, can inhibit the growth of yeast and fungi during their germination. Two vicilin orthologs, designated as PaVIC1 and PaVIC2, were identified from the transcriptome of Phalaenopsis aphrodite protocorm. In this study, characterized these two Phalaenopsis vicilin orthologs are characterized. With six-nucleotide deletion, PaVIC2 were divided into PaVIC2.1 and PaVIC2.2. All PaVICs have similar predicted molecular weight around 50 kDa. The expression patterns of two genes showed that PaVIC1 was constitutively and highly expressed at different embryonic developmental stages, while the expression of PaVIC2 was much less and developmentally regulated. High expression level of PaVIC2 was detected in reproductive organs such as sepal, petal and lip. SDS-PAGE analysis showed that there were large amount of proteins present at the molecular weight of 40-55 kDa. The existence of PaVICs protein in the extract of Phalaenopsis aphrodite seeds was confirmed by using MALDI-TOF MS, and strongly supported the existence of PaVIC2.2 in protocorm. Confocal images showed that the fusion protein of PaVIC1-GFP, PaVIC2.1-GFP and PaVIC2.2-GFP were localized in cytosol and forming punctate bodies. Antifungal activity of total extract protein of Phalaenopsis protocorm against phytopathogenic fungus was detected in agar disc diffusion assay. Furthermore, antifungal property of Phalaenopsis vicilins were further confirm by treating phytopathogenic fungus with proteins extracted from PaVICs-overexpressed transgenic Arabidopsis. Total protein from Phalaenopsis protocorm also exhibited inhibition of Candida species in minimum inhibitory concentration assay. Thus, as a large amount of protein in protocorm development, PaVICs involved in defense of fungal pathogens. The antifungal activity of total protein from P. aphrodite protocorm against human pathogenic fungi provided a possibility to develop a new biofungicides from Phalaenopsis orchids.

摘要....................................................i
Abstract..............................................iii
致謝....................................................v
Table of Contents.....................................vii
List of Tables.........................................xi
List of Figures.......................................xii
List of Appendixs Figures..............................ix

1.	Introduction
1.1	Orchid seeds
1.1.1	The feature of orchid seed morphology...........1
1.1.2	Germination of orchid seed......................2
1.2	Antimicrobial proteins
1.2.1	The role of antimicrobial proteins played in defense response........................................4
1.2.2	Thaumatin-like proteins.........................5
1.2.3	Chitinases......................................6
1.2.4	β-1,3-glucanases................................6
1.2.5	Lipid-transfer proteins.........................7
1.2.6	Protease inhibitors.............................7
1.2.7	Defensins.......................................8
1.2.8	2S albumins.....................................8
1.2.9	Lectins.........................................9
1.3	Vicilin family
1.3.1	Vicilin family in plant.........................9
1.3.2	Antifungal activity of vicilin.................10

2.	Specific Aim...................................12

3.	Materials and Methods
3.1	Plant materials and growth conditions..........13
3.2	RNA preparation................................13
3.3	RT-PCR and real-time quantitative PCR..........14
3.4	Sequence alignments and phylogenetic analysis..14
3.5	Total protein preparation
3.5.1	Crude protein extraction.......................16
3.5.2	Protein concentration and buffer exchange......16
3.5.3	Protein lyophilization and re-dissolve.........17
3.6	SDS-PAGE of total protein......................17
3.7	In-gel digestion...............................18
3.8	MALDI-TOF MS analysis..........................18
3.9	Subcellular localization of PaVIC1-GFP, PaVIC2.1-GFP and PaVIC2.2-GFP fusion protein....................19
3.10	Arabidopsis transformation.....................21
3.11	Agar disc diffusion assay......................22
3.12	Minimum inhibitory concentration assay.........22

4.	Result
4.1	Identification of PaVIC1 and PaVIC2 in P. Aphrodite..............................................23
4.2	Phylogenetic analysis of PaVIC1, PaVIC2 and other putative vicilin sequences.............................24
4.3	Temporal expression of PaVIC1 and PaVIC2 during protocorm development and different organs.............24
4.4	Identification of PaVIC1, PaVIC2.1 and PaVIC2.2 in total protein from 0 DAI protocorm..................25
4.5	Subcellular localization of PaVIC1, PaVIC2.1 and PaVIC2.2...............................................26
4.6	Total proteins extracted from various stages of protocorm displayed activities against phytopathogenic fungi..................................................26
4.7	Agar disc diffusion assay of total protein from transgenic Arabidopsis thaliana against phytopathogenic fungi..................................................27
4.8	Minimum inhibitory concentration test of total protein from various stages of protocorm against human pathogenic fungi.......................................27

5.	Discussion
5.1	Phalaenopsis aphrodite vicilin genes...........29
5.2	PaVIC2.2 presented in total protein from 0 DAI protocorm..............................................29
5.3	Localization of vicilins showed punctate structures in cytosol..................................30
5.4	Crude proteins from protocorm exhibited antifungal activity against phytopathogenic fungi......31
5.5	Crude protein from transgenic Arabidopsis thaliana exhibited antifungal activity against phytopathogenic fungi..................................32
5.6	Crude protein from protocorm exhibited antifungal activity against Candida species.......................33

6.	Reference......................................35

List of Tables
Table 1. List of primers used in this study............43
 
List of Figures
Figure 1.	Alignment of nucleotides sequence of PaVIC1, PaVIC2.1 and PaVIC2.2..........................44
Figure 2.	Alignment of amino acid sequence of PaVIC1, PaVIC2.1 and PaVIC2.2..........................46
Figure 3.	Phylogenetic analysis of PaVIC1, PaVIC2.1, PaVIC2.2 and other putative vicilin protein sequences by neighbor-joining method...................47
Figure 4.	Temporal expression of PaVIC1, PaVIC2.1 and PaVIC2.2 during protocorm development..............48
Figure 5.	Temporal expression of PaVIC1, PaVIC2.1 and PaVIC2.2 in different organs.......................49
Figure 6.	SDS-PAGE analysis of total protein from various stages of protocorm............................50
Figure 7.	Matching peptides of PaVIC1 MOLDI-TOF MS analysis...............................................51
Figure 8.	Matching peptides of PaVIC2.1 MOLDI-TOF MS analysis............................................52
Figure 9.	Matching peptides of PaVIC2.2 MOLDI-TOF MS analysis............................................54
Figure 10.	Subcellular localization of PaVIC1, PaVIC2.1 and PaVIC2.2..................................56
Figure 11.	Agar disc diffusion assay of total protein from various stages of protocorm against phytopathogenic fungi..................................57
Figure 12.	Agar disc diffusion assay of total protein from transgenic Arabidopsis thaliana against phytopathogenic fung...................................58
Figure 13.	Minimum inhibitory concentration test of total protein from various stages of protocorm against human pathogenic fungi.................................59
 
List of Appendixs Figures
Appendixs Figure 1.	pBI121 map.....................61
Appendixs Figure 2.	pDONR221 map...................62
Appendixs Figure 3.	p2FGW7 map.....................63
 
 

                                    

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