臺灣博碩士論文加值系統

番茄黃化捲葉泰國病毒 (Tomato yellow leaf curl Thailand virus, TYLCTHV) 屬於雙生病毒科 (Geminiviridae)、豆類金黃嵌紋病毒屬 (Begomovirus)，其感染所引起的番茄黃化捲葉病 (tomato yellow leaf curl disease) 疫情遍及中國、緬甸、泰國和臺灣。番茄黃化捲葉病是近年來新興的病毒性病害，常帶來番茄產量 20-100% 的損失，尤以熱帶與亞熱帶地區為甚。TYLCTHV的基因體具有兩條單股環狀DNA，稱之為DNA-A與DNA-B。DNA-A上有六個基因：AV1、AV2、AC1、AC2、AC3與AC4，分別表現CP，AV2，Rep，TrAP，REn及AC4蛋白。DNA-B上則有兩個基因：BV1與BC1，分別表現NSP與MP蛋白。為瞭解TYLCTHV的病毒蛋白之功能，我建構了蛋白質表現載體將使植物分別表現TYLCTHV的八個病毒蛋白與綠螢光蛋白 (green fluorescence protein, GFP) 相接形成的融合蛋白，將建構好的載體藉由農桿菌浸潤法 (agroinfiltration) 送到圓葉菸草 (Nicotiana benthamiana) 的細胞內，使 GFP-病毒蛋白融合蛋白得以在菸草的表皮細胞中表現。為進一步瞭解TYLCTHV病毒蛋白之間的交互作用，我利用雙分子螢光互補 (bimolecular fluorescence complementation, BiFC) 技術檢測TYLCTHV病毒蛋白的交互作用與作用的細胞內定位。結果發現在健康菸草細胞中，GFP-CP融合蛋白與GFP-NSP融合蛋白都表現在植物細胞核，且在核仁中有較多的累積。GFP-Rep融合蛋白表現於細胞核中，但沒有進入到核仁。其他五個GFP-病毒蛋白融合蛋白GFP-AV2、GFP-TrAP、GFP-REn、GFP-AC4及GFP-MP則遍布細胞質與細胞核。此外，結果也發現TYLCTHV的病毒蛋白在感染TYLCTHV的菸草病株中的蛋白質定位與在健康菸草細胞中相同。以BiFC技術偵測病毒蛋白間的交互作用的結果顯示沒有任何TYLCTHV蛋白質交互作用。病毒蛋白於菸草病株細胞中的蛋白質定位與在健康菸草細胞中相同的結果與BiFC偵測到沒有蛋白質交互作用的結果相互呼應。本研究結果所得到的TYLCTHV病毒蛋白的細胞內定位，提供了病毒蛋白的功能之相關資訊。未來仍需要更多有關TYLCTHV蛋白功能和病毒蛋白與寄主蛋白相互關係等方面的研究，以增進我們對植物病毒之複製、轉移與感染等機制的瞭解。

Tomato yellow leaf curl Thailand virus (TYLCTHV) is a member of the genus Begomovirus in the family Geminiviridae, and it is associated with tomato yellow leaf curl disease (TYLCD) in China, Myanmar, Thailand, and Taiwan. TYLCD is one of the major constraint on tomato production worldwide and often leads to 20-100% reduction in the yield of tomato particularly in tropical and subtropical areas. The genome of TYLCTHV is composed of bipartite DNA that are designated DNA-A and DNA-B. DNA-A carries six genes that encode the CP, the AV2 protein, the Rep protein, the TrAP, the REn protein, and the AC4 protein. DNA-B encodes two viral proteins, the NSP and the MP. To better understand the functions of the TYLCTHV proteins, green fluorescence protein (GFP) fused with each viral protein was expressed in planta by agroinfiltration. Further, the protein-protein interaction was examined by bimolecular fluorescence complementation (BiFC) assay which provides simultaneous protein interaction and localization data in living cells. The results showed that the GFP-CP fusion and the GFP-NSP fusion both localized exclusively to the nucleus with an accumulation in the nucleolus. The GFP-Rep fusion distributed throughout the nucleus excluding the nucleolus. The GFP-AV2, GFP-TrAP, the GFP-AV2, GFP-TrAP, GFP-REn, GFP-AC4, and GFP-MP fusions localized to both the cytoplasm and the nucleus. My results demonstrated that the subcellular localizations of the TYLCTHV proteins in the virus-infected plant leaves were identical to those in the healthy plant leaves. Besides, none of the protein-protein interactions of TYLCTHV were detected by BiFC assay. The results of the unchanged subcellular localizations of the TYLCTHV proteins in the healthy and in the TYLCTHV-infected N. benthamiana leaves are in agreement with the results of no protein-protein interactions examined by BiFC assay. Viral protein localizations in host cells provides valuable information about the biological roles of viral proteins. More studies are needed to further understand the relationship between the TYLCTHV proteins and their interactions with host proteins. The information will help us to further understand the molecular mechanisms that underlie the processes of virus replication, movement, and infection.

誌謝 i
中文摘要 ii
ABSTRACT iv
CONTENTS vi
LIST OF TABLES viii
LIST OF FIGURES ix
1. INTRODUCTION 1
2. MATERIALS AND METHODS 6
2.1 Virus and plant 6
2.2 Sequence analysis 6
2.3 Cloning of viral genes for protein expression 7
2.4 Protein expression in plant cells 8
2.5 Bimolecular fluorescence complementation (BiFC) 9
2.6 Confocal microscopy 10
3. RESULTS 11
3.1 Sequence analysis 11
3.2 Subcellular localizations of the TYLCTHV proteins 12
3.3 TYLCTHV protein interactions in plant cells 14
4. DISCUSSION 15
5. REFERENCES 21

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