http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87 2026-04-03T23:50:07Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55957 標題: 齒舌蘭輪斑病毒鞘蛋白具交互作用之寄主蛋白之研究; Study of host factors interacting with the capsid protein of Odontoglossum ringspot virus 作者: Po-Yen Chen; 陳柏諺 摘要: 蘭花為我國重要之外銷花卉作物之一，其在栽培過程中易遭受齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)之侵染而造成經濟損失。ORSV為 Tobamovirus屬病毒，其在寄主中的系統性移動需要鞘蛋白(capsid protein)的協助，然而詳細的移動機制仍尚待釐清。本實驗室先前發現將ORSV鞘蛋白的第100個胺基酸glutamic acid (E)置換為glycine (G)，或點突變為alanine (A)，則ORSV鞘蛋白突變株CPE100G和CPE100A會喪失於圓葉菸草(Nicotiana benthamiana)中系統性感染的能力。欲尋找可能參與病毒移動，或啟動植物防禦反應的寄主分子，本實驗室之前進行了免疫共沉澱法(co- immunoprecipitation, co-IP)，找出與鞘蛋白有交互作用的寄主因子。接著經由後續質譜分析，找到了一個與鞘蛋白突變株CPE100A有較強交互作用的寄主蛋白，其為一putative proteinase inhibitor，因此將其命名為NbPI。本研究首先對此基因進行了5’ RACE，確認其mRNA的5’ UTR共有33個核苷酸。為了進一步確認NbPI與ORSV鞘蛋白之間的交互作用，本研究以大腸桿菌系統表現NbPI與ORSV鞘蛋白後，進行生體外蛋白結合實驗(in vitro pull down)，結果發現NbPI與ORSV-CPE100A具有交互作用。此外，以圓葉菸草短暫共同表現NbPI及ORSV鞘蛋白，結果發現在植物體內(in planta)，ORSV鞘蛋白(CPWT與CPE100A)與NbPI也具有交互作用。在核酸層次，為了解NbPI在ORSV侵染過程中是否受到影響，本研究以北方雜合法偵測NbPI的表現量，結果圓葉菸草在接種緩衝液和ORSV後，NbPI表現量皆顯著地上升。最後，為了探討NbPI在ORSV感染圓葉菸草的過程中的重要性，本研究在圓葉菸草建立了病毒誘導基因靜默(virus-induced gene silencing, VIGS)系統。當NbPI產生基因靜默之後，先接種ORSV，數日後以ELISA偵測ORSV；結果發現ORSV在NbPI基因靜默植物的上位葉的累積量相較於對照組顯著地增加。依據上述研究結果，圓葉菸草受到ORSV侵染時，NbPI基因被快速誘導表現，並且NbPI蛋白可能會與ORSV鞘蛋白結合。而VIGS實驗結果顯示NbPI可能參與在圓葉菸草的抗病反應中，或許具有減緩ORSV系統性移動的能力。; Orchid is one of the most important export crops in the floral industry in Taiwan. However, the quality and yield of orchid frequently reduce due to the threat of Odontoglossum ringspot virus (ORSV). ORSV belongs to the genus Tobamovirus and requires capsid protein (CP) for systemic movement, but not for cell-to-cell movement in Nicotiana benthamiana. Nevertheless, the detailed movement mechanism of ORSV is still needed to be further studied. In our previous studies, when glutamic acid (E) at the 100th amino acid of ORSV CP mutated to glycine (G) or alanine (A), the mutant virus lost its ability to systemically infect N. benthamiana. Co- immunoprecipitation (co-IP) assay was tried previously to identify host factors involved in the movement of ORSV or host defense. In co-IP assay, we identified a putative proteinase inhibitor, NbPI, which was highly accumulated in CPE100A co-IP products. In this strudy, we first conducted 5’ RACE assay, and the 5’ untranslated region (5’ UTR) of NbPI mRNA was proved to be 33 nucleotides. For further confirmation of the interaction of NbPI and ORSV capsid proteins, we utilized Escherichia coli to express proteins above. NbPI co-precipitated with ORCPE100A in the in vitro pull down assay. Moreover, we transiently expressed NbPI and ORSV capsid proteins in N. benthamiana. NbPI co-precipitated with ORSV capsid proteins (CPWT and CPE100A) in planta as well. In nucleotide level, Northern blot assay showed that the mRNA level of NbPI gene was rapidly elevated after buffer and ORSV inoculation in N. benthamiana. To study the role of NbPI during ORSV infection, we established a virus-induced gene silencing (VIGS) system in N. benthamiana. After inoculating ORSV to NbPI-silenced plants for several days, ELISA assays were performed to detect ORSV. The result showed that the accumulation levels of ORSV capsid protein in the systemic leaves of NbPI-silenced plants were higher than those of control one. Accordingly, these results suggested that NbPI gene expression level was rapidly elevated after ORSV infection, and then NbPI proteins might be associated with ORSV capsid proteins. As to the result of VIGS, NbPI might participate in N. benthamiana defense response and involve in retarding the systemic movement of ORSV. 2014-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55443 標題: 齒舌蘭輪斑病毒缺失性RNA與鞘蛋白次基因體啟動子之分析與應用; Characterization and Application of Odontoglossum ringspot virus Defective RNA and Capsid Protein Subgenomic Promoter 作者: YU-JUNE WANG; 王昱珺 摘要: 蘭花為台灣重要之經濟花卉，其中又以蝴蝶蘭種苗為出口的最大宗。目前已知紀錄之蘭花病毒至少有28種，其中菸草嵌紋病毒屬(Tobamovirus)中的齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)為對蘭花最具威脅的病毒之一。缺失性RNA (defective RNA, dRNA)是RNA病毒經由基因體缺失重組後所形成的次病毒分子，此類型RNA必需有輔助病毒(helper virus)的協助，才能在植物體內進行複製、移行或包被；有些dRNA的高增殖力甚至能干擾輔助病毒的感染，使植物病徵減弱，因為這些特性，使得dRNA常成為病毒學家研究的對象或工具。本研究嘗試以缺失性RNA的形式製備ORSV之病毒載體，並利用鞘蛋白次基因體啟動子(subgenomic promoter)來表現綠螢光蛋白(enhanced green fluorescent protein, EGFP)。利用實驗室先前所構築的ORSV感染性選殖株，建立人為的ORSV缺失性RNA，接種於菸草原生質體中並篩選出感染性較佳的選殖株。結果顯示在人為構築的5種缺失性RNA中，以保留5’端1444個核苷酸及3’端1173個核苷酸之突變株lc的RNA累積量較多。另外藉由構築一系列ORSV刪除突變株，分別感染菸草原生質體以分析ORSV鞘蛋白基因的次基因體啟動子區域。實驗結果顯示，以鞘蛋白基因轉錄起始點為基準，-78到+32的序列位置為可能的啟動子區域，而-38到+12之序列為核心區域。另外，此區域序列在二級結構預測中會形成stem-loop的結構，而此二級結構對鞘蛋白次基因體啟動子之功能是必要的。綜合以上結果，將複製能力良好、並保留鞘蛋白次基因體啟動子的dRNA選殖株作為載體，攜帶EGFP基因，並測試其在植物單細胞層次的表現情形，經實驗確定此載體能成功表現EGFP。另外也嘗試接種於菸草植株，以分析此病毒載體之基本特性，並評估其應用潛力。; Orchids are important floral plants in Taiwan, and the Phalaenopsis is the most cultivated and exporting one. There are more than 28 viruses that have been reported to infect orchids. One of the most important viruses affecting orchid industry is the Odontoglossum ringspot virus (ORSV), belonging to the genus Tobamovirus. Defective RNAs (dRNAs) are RNA molecules that arise through the deletion and rearrangement of internal sequences from the genome of RNA viruses. Therefore, dRNAs require helper virus for their replication, movement or encapsidation. Some dRNAs with higher replication capability can even attenuate the symptoms caused by helper virus. Because of these unique traits, dRNAs become useful research tools for virologists. In this study, we try to develop viral vector base on ORSV artificial dRNA clone, and use capsid protein (CP) subgenomic RNA promoter to express green fluorescent protein. At first, we used previously constructed ORSV cDNA clone to create artificial dRNAs. The replication capability of the dRNA clones were then assayed in Nicotiana benthamiana protoplasts. One of dRNA clone lc which contains the ORSV genomic sequence of 5’ 1444 nts and 3’ 1173 nts showed high RNA accumulation in protoplast assays. In addition, we mapped the ORSV CP subgenomic RNA promoter using a series of deletion mutations. According to the results, CP subgenomic RNA promoter is located between -78 to +12 and the core active promoter is between -38 to +12, relative to the +1 transcription start site. In addition, computer predicted folding of these region revealed a unique stem-loop structure. This secondary structure is required for the normal function of CP subgenomic RNA promoter. Finally, we tried to use the ORSV artificial dRNA clone containing the CP subgenomic RNA promoter as a viral vector and the expression of EGFP in protoplasts was confirmed. We will evaluate basic characteristic and application possibility of this viral vector. 2014-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56569 標題: 齒舌蘭輪斑病毒及蕙蘭嵌紋病毒協力感染之研究; Synergism in infectivity between Odontoglossum ringspot virus and Cymbidium mosaic virus 作者: Yu-Wen Huang; 黃裕雯 摘要: 當植物病毒複合感染於同一植株時，所導致的寄主病徵加重、其中一個或雙方病毒累積量上升之現象稱為協力作用(synergistic interaction)。蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV) 及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)為兩種感染蘭科植物之重要病毒，其複合感染之情形在人工栽培之蘭園十分常見，並導致植株病徵加重的情況。前人研究顯示，當CymMV及ORSV複合感染蘭花的原生質體時，病毒累積量會較單獨感染高，然而其協力作用的分子機制仍尚未了解。本研究以圓葉菸草(Nicotiana benthamiana)作為寄主，當CymMV及ORSV複合感染於菸草原生質體時，仍保有協力作用的現象，且CymMV在複合感染時的累積量增加幅度較ORSV大；推論此現象係由病毒之基因靜默抑制子(silencing suppressor)參與調控。CymMV的triple gene block 1 (TGB1) 蛋白或ORSV的126 kDa (p126)蛋白為具有基因靜默抑制子潛力之蛋白，在16c轉基因菸草上同時表現綠色螢光蛋白與這兩種病毒蛋白時，發現p126具有較強之基因靜默抑制活性。當接種CymMV於短暫表現具基因靜默能力的p126之菸草植株時，發現p126對CymMV有提升病毒累積量的效果；但基因靜默活性低的TGB1亦能提升ORSV病毒累積量。由此推論p126與TGB1可能藉由不同的機制來達到提升病毒累積量的效果。進一步探討p126可能具有抑制子功效的功能區塊(domain)，發現此些功能區塊均無法獨立的抑制基因靜默，也無法幫助CymMV協力作用的進行。因此我們認為ORSV的p126蛋白藉由抑制轉錄後基因靜默，使得CymMV在單細胞層次的協力作用中得到較大的利益。; Synergistic interaction refers to a facilitating effect that the symptom and replication of one or both virus partners increase on mixedly infected host plant. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are two economically important viruses infecting orchids. Mixed infection by these two viruses is a common phenomenon and leads to severe symptoms. Besides, when CymMV and ORSV were inoculated to orchid protoplasts simultaneously, the accumulation of virus increased as compared to the singly infected protoplasts. However, the molecular mechanism of the synergistic interaction is still unknown. In this study, CymMV and ORSV were proved to have synergistic interaction on Nicotiana benthamiana protoplasts which were mixedly inoculated. Additionally, the accumulation of CymMV increased much more than that of ORSV. We proposed that this interaction may be mediated by silencing suppressor of the viruses. Triple gene block 1 protein (TGB1) of CymMV and p126 protein of ORSV which are potential PTGS suppressors were assayed on 16c line GFP transgenic N. benthamiana plants. Our results indicated that p126 possessed stronger suppressor activity than TGB1. When CymMV was inoculated onto N. benthamiana plants transiently expressed with p126, the accumulation of CymMV increased. In contrast, TGB1, which does not possess the ability of silencing suppression, could also enhance the accumulation of ORSV. Accordingly, p126 and TGB1 could mediate the synergistic interaction between CymMV and ORSV but through different mechanisms. Furthermore, we dissected the potential silencing suppression function of each p126 domain. The results indicated that none of these domains could independently suppress PTGS and facilitate the synergistic interaction with CymMV. Hence, we concluded that the ability of silencing suppression was required for ORSV p126 to facilitate CymMV accumulation during mixed infection in N. benthamiana protoplasts. 2014-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96935 標題: 高葡萄糖所誘導脂肪細胞外泌體在腎臟病變之作用機制的探討; Mechanistic study of kidney pathogenesis attributed to adipocyte exosomes induced by high glucose 作者: 高家渝; Chia-Yu Kao 摘要: 糖尿病是現代人群當中，最常見的慢性疾病之一，長期的高血糖會導致各種併發症，包括像是心臟、腦血管、周邊血管疾病和腎臟病變。我們的研究重點是糖尿病高血糖導致的腎臟併發症，因為大約 40% 的第 2 型糖尿病患者，具有糖尿病的腎臟病變，這種疾病的發生和死亡率很高。和肥胖有關的脂肪細胞功能障礙，是第 2 型糖尿病重要的危險因子。除了在儲存能量方面的作用之外，脂肪細胞還發揮內分泌細胞的作用，分泌一系列激素和細胞激素，它們是代謝和平衡的重要調節劑。最近的研究顯示，功能失調的脂肪組織會分泌病理訊號，直接損害其他組織，可能也會導致腎臟疾病的發展。近年來，外泌體已經成為，細胞之間通訊的重要介質，這些細胞外囊泡可以將許多的生物活性分子（如RNA和蛋白質）運送到遠處的目標細胞，從而影響遠端細胞和組織的功能。本研究是在探討，高糖條件下所誘導的脂肪細胞，分泌的外泌體，是否帶有影響遠端腎臟細胞的異常分子訊號，進而導致糖尿病腎臟病變的發病機制。為了去驗證我們的假設，利用模擬第 2 型糖尿病的環境，培養脂肪細胞，分別將脂肪細胞培養在低糖環境（5.5 mM）模擬正常人血糖環境和高糖環境（33 mM），並且收集培養脂肪細胞的培養液。隨後，透過差速離心從這些脂肪細胞中分離外泌體，確保外泌體的純度和質量，並使用奈米粒子追蹤分析（NTA）、電子顯微鏡(TEM)和蛋白質印跡進行表徵，以確認其大小和蛋白質的量。外泌體PKH26染色結果證實脂肪細胞來源的外泌體可被CIHP-1和HK-2細胞內化。此外，高糖條件下的外泌體處理的CIHP-1、HK-2細胞活力顯著降低。以高糖條件下來源的外泌體處理CIHP-1和HK-2細胞的形態學結果顯示，細胞形態明顯轉變為長條狀，表明HK-2已成為上皮間質轉化（EMT）類型。CIHP-1細胞經過高糖來源的外泌體處理後，鬼筆環肽F-肌動蛋白染色結果顯示細胞骨架紊亂，足突消失，白蛋白內流實驗結果顯示狹縫隔膜的完整性受到一定破壞，損害了狹縫隔膜的濾過屏障功能。為了探究外泌體糖尿病併發症的分子機制，採用蛋白質體學對外泌體蛋白進行蛋白質體學分析。研究結果表明，外泌體蛋白與 ECM 積聚、纖維化途徑密切相關，可能導致糖尿病腎臟併發症。未來的實驗將對透過蛋白質體學分析確定的目標路徑進行，以確認這些假設的準確性。; Diabetes is among the most prevalent chronic diseases in modern populations. Prolonged hyperglycemia can lead to various complications, including Cardiovascular diseases, cerebrovascular diseases, peripheral vascular diseases and nephropathy. Our research focuses on diabetic kidney complications, as approximately 40% of patients with type 2 diabetes (T2D) develop diabetic nephropathy, a condition associated with high morbidity and mortality rates. Obesity-related adipocyte dysfunction is a significant risk factor for T2D. Beyond their role in energy storage, adipocytes function as endocrine cells, secreting a range of hormones and cytokines collectively referred to as adipokines, which are critical regulators of metabolic homeostasis. Recent studies have demonstrated that dysfunctional adipose tissue can secrete pathological signals that directly damage other tissues, contributing to the development of kidney disease. In recent years, exosomes have emerged as key mediators of intercellular communication. These extracellular vesicles can transport bioactive molecules, such as proteins, lipids, and RNAs, to distant target cells, thereby influencing the function of remote cells and tissues. This study aims to investigate whether exosomes secreted by adipocytes under high-glucose conditions carry abnormal molecular signals that affect distal renal cells, contributing to the pathogenesis of diabetic kidney complications. To test this hypothesis, we cultured adipocytes by simulating the environment of type 2 diabetes, isolating the exosomes they secreted during the adipogenesis process of cells under both low (5.5 mM) and high (30 mM) glucose concentrations. Following this, exosomes isolated from these adipocytes through differential centrifugation, ensuring the purity and quality of the exosomes and characterized using Nanoparticle Tracking Analysis (NTA), transmission electron microscopy and western blot, which confirmed their size and protein content. The result of exosomes PKH26 staining confirmed that adipocyte-derived exosomes could be taken up by CIHP-1 and HK-2 cell lines. In addition, CIHP-1, HK-2 cells treated with exosomes derived from high glucose conditions exhibited a significant reduction in viability. Morphology of CIHP-1 and HK-2 cells treated with exosomes derived from high glucose conditions, the results showed that the morphology obviously changed into a long strip shape, indicating that HK-2 has become an Epithelial–mesenchymal transition (EMT) type and CIHP-1 also showed an EMT type and gradually develops into renal fibrosis. After CIHP-1 cells were treated with high glucose-derived exosomes, phalloidin F-actin staining results showed cytoskeleton disorder , the loss of foot processes and the results of the albumin influx assay clearly indicate compromised slit diaphragm integrity, leading to impaired filtration barrier function in the podocyte monolayer. This disruption ultimately contributes to proteinuria and glomerulosclerosis. To find out the molecular mechanism of exosome-diabetic complication, exosome protein was performed by LC MS/MS for proteomics analysis. The result show that exosome proteins were high relative with excessive extracellular matrix (ECM) accumulation, fibrosis pathway which could lead to diabetic kidney complications. Future experiments will be conducted on the target pathways identified through proteomic analysis to confirm the accuracy of these hypotheses. 2025-01-01T00:00:00Z