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研究生:陳鈺旻
研究生(外文):Chen,Yu-Min
論文名稱:分析誘變之番茄捲葉臺灣病毒 C2 蛋白對宿主泛素化防禦系統的干擾
論文名稱(外文):Mutagenesis of tomato leaf curl Taiwan virus C2 protein revealed its role in pathogenesis by interference with host ubiquitin-proteasome pathways
指導教授:傅士峯
指導教授(外文):Fu, Shih-Feng
口試委員:傅士峯蕭乃文李勇毅
口試委員(外文):Fu, Shih-FengHsiao, Nai-WanLee, Yung-I
口試日期:2020
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:72
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外文關鍵詞:
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番茄捲葉台灣病毒(Tomato leaf curl Taiwan virus; ToLCTWV)為一種雙生病毒,此病毒造成了台灣茄科經濟作物嚴重的損失。其所編碼的 C2 蛋白於本實驗室先前研究結果已知會累積在植物的細胞核中並抑制植物的轉錄後基因靜默功能,是決定宿主病徵的致病因子。但對於 ToLCTWV-C2 於植物泛素-蛋白酶體系統(Ubiquitin-proteasome pathways; UPP)的作用以及 C2 胺基酸序列之功能皆尚無深入的研究。我們分別突變了 ToLCTWV-C2 蛋白的核定位訊號(Nuclear Localization Signal mutation; NLSm)和抑制植物 PTGS (Post-Transcriptional Gene Silencing mutation; PTGSm)功能的序列。發現此三種突變皆會使 C2 蛋白無法累積在細胞核中,而相較於野生型 C2 蛋白,PVX/ToLCTWV C2NLSm (PVX/ToLCTWV-C2NPm)會減少病毒誘導的病徵出現,因此推論 ToLCTWV C2 蛋白的核定位會增強病毒誘導的病徵。接著檢測穩定表現 ToLCTWV-C2 圓葉菸草(Nicotiana benthamiana)轉植株中的泛素和 20S proteasome 之表現量,與野生型 Nicotiana benthamiana 相比發現ToLCTWV C2 轉植株有較低的泛素鏈累積,推測是因為 ToLCTWV C2 蛋白會影響泛素系統上游的相關蛋白,造成泛素化途徑中的目標蛋白無法有效形成泛素鏈。除此之外,ToLCTWV C2 轉植株有較多的 20S proteasome表現量,可能是因為 ToLCTWV-C2 蛋白使植物無法形成泛素鏈,減少 26S蛋白酶體的水平,且突變後的 ToLCTWV C2 也會干擾植物的 UPP。從本研究結果得知 ToLCTWV C2 蛋白會擾亂植物宿主泛素-蛋白酶體系統,因而影響植物的防禦與生長系統,達成反防禦功能。希望研究成果有助於未來農業上防治雙生病毒危害之問題與番茄抗病育種,減少因番茄捲葉台灣病毒所造成的農業損失。
Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the Geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The Geminivirus-encoded C2 proteins are pathogenic factors between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV in ubiquitination system in host have not been investigated. We analyzed the molecular function(s) in ubiquitination system of the C2 protein by transient or stable expression in Nicotiana benthamiana as host plants. The C2 protein of ToLCTWV accumulates in the nucleus of plant hosts and inhibits PTGS. We mutated the NLS (nuclear localization signal) in C2 protein of ToLCTWV or the sequence that inhibits plant PTGS (post-transcriptional gene silencing) functions. After recombination with GFP, it was found that GFP-ToLCTWV C2NLSm, GFP-ToLCTWV C2PTGSm or GFP-ToLCTWV C2NPm (double mutations) can prevent C2 protein of ToLCTWV from accumulating in the nucleus. PVX/ToLCTWV C2NLSm or PVX/ToLCTWV C2NPm exhibited less virus-induced symptoms than wild-type C2. Therefore, nuclear localization of ToLCTWV C2 protein may enhance virus-induced symptoms. Subsequently, we examined the steady-state level of ubiquitin and proteasome 20S protein in transgenic ToLCTWV-C2 Nicotiana benthamiana plants as compared with wild-type. Western blot analysis showed that the content of ubiquitin-related complex in the ToLCTWV-C2 transgenic plants was less than that of wild-type plants. However, the proteasome 20S protein in the ToLCTWVC2 transgenic plants was more than that of wild-type plants. It is suggested that the ToLCTWV-C2 protein may inhibit the ubiquitination processes of the host plants. The protein level of ubiquitination in the mutations of C2 was more than that of wild-type plants. In conclusions, the ToLCTWV C2 protein can interfere with the defense mechanisms of the host plants by inhibition of the ubiquitination processes.
中文摘要 1
Abstract 2
致謝 3
目錄 5
圖目錄 7
表目錄 8
縮寫表 9
1. 前言 12
1.1雙生病毒種類與蛋白特性 12
1.2雙生病毒C2蛋白質之功能 12
1.3植物以泛素蛋白酶體系統作為防禦機制 13
1.4病毒反防禦宿主泛素蛋白酶體之分子機制 14
1.5雙生病毒C2胺基酸序列功能 15
1.6實驗目的 15
2.材料方法 17
2.1植株培養(plant cultivation) 17
2.2番茄捲葉台灣病毒(tomato leaf curl Taiwan virus, ToLCTWV) 17
2.3 DNA萃取(DNA extraction) 18
2.4 DNA純化(DNA purification) 19
2.5 DNA濃度測定(nucleic acid quantitative analysis) 20
2.6聚合酶連鎖反應(polymerase chain reaction, PCR) 20
2.7瓊脂膠體電泳(gel electrophoresis) 22
2.8蛋白質萃取(protein extraction) 22
2.9蛋白質電泳與西方墨點法(SDS-PAGE and Western blot) 23
2.10載體建構(TOPO-D/GATEWAY cloning) 25
2.11載體抽取(plasmid miniprep) 27
2.12電穿孔(electroporation) 28
2.13菌液保存(preparation A. tumefaciens stock) 29
2.14農桿菌回溶與感染(agro-infiltration) 30
2.15圓葉菸草基因轉殖(gene transformation of Nicotiana tabacum) 31
2.16突變載體建構 32
3.結果 34
3.1分析番茄捲葉台灣病毒之C2蛋白的圓葉菸草轉殖株表現型 34
3.2分析番茄捲葉台灣病毒C2蛋白對幼苗子葉氣孔開閡之影響 36
3.3分析番茄捲葉台灣病毒C2突變蛋白之胞器定位 37
3.4分析番茄捲葉台灣病毒C2突變蛋白之致病性 38
3.5分析番茄捲葉台灣病毒C2突變蛋白對於宿主轉錄後基因靜默之影響40
3.6分析番茄捲葉台灣病毒之C2蛋白對宿主泛素-蛋白酶體過程之干擾42
3.7分析番茄捲葉台灣病毒之C2突變蛋白對泛素蛋白酶體過程之干擾44
4.討論 45
4.1分析番茄捲葉台灣病毒之C2轉殖株產生側枝與延長葉片老化原因45
4.2分析番茄捲葉台灣病毒之C2蛋白對幼苗子葉氣孔之影響 46
4.3比較突變ToLCTWV C2與TYLCV-C C2於宿主中的細胞定位47
4.4比較突變ToLCTWV C2與TYLCV-C C2之致病性差異 47
4.5比較突變ToLCTWV C2與TYLCV-C C2對宿主轉錄後基因靜默差異50
4.6確認番茄捲葉台灣病毒之C2PTGSm之突變點位於鋅指結構 51
4.7分析番茄捲葉台灣病毒之C2蛋白對植物宿主泛素化系統之反防禦52
4.8分析番茄捲葉台灣病毒之C2突變蛋白對宿主泛素化系統之反防禦53
5.結論 55
6.參考文獻 56
7.實驗結果圖 59
8.附錄 70
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