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研究生:陳禮弘
研究生(外文):Li-Hung Chen
論文名稱:葉綠體磷酸甘油酸激酶對於竹嵌紋病毒感染可能影響機制之研究
論文名稱(外文):The Possible Functions of Chloroplast Phosphoglycerate Kinase in the Life Cycle of Bamboo Mosaic Virus Infection
指導教授:蔡慶修蔡慶修引用關係
指導教授(外文):Ching-Hsiu Tsai
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:30
中文關鍵詞:葉綠體磷酸甘油酸激酶竹嵌紋病毒
外文關鍵詞:Chloroplast Phosphoglycerate KinaseBamboo Mosaic Virus
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竹嵌紋病毒(Bamboo mosaic virus)是一單股正向的RNA病毒,在病毒的生活史中需要寄主裡的各種因子來幫助病毒生存。根據先前的研究得知葉綠體磷酸甘油酸激酶(PGK)可以影響BaMV的複製,而PGK是一個由細胞核轉錄的葉綠體蛋白,具有導引訊息肽鍵(transit peptide),在細胞質轉譯後後會移動到葉綠體裡。我們認為它有可能參與了病毒RNA的移動。為了要證明這個假設,設計了不同的PGK突變蛋白並且接上T7 tag,包括了去除導引訊息肽鍵突變蛋白(NOTP)和去除導引訊息肽鍵改接上核定位訊號突變蛋白(Nucleus location signal; NLS)。利用短暫表現技術(transient expression)於菸草中表現正常的PGK與另外兩個PGK突變蛋白。由於訊息肽鍵的改變,我們利用共軛焦顯微鏡來確定蛋白位置,PGK位於葉綠體中,NLS-PGK位於細胞核內,而NOTP-PGK則位於細胞質。接著利用短暫表現技術了解病毒與蛋白間的交互關係,根據我們初步研究結果,於菸草葉片或原生質體中大量表現不同位置的PGK,並不會影響到病毒鞘蛋白的累積量,PGK於病毒生活史中真正扮演的腳色有待進一步分析研究。
Bamboo mosaic virus (BaMV) is a positive-sense single-stranded RNA virus. In previous study shows that the Nicotiana benthamiana chloroplast phosphoglycerate kinase (PGK) is one of the host factors in the life cycle of BaMV infection. Due to its characteristics of being transported to chloroplast, we hypothesize that chloroplast PGK might be involved in virus localization to chloroplast for replication. We make two chloroplast PGK mutants, one is transit peptide deletion mutant (NOTP) and the other is nucleus location signal fused PGK mutant without transit peptide (NLS). We transiently express two mutant and wild type PGKs in N. benthamiana. We expected that wild type PGK should be localized in chloroplast, NLS-PGK mutant should be localized in nucleus, and NOTP-PGK mutant should stay in cytoplasm. We use confocal microscopy to inspect their localization. By over-expression study, our preliminary data show that over-expression of different subcellular localization of PGK would not be able to change the amount of BaMV coat protein accumulation in protoplast or plant. The actual function of chloroplast PGK for BaMV needs further analysis.
中文摘要........................................ i
Abstract…………………………………………………… ii


Introduction

Bamboo mosaic virus……………………………………………… 1
Replication of BaMV……………………………………………… 2
Involvement of host factors in viral replication……… 2
Chloroplast phosphoglycerate kinase involved in BaMV replication....... 3

Materials and Methods

Construct for transient expression of wild type and mutant PGK.................................... 5
PGK-fused GFP construct…………………… 5
Argroinfiltration based transient expression…………… 6
Immunoprecipitation and Western blot analyses………… 6
Protoplast isolation and inoculation……………………… 7
Fluorescence Microscopy……………………………………… 8

Result

Transient expression of the wild type and mutant PGK in N. bethamiana plants……… 9
The subcellular localization of PGK, NOTP-PGK, and NLS-PGK…………………… 10
Overexpression of PGK, NOTP-PGK, and NLS-PGK in N. benthamiana plants or
protoplasts does not affect BaMV coat protein accumulation……… 10

Discussion………………………………………………… 12

Table and Figures…………………………………………… 15

References……………………………………………………… 26
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