Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36252
標題: 分子選殖菸草熱休克蛋白 90 (Hsp90) 基因並分析 Hsp90 於竹嵌紋病毒感染週期中之表現量
Molecular cloning of Hsp90 gene from Nicotiana benthamiana and analysis of Hsp90 expression level in infection cycle of Bamboo mosaic virus
作者: 賴彥君
Lai, Yan-Jun
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;Heat shock protein 90;transgenic N. benthamiana;熱休克蛋白 90;轉基因菸草
出版社: 生物科技學研究所
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摘要: 
竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為單股正極性 RNA 病毒,屬於Flexiviridae 科Potexvirus屬的成員。病毒為絕對寄生的微生物,由於病毒基因體大小的限制,導致病毒本身所轉譯的蛋白是有限的,因此在病毒複製時,需要許多寄主因子的幫助,才能完成整個生活史。先前研究發現菸草熱休克蛋白 90 (Hsp90) 會專一性地與 BaMV 病毒基因組之 3’ UTR 直接結合,顯示 Hsp90 可能參與 BaMV 的複製。 Hsp90 是一種伴隨蛋白 (chaperone),其生物功能是幫助特定類群的蛋白摺疊、組合、或穩定其結構,這是和其他的伴隨蛋白的生理功能有非常大的不同。此特定類群蛋白種類包括有訊息傳遞相關的蛋白、蛋白激酶、轉錄因子等。且 Hsp90 也參與許多細胞內重要的代謝過程。先前已有許多文獻指出 Hsp90 參與 DNA 或 RNA 病毒的生活史,如病毒的侵入、複製、轉譯、穩定病毒複製酵素、病毒蛋白的定位等。目前 Hsp90 參與病毒生活史所扮演的功能皆是藉由其與病毒蛋白質之間有交互作用,目前尚無報導指出 Hsp90 與病毒核酸之間具有直接的交互作用。Hsp90 基因家族內存在許多基因異構體 (isoform),以阿拉伯芥 (Arabidopsis thaliana) 為例,共有七個基因異構體。由於目前 NCBI 資料庫中,菸草 (Nicotiana benthamiana) Hsp90 基因家族尚未有完整序列資料,為探究 BaMV 在菸草中感染與 Hsp90 之間的關係,本實驗將分子選殖 Hsp90 之基因。實驗結果發現四個屬於細胞質次家族成員 (cytosolic subfamily) 的 Hsp90 基因異構體,目前已得到一個 Hsp90 cDNA 之全長序列。為了探究細胞受到 BaMV 感染時,是否會造成 Hsp90 的表現異常,利用北方雜配法 (Northern hybridization) 與即時定量聚合酶連鎖反應 (Real-time quantitative PCR) 分析 Hsp90 mRNA 表現量,結果顯示 Hsp90 在竹嵌紋病毒的感染週期中其表現量無明顯增加。另一方面,本實驗構築 Hsp90 轉基因菸草,探討大量表現 Hsp90 的轉基因菸草對 BaMV 複製情形之影響。分別構築 Hsp90 及 satHsp90 轉基因菸草,其中satHsp90 轉基因菸草可以利用竹嵌紋病毒感染後,大量增加 Hsp90 表現量。本實驗也利用 satHsp90 F1 轉基因菸草進行竹嵌紋病毒接種測試,再以北方雜配法分析,結果發現本實驗測試之 F1 轉基因菸草中 Hsp90 表達沒有明顯增加,而此轉基因菸草對 BaMV 複製情形也沒有明顯影響,利用西方墨點法偵測竹嵌紋病毒外鞘蛋白,呈現結果與北方雜配法一致。

Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus, belongs to genus Potexvirus of the family Flexiviridae. Viruses are all obligate parasites. Because of the restriction of the size of viral genome, efficient viral replication is dependent on many host factors. Recently, study has shown that Nicotiana benthamiana 90 kDa heat shock protein (Hsp90) specifically binds to 3' untranslated region (3' UTR) of BaMV genomic RNA, but not to its satellite RNA of BaMV (satBaMV). It indicates that Hsp90 is involved in life cycle of BaMV in N. benthamiana. At present, Hsp90 is only known involved in viral life cycle via interactions with viral proteins, no evidence has been reported about Hsp90 interacting with viral RNA or DNA. The Hsp90 is a member of molecular chaperones found in prokaryotes and all eukaryotes. In cells, Hsp90 is required for the folding, assembly, and stability of a specific subset of proteins, including cellular signaling proteins, protein kinases, and transcription factors. Many evidences suggest that Hsp90 participates in the life cycle of DNA and RNA viruses, such as viral entry, replication, translation, viral protein stabilization, localization, etc. This study intends to investigate the role played by Hsp90 in the life cycle of BaMV. In Arabidopsis thaliana, there are 7 Hsp90 gene isoforms. Currently, the full-length Hsp90 genes in N. benthamiana have not yet been fully understood. We have cloned the Hsp90 genes from N. benthamiana and four cytosolic isoforms in N. benthamiana were found. Using northern hybridization and real-time quantitative PCR, we analyzed accumulation of the Hsp90 genes in infection cycle of BaMV. Our results showed that Hsp90 accumulation in BaMV infected tissues was at similar level as non-infected tissues. Additionally, we have established two types of Hsp90 transgenic N. benthamiana, Hsp90 and satHsp90, respectively, in which the Hsp90 gene is expressed by CaMV 35S promoter. The satHsp90 F1 transgenic plants were used to investigate the effect of Hsp90 over-expression on BaMV life cycle. However, no significant effects were found by northern hybridization and western blot analysis.
URI: http://hdl.handle.net/11455/36252
其他識別: U0005-2007201012310700
Appears in Collections:生物科技學研究所

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