Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90079
標題: The study of NbLTP1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus
菸草蛋白NbLTP1參與 竹嵌紋病毒複製機制之研究
作者: 邱齡瑩
Ling-Ying Chiu
關鍵字: 竹嵌紋病毒;差異性表現基因;脂質傳送蛋白;攜鈣素結合蛋白;bamboo mosaic virus;differentially expressed host gene;lipid transfer protein;calmodulin binding protein
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摘要: 
竹嵌紋病毒(Bamboo mosaic virus, BaMV)為一隸屬於Flexiviridae科Potexvirus屬的正股RNA病毒,是感染竹子的主要病毒。本研究的主要目的是找到與竹嵌紋病毒感染過程中有關的宿主蛋白質,藉此了解病毒的繁殖機制。根據前人研究之cDNA-amplified fragment length polymorphism (cDNA-AFLP)結果,我們在菸草中找到一個宿主基因ACGT12,會在感染竹嵌紋病毒後表現量降低。利用菸草脆裂病毒(Tobacco rattle virus, TRV)誘導基因靜默(virus-induced gene silencing; VIGS)以降低基因的表現,進而以西方墨點法(western blot)偵測這些基因對於BaMV在宿主內繁殖的影響,結果發現靜默宿主基因ACGT12後,病毒鞘蛋白(coat protein)的累積量下降。再以西方墨點法偵測基因靜默後的原生質體(protoplast),發現病毒的累積量也同樣下降。因此推斷宿主基因ACGT12可能與病毒的複製而非移動有關,且ACGT12的基因表現下降時,病毒的累積量也會隨之減少。為進一步了解此基因在宿主內可能的用途,利用rapid amplification of cDNA ends (RACE)技術,延長宿主基因的上下游片段,得到全長的cDNA,將其序列與Biology WorkBench資料庫之BLASTP比對後,推測此基因應為non-specific lipid transfer protein 1 (nsLTP1),因此我們將此基因命名為NbLTP1。利用融合橘螢光蛋白(Orange fluorescent protein)的表現,觀察其於細胞的位置主要分佈於胞外(extracellular matrix),然而如將分泌的signal peptide去除,則發現大部分的蛋白會與葉綠體的自體螢光重疊。此外,在大量表現NbLTP1後感染病毒,也發現病毒鞘蛋白表現量會上升。總結以上的結果,我們可以推測NbLTP1在病毒的感染週期中扮演幫助病毒核酸的複製。與其他LTP比對後也發現,NbLTP1具有可與calmodulin (CaM)結合的保守區域,此一特性也可能為影響病毒複製的原因。

Bamboo mosaic virus (BaMV) is a single-stranded positive sense RNA virus belonging to genus Potexvirus of the family Flexiviridae. The objective of this study is to understand the relationship of the host proteins involved in the replication mechanism of BaMV. The results derived from a previous study, a downregulated gene fragment ACGT12 from Nicotiana benthamiana post BaMV infection identified by cDNA-AFLP technique is further characterized. We used the Tobacco rattle virus (TRV)-based silencing system (virus-induced gene silencing; VIGS) to knock down the expression of ACGT12 and infected the BaMV on the knockdown plants. The results indicate that the accumulation levels of BaMV coat protein in the knockdown plants are lower than that in the control plants. Similar results are observed in the knockdown protoplasts that less coat protein accumulated than that in the control protoplasts. These results suggest that ACGT12 may be involved in the replication process rather than in viral movement. The full-length cDNA of ACGT12 is obtained by rapid amplification of cDNA ends (RACE) technique. The sequence of ACGT12 is blasted to the Biology WorkBench database and matches to that of non-specific lipid transfer protein 1 (nsLTP1) and designated as NbLTP1. Furthermore, the cellular localization of NbLTP1-OFP (fused with Orange fluorescence protein) is mainly associated with the extracellular matrix. However, when the signal peptide is removed, the majority of the expressed mutant protein is associated with chloroplasts. The accumulation of BaMV coat protein is enhanced when NbLTP1 is transiently expressed in plants. Overall, the results indicate that the newly identified host protein NbLTP1 is a positive regulator for the replication of BaMV RNA. In addition to the hydrophobic pocket to accommodate the lipid, NbLTP1 has a conserved calmodulin
(CaM)-binding site at the very C-terminus. Therefore, the lipid binding and the CaM-binding properties involving in the replication of BaMV is needed to be further characterized.
URI: http://hdl.handle.net/11455/90079
Rights: 同意授權瀏覽/列印電子全文服務,2014-08-31起公開。
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