Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36144
標題: 利用蛋白質體學研究菸草感染竹嵌紋病毒後具有差異性表現之蛋白
Proteomic Analysis of Differential Protein Expression of Bamboo mosaic virus Infected Nicotiana benthamiana
作者: 吳麗琴
Wu, Li-Chin
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;host factors;proteomic;寄主因子;蛋白質體學
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),為彎曲絲狀外型,單股正極性的RNA病毒。當病毒感染其寄主後,除了病毒本身表現的蛋白質外,也會利用寄主蛋白質來參與病毒的複製,同時寄主也會產生抵抗病毒複製及傳播的蛋白質。本實驗主要是利用蛋白質體學,分析當菸草被竹嵌紋病毒感染後,有哪些寄主蛋白質的表現量會與未接種菸草產生差異性。由於病毒進入寄主細胞後會嵌入特定胞器膜中進行複製,因此我們先分離出菸草細胞之膜層,接著利用二維電泳將健康及被BaMV感染的菸草膜層蛋白分開,分別比較兩者之間蛋白質分佈的差異,發現存在許多表現量不同的蛋白質,再利用基質輔助雷射脫附離子化-飛行時間質譜儀(MALDI-TOF MS)來鑑定這些蛋白質的身分。找到之差異表現蛋白分別有ribosomal protein L25、RubisCO small chain、calmodulin-1及glycoprotein endopeptidase-like protein。其中ribosomal protein L25只在被BaMV感染的膜層蛋白出現,L25和逆境下會產生的CTC蛋白序列具有同源性。為了暸解L25是否會影響BaMV在植物體內的累積,我們利用菸草脆裂病毒(Tobacco rattle virus, TRV)為載體進行此基因的靜默,經由即時定量聚合酶連鎖反應偵測結果顯示,利用此靜默系統確實可以將L25 mRNA含量降到對照組之25至40 %左右。將此L25基因靜默植物接種病毒,在接種七天後,以西方漬染免疫偵測及北方雜配分析偵測病毒鞘蛋白及RNA的累積,由結果發現L25基因靜默確實會降低竹嵌紋病毒在植物中的累積,但若以狐尾草嵌紋病毒(Foxtail mosaic virus, FoMV)接種此L25基因靜默植物,發現對於狐尾草嵌紋病毒在植物中的累積並無顯著影響。綜合上述結果顯示L25蛋白會影響BaMV在植物中的累積能力,但對FoMV則較並不重要。

關鍵字:竹嵌紋病毒/寄主因子/蛋白質體學

Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. A number of studies have been devoted to analyze the replication of plus-stranded RNA viruses; several host proteins are known to be involved in assembling the viral RNA replication complex, activating the complex for RNA synthesis, and other steps. However, the host has the defense system to against viral replication and spreading which are also mediated through proteins. In this study, we have tried to identify the differentially expressed proteins between mock and BaMV inoculated N. benthamiana leaves by proteomics approach. Since positive-sense RNA virus replication is usually associated with rearrangements of membranous organelles, we first isolated the membrane fraction (P30) of the N. benthamiana leaves. Proteins associated with P30 fraction were resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE) and identified by MALDI-TOF MS. Comparison of protein patterns from P30 fractions of mock and BaMV-infected N. benthamiana in 2-D gels revealed several differential expressed proteins. The differential expression of ribosomal protein L25, RubisCO small chain, calmodulin-1 and glycoprotein endopeptidase-like protein. Among these proteins, ribosomal protein L25 was only found in P30 fractions sample from BaMV-infected plants. It had been reported that ribosomal protein L25 is homologous to general stress proteins CTC. We utilized TRV- based virus-induced gene silencing (VIGS) system to generate L25-knockdown plants and to investigate the effect of this protein on BaMV accumulation. Real-time PCR results showed that the levels of the L25 mRNA in the various L25 knock-down plant were reduced to about 25 and 40 % to those of the control plants. Western blots and northern blots were used to analyze the accumulation of viral coat protein and viral RNA at seven day post-inoculation of BaMV virions. Results showed that the accumulation of BaMV coat protein and viral RNA in L25-knockdown plant were reduced. But no interference on the accumulation of FoMV coat protein was observed in all L25-knockdown plants. Together, these data suggest that L25 likely plays an important role in the BaMV accumulation.

Keywords: Bamboo mosaic virus / host factors / proteomic
URI: http://hdl.handle.net/11455/36144
其他識別: U0005-1607200713585600
Appears in Collections:生物科技學研究所

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