菸草蛋白NbLTP1參與 竹嵌紋病毒複製機制之研究

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.中文摘要 i Abstract ii Introduction 1 Materials and Methods 5 ACGT12 knockdown and virus infection 5 Viral particle purification 5 Viral RNA extraction 6 Protoplast prepared and viral RNA inoculation 6 Protein extraction 7 Western blotting assay 8 Total RNA extraction 9 Semi-quantitative RT-PCR 9 Transient expression of Orange fluorescent protein (OFP)-fusion protein and virus inoculation 10 Confocal microscopy 10 Results 12 The replication of BaMV is enhanced by ACGT12 12 ACGT12 is a homolog of the nonspecific lipid transfer protein 1 (nsLTP1) in Nicotiana tabacum 13 The fusion protein NbLTP1-OFP is a secretary protein 13 Transiently expressed NbLTP1-OFP enhanced the accumulation of BaMV 14 Discussion 15 Tables and Figures 18 Fig 1. The morphology and silencing efficiency of the Luc- and ACGT12-knockdown plants 18 Fig 2. BaMV coat protein accumulation in ACGT12-knockdown leaves 19 Fig 3. The accumulations of BaMV coat protein and viral RNA in ACGT12-knockdown protoplasts 20 Fig 4. The illustration of the cDNA sequence of NbLTP1 and the alignment with NtLTP1 21 Fig 5. The alignment of nsLTPs from different species 22 Fig 6. Localization of NbLTP1-OFP and NbLTP1/ΔSP-OFP in N. benthamiana by confocal microscopy 23 Fig 7. The accumulation level of BaMV coat protein in NbLTP1-OFP transiently expressed N. benthamiana 25 References 2