臺灣博碩士論文加值系統

竹嵌紋病毒(Bamboo mosaic virus, BaMV)為單一正股RNA病毒，隸屬於Alphaflexiviridae科Potexvirus屬。在先前的研究中，我們利用cDNA-amplified fragment length polymorphism分離出菸草(Nicotiana benthamiana)的90個在BaMV感染後具有差異性表現的基因片段。宿主基因的表現可能會因為受到病毒的感染而產生改變，這些具有差異性表現的基因可能扮演著幫助或是抵抗病毒的角色。其中一條基因片段ACCT7-1的表現量在病毒感染後有增加的趨勢。為了進一步的研究，以rapid amplification of cDNA ends (RACE)技術延長ACCT7-1上下游的序列，並經過National Center for Biology Information (NCBI)資料庫比對，推測ACCT7-1是DELLA家族的成員之一，與菸草(N. sylvestris)的GA-INSENSITIVE (GAI)基因有高度相似性，因此將ACCT7-1命名為NbGAI。接著我們利用病毒引發基因靜默技術(virus-induced gene silencing, VIGS)使NbGAI表現量下降，發現BaMV的外鞘蛋白在菸草的葉子上的累積量有明顯的下降。接著將處理過VIGS的原生質體接種病毒RNA，進一步探討NbGAI影響是在病毒複製還是移動的層面，結果顯示BaMV的外鞘蛋白的累積量也有顯著的下降，從上述兩個結果可以推測NbGAI可能扮演幫助病毒複製的角色。隨後再利用以 RACE技術延長所得到的全長基因表現於菸草後感染病毒，並發現BaMV的外鞘蛋白的累積量有明顯的上升，這個結果不僅能更確定NbGAI可能扮演幫助病毒的角色並且也驗證了上述基因靜默技術的結果。由於DELLA家族成員之一的NbGAI，會利用核定位訊號將蛋白帶入細胞核後參與並調節許多植物賀爾蒙路徑。因此我透過共軛焦顯微鏡及過表現NbGAI融合螢光蛋白來確定NbGAI在細胞的表現位置，也利用網路軟體的篩選，成功的預測到NbGAI的核定位訊號，再一次確認NbGAI的表現位置位於細胞核中並進一步的探討NbGAI對BaMV所產生影響。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, a member of Potexvirus genus of Alphaflexiviridae family. In a previous study, 90 differentially expressed genes were identified from BaMV-inoculated N. benthamiana plants using the technique cDNA-amplified fragment length polymorphism (cDNA-AFLP). One of the differentially expressed genes ACCT7-1 was upregulated in BaMV-inoculated N. benthamiana plants. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends (RACE) and sequence. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, ACCT7-1 is then designated as NbGAI. NbGAI was characterized by BaMV inoculated on virus-induced gene silencing (VIGS) leaves and protoplasts. The results showed that the accumulation of BaMV coat protein was decreased significantly in both knockdown leaves and protoplasts compared to the controls. These results can be inferred that NbGAI may play a positive role in assisting the virus replication. Furthermore, the fusion protein NbGAI with Orange fluorescent protein (OFP), NbGAI-OFP is expressed in plants and BaMV is then inoculated. The accumulation of BaMV coat protein is increased significantly compared with the control plants with the expression of OFP only. The results indicate that NbGAI play a role in assisting BaMV accumulation. NbGAI is a member of DELLA family and contains a nuclear localization signal (NLS). In this study, I have used confocal microscopy to confirm the nuclear localization of NbGAI in cell. In addition, the NLS of NbGAI is predicted by screening online software: cNLS Mapper. These results indicated that NbGAI could play an assisting role in BaMV replication.

中文摘要 i
Abstract ii
Content iii
Introduction 1
Bamboo mosaic virus 1
NbGAI and BaMV replication 2
Gibberellic acid insensitive and gibberellin signaling pathway 3
Materials and Methods 4
Plant growth and virus purification 4
Viral RNA extraction 4
Virus-induced gene silencing (VIGS) 5
Protoplast preparation and viral RNA inoculation 5
Total RNA extraction 6
Reverse transcription and real-time PCR 6
Transient expression of NbGAI-OFP fusion protein and its cellular localization 7
Western blot analysis 8
Northern blot analysis 9
Results 10
The accumulation of BaMV was decreased in NbGAI-knockdown plants 10
NbGAI is involved in BaMV replication 10
NbGAI plays a positive role in BaMV infection cycle 11
NbGAI-OFP was localized in nucleus of N. benthamiana cells 11
Discussion 12
Figures 14
Figure 1. The expression of NbGAI and BaMV accumulation in NbGAI-knockdown Nicotiana benthamiana protoplasts. 14
Figure 2. The accumulation of BaMV coat protein in NbGAI-OFP transiently expressed N. benthamiana. 16
Figure 3. The localization of NbGAI-OFP in N. benthamiana protoplasts. 17
Appendix 18
Appendix Figure 1. The accumulation of BaMV in ACCT7-1-knockdown plants analyzed by western blotting. 18
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