臺灣博碩士論文加值系統

竹嵌紋病毒(Bamboo mosaic virus)是一正極單股核糖核酸的病毒，被分類於alpha-flexiviridae科Potexvirus屬。我們利用cDNA-AFLP技術篩選出在本氏菸草(Nicotiana benthamiana)中在經過竹嵌紋病毒感染後，針對具有不同表現量的宿主基因進行研究。其中有一感染後表現量下降的基因片段ACTG7-1，與阿拉伯芥(Arabidopsis)中的REVERSION-TO-ETHYLENE SENSITIVITY (RTE1)具有59%的相同。我們利用基因靜默技術(virus-induced gene silencing, VIGS)探討NbRTE1在竹嵌紋病毒感染過程中扮演的功能。前人以NbRTE1-靜默的原生質體進行竹嵌紋病毒的接種實驗指出NbRTE1在竹嵌紋病毒複製過程中可能扮演抑制病毒複製的角色。因此為釐清NbRTE1是如何參與病毒的複製，我將針對ethylene的訊息傳遞路徑進行探討，首先根據前人的研究指出阿拉伯芥中的RTE1是位於內質網膜(ER membrane)上，因此我們利用共軛焦顯微鏡及過表現目標蛋白-螢光蛋白的技術去證實菸草的NbRTE1確實是位於內質網膜上。由於RTE1在阿拉伯芥中是參與了乙烯的訊息途徑，會專一的與膜上的乙烯接受器ETR1結合，因此我利用了VIGS的技術將NbETR1的表現量下降至18%時竹嵌紋病毒之鞘蛋白降低至75%。這與我們所期待中的結果不同，如果乙烯的訊息途徑參與了竹嵌紋病毒的複製，降低NbETR1的結果應該會與NbRTE1的結果相似。更進一步的，我利用乙烯抑制劑去減少內生性的乙烯訊息分子，發現施加抑制劑後，竹嵌紋病毒之鞘蛋白與控制組並無太大差異。因此我們推測NbRTE1並非是經由乙烯途徑去影響竹嵌紋病毒之複製。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus which belongs to the Potexvirus of alphaflexiviridae. We used cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes of Nicotiana benthamiana post BaMV inoculation. One of downregulated genes, ACTG7-1, sharing 59% identify to REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) of Arabidopsis was further investigated. We used virus-induced gene silencing (VIGS) technique to knock down the expression of RTE1 and investigate the involvement of NbRTE1 in BaMV infection cycle. The results derived from previous studies revealed that NbRTE1 could negatively regulate the replication of BaMV. My research goals are aiming at the localization of NbRTE1 in cell by using confocal microscopy and clarifying whether the ethylene response pathway is involved in BaMV infection. The RTE1 in Arabidopsis was reported to associate with endoplasmic reticulum (ER), accordingly the localization of NbRTE1 on the ER needs to be confirmed. Because RTE1 is one of the components in the ethylene response pathway and is involved in BaMV replication in N. benthamiana, we would like to examine more components in the pathway and inspect if they are also involved in BaMV infection. NbETR1, the ethylene receptor, specifically interacts with RTE1 to transduce the signaling. The results derived from VIGS indicated that the expression level of ETR1 was decreased to 18% that of the control plants, the accumulation of BaMV coat protein was reduced to 75%. To further inspect if ethylene is involve in the BaMV infection cycle, we have treated the protoplasts with the ethylene inhibitor to block the signaling pathway. The results revealed no significant difference of the BaMV accumulation. Overall, the results suggest that NbRTE1 involved in BaMV replication might not go through the ethylene signaling pathway. The mechanism of NbRTE1 involved in BaMV replication is still need to be clarified

Content
中文摘要 i
Abstract ii
Introduction 1
Bamboo mosaic virus 1
REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) in ethylene pathway 2
NbRTE1 negatively regulated the replication of BaMV 3
Aim 4
Materials and Methods 5
Virus-induced gene silencing 5
BaMV isolation 5
Protoplasts preparation 6
Transient expression of NbRTE1-OFP fusion protein 6
Western blotting assay 7
Total RNA extraction and real-time PCR 8
Ethylene treatment 8
Results 9
The replication of BaMV is promoted in NbRTE1-knockdown plants. 9
The fusion protein OFP-NbRTE1 is localized at endoplasmic reticulum of N. benthamiana 9
The ethylene pathway is not involved in the accumulation of BaMV. 9
Transient expression of NbRTE1 decreases the accumulation of BaMV. 11
Discussion 12
Figure 14
Appendix Figure. 1. Model for the ethylene signaling pathway (Ju and Chang, 2015).. 14
Fig. 1. The expression levels of NbRTE1 in Luc- and NbRTE1-knockdown plants. 15
Fig. 2. Localization of OFP-NbRTE1 in N. benthamiana protoplasts examined by confocal microscopy.. 16
Fig. 3. The full-length cDNA sequence of NbETR1 retrieved from the data base of Nicotiana benthamiana provided by The University of Sydney. 18
Fig. 4. The amino acid sequence alignment of NbETR1 and NtETR1.. 20
Fig. 5. The expression levels of NbETR1 in control and knockdown N. benthatmiana plants.. 21
Fig. 6. The accumulation levels of BaMV coat protein in control and NbETR1-knockdown plants.. 22
Fig. 7. The effect of silver thiosulfate (STS) on the accumulation of BaMV.. 23
Fig. 8. The accumulation levels of BaMV coat protein in NbRTE1-OFP or OFP-NbRTE1 transiently over-expressed plants. 24
Reference 25

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