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dc.contributor.authorYan-Cheng Linen_US
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Plant Physiology 154: 567-570 Sun TP (2011) The Molecular Mechanism and Evolution of the GA-GID1-DELLA Signaling Module in Plants. Current Biology 21: R338-R345 Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu GH, Nomura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCFCO11 complex during jasmonate signalling. Nature 448: 661-U662 Tsai CW (2014) The study of the differentially expressed gene ACCT7-1 of Nicotiana benthamiana is involved in Bamboo mosaic virus infection cycle. Tyler L, Thomas SG, Hu JH, Dill A, Alonso JM, Ecker JR, Sun TP (2004) DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiology 135: 1008-1019 Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh H, Katoh E, Kobayashi M, Chow TY, Hsing YIC, Kitano H, Yamaguchi I, Matsuoka M (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. 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dc.description.abstract竹嵌紋病毒(Bamboo mosaic virus, BaMV)是單一正股RNA病毒,隸屬於Flexiviridae科Potexvirus屬。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。在先前的研究中,本實驗室利用cDNA-amplified fragment length polymorphism (cDNA-AFLP) 分離出90個在BaMV感染後具有差異性表現的菸草(Nicotiana benthamiana) 基因片段。其中一條具有差異性表現的基因片段ACCT7-1,在病毒感染後表現量有增加的趨勢,並且利用病毒引發基因靜默技術(virus-induced gene silencing, VIGS)使ACCT7-1表現量下降,發現BaMV的外鞘蛋白在菸草的葉子上累積量有明顯的下降。接著進一步探討ACCT7-1是影響在病毒複製還是移動的層面,將處理過VIGS的原生質體接種病毒RNA,結果顯示BaMV的外鞘蛋白的累積量也有顯著的下降,從這兩個結果可以推測ACCT7-1可能扮演幫助病毒複製的角色。為了做更進一步的研究,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應該是GA-INSENSITIVE (GAI),並將ACCT7-1命名為NbGAI。接著利用得到的全長大量表達於菸草後感染病毒,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ACCT7-1可能扮演幫助病毒的角色並且也驗證了病毒引發基因靜默技術的結果。NbGAI是DELLA家族的成員之一,同時也是植物生長賀爾蒙吉貝素(gibberellin, GA)的抑制因子,除此之外,DELLA也參與並調節許多植物賀爾蒙路徑,包括茉莉酸(Jasmonates, JA),已有很多研究指出JA跟植物的防禦有關。本篇研究將進一步探討是否NbGAI會透過JA而對BaMV而產生影響。zh_TW
dc.description.abstractBamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The RNA genome comprises 6366 nts with a 5 cap and a 3 poly (A) tail. In general, the expression profile of host genes could be altered when infected by viral pathogens. These differentially expressed genes might play positive or negative roles in regulating BaMV infection cycle. In a previous study, our lab isolated 90 differentially expressed genes from BaMV-inoculated N. benthamiana plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. One of the upregulated genes post BaMV infection ACCT7-1 was found to involve in BaMV replication since the accumulation of BaMV coat protein was reduced when the expression of ACCT7-1 was knocked down by virus-induced gene silencing (VIGS) in the inoculated leaves and protoplasts. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends technique and sequenced. 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, we then designated this gene as NbGAI. To localize NbGAI in cell, I subcloned NbGAI into pEpyon vector with which can produce the fusion protein of NbGAI with Orange fluorescent protein (OFP) when expressed in plant cells. Under the circumstance of the fusion protein expression, the accumulation of BaMV coat protein is increased compared to the control plants with the expression of OFP only. The results indicate that NbGAI plays a positive role in assisting BaMV accumulation. Since NbGAI is one of the DELLA proteins that regulate plant hormones including gibberellin (GA) and Jasmonate (JA), the involvement of regulating in BaMV replication could be through the JA pathway. In applying JA or GA in plants and followed BaMV inoculation revealed that JA has either no effect or negative on the accumulation of BaMV, whereas the GA plays a positive role on that of BaMV. These results are conflict on the effect of NbGAI in BaMV. Therefore, we conclude that NbGAI, although it is a DELLA protein, may not go through the signaling pathway.en_US
dc.description.tableofcontents誌謝辭 I 中文摘要 II Abstract III Content V Introduction 1 Bamboo mosaic virus 1 GAI-homologs and gibberellin signaling pathway 2 Gibberellin crosstalk with Jasmonate via DELLAs and JAZs interaction 3 Materials and Methods 5 Plant and viruses 5 Viral RNA extraction 5 Protoplast preparation and viral RNA inoculation 6 Total RNA extraction 7 Rapid Amplification of cDNA Ends (RACE) 7 Virus induced gene silencing (VIGS) 8 Transient expression of NbGAI-OFP fusion protein 9 Semi-quantitative RT-PCR 9 Northern blotting assay 10 Western blotting assay 11 Methyl Jasmonate treatment 12 Nuclear localization of NbGAI-OFP 12 Gibberellic acid treatment 12 Result 14 The full length of ACCT6-1 14 NbGAI plays a positive role in BaMV infection cycle 14 The accumulation of CMV and PVX coat protein are decreased in NbGAI-knockdown plants. 15 The effect of NbGAI in BaMV replication is not through the JA signaling pathway. 16 Discussion 17 A unique gene of ACCT6-1 17 The role of NbGAI in BaMV infection cycle 17 Figures 19 Appendix Figure 1. The accumulation of BaMV in ACCT7-1-knockdown plants and protoplasts analyzed by western blotting. 19 Appendix Figure 2. Current model of the GA-GID1-DELLA signaling module network (Sun 2011). 21 Figure 1. The sequence of ACCT6-1 derived from the RACE clones and EST. 22 Figure 2. The relative accumulation of BaMV coat protein in NbGAI-OFP-transiently expressed N. benthamiana. 23 Figure 3. The expression of NbGAI gene in NbGAI-knockdown plants and the relative accumulation level of CMV and PVX coat protein in the knockdown N. benthamiana. 24 Figure 4. The relative accumulation of BaMV coat protein in Jasmonic acid and Gibberellic acid treated plants. 26 References 27zh_TW
dc.subjectBamboo mosaic virusen_US
dc.subjectNicotiana benthamianaen_US
dc.titleThe study of NbGAI involved in the accumulation of Bamboo mosaic virus in Nicotiana benthamianaen_US
dc.typeThesis and Dissertationen_US
item.openairetypeThesis and Dissertation-
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