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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭石通 | zh_TW |
| dc.contributor.advisor | Shih-Tong Jeng | en |
| dc.contributor.author | 金禹圻 | zh_TW |
| dc.contributor.author | Yu-Chi King | en |
| dc.date.accessioned | 2021-07-10T21:52:53Z | - |
| dc.date.available | 2024-08-15 | - |
| dc.date.copyright | 2019-08-19 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Ang, L.H., and Deng, X.W. (1994). Regulatory hierarchy of photomorphogenic loci: Allele-specific and light-dependent interaction between the HY5 and COP1 loci. Plant cell 6: 613-628.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77254 | - |
| dc.description.abstract | 3’,5’-cyclic guanyl monophosphate（cGMP）是一個重要的二級訊息傳遞者（secondary messenger），在動物和真菌中已經發現一系列位在cGMP下游的作用者 （effectors），而在植物中，cGMP也被發現參與許多逆境的訊息調控。利用Dröge-Laser實驗室所建構的過量表現bzip轉錄因子的阿拉伯芥種子庫，藉由處理guanylyl cyclase抑制劑1H- (1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one （ODQ），發現轉錄因子ELONGATED HYPOCOTYL 5（HY5）可能為cGMP下游調控的轉錄因子之一，在根的延長中扮演正調控者。利用已發表的文獻，以cGMP下游的轉錄因子微陣列晶片（microarray）資料和受HY5調控的轉錄因子ChIP-chip資料交叉比對，發現可能的下游轉錄因子為GIS3）、OZF1、OBP1、WRKY22以及RGL3等。藉由quantitative real-time PCR（qRT-PCR）檢驗這些基因與cGMP和HY5的相關性，發現GIS3可能是cGMP調控根部生長發育途徑的正調控因子。GIS3和HY5的表現都會受到ODQ的抑制及cGMP的誘導。前人研究指出HY5蛋白質的穩定性會受到磷酸化影響，且未磷酸化的HY5蛋白質活性也比較強。本研究發現GIS3的表現也會受到okadaic acid（OKA，protein phosphatase抑制劑）的抑制及staurosporine（STA，protein kinase抑制劑）的誘導，符合之前對HY5活性的研究結果。同時發現HY5和GIS3都會受到外加一氧化氮（nitric oxide，NO）的誘導，而在NO缺乏的情況下，HY5和GIS3的表現也都會受到抑制，因此NO可能參與在cGMP-HY5-root的調控路徑。同時外加cGMP可以回復Atnoa1（Arabidopsis nitric oxide associated 1，內生NO缺乏突變株）中因為NO缺乏所導致的HY5和GIS3表現抑制，此結果證明NO在cGMP調控的根生長發育的上游。而在處理hydrogen peroxide（H2O2）和diphenylene iodonium（DPI，NADPH oxidase抑制劑）之後，HY5的表現不會被影響，然而GIS3的表現會被抑制，而在HY5OE轉殖株中H2O2和DPI的處理也會抑制GIS3表現，ODQ則不會抑制GIS3表現，這個結果表示NADPH oxidase所生成的H2O2可能位於HY5的下游和GIS3上游, 進而參與NO-cGMP-HY5-GIS3所調控的根生長訊息傳遞路徑。 | zh_TW |
| dc.description.abstract | 3’,5’-cyclic guanyl monophosphate (cGMP) is an important secondary messenger in both animals and plants. In the presence of 1H- (1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, the root lengths of Arabidopsis become short, indicating the requirement of cGMP for root development. By screening Arabidopsis seeds overexpressing bZIP transcription factors from Dröge-Laser’s Lab in the presence of ODQ, the root lengths of ELONGATED HYPOCOTYL 5 (HY5) overexpression transgenic plants were longer than that of wild type. HY5 expressed was significantly induced after cGMP treatment and was inhibited after ODQ treatment, demonstrating that the expression of HY5 was stimulated by cGMP. Arabidopsis genes induced by cGMP and induced by HY5 were published separately. Conbimed these two microarray data, five genes GIS3, RGL3, WRKY22, OBP1, and OZF1 were chosen for investigating the downstream signal of HY5. GIS3 were down-regulated in hy5 plant and up-regulated in HY5OE plant. GIS3 was also induced after cGMP treatment and inhibited by ODQ. Furthermore, the expression of GIS3 was inhibited after okadaic acid (OKA), a protein phosphatase inhibitor, treatment and induced after staurosporine (STA), a protein kinase inhibitor, application. These results agree with the previous researches that unphosphorylated HY5 interacting strongly with COP1 results in the degradation of HY5, and it also interacts with target promoters. In addition, HY5 and GIS3 expression increased significantly after nitric oxide (NO) donors treatment such as sodium nitroprusside dehydrate (SNP) and S-Nitrosoglutathione (GSNO), while in Arabidopsis nitric oxide associated 1 (Atnoa1) mutant, whose NO production was deficient, the expression of HY5 and GIS3 decreased. Besides, in nox1, an excess endogenous NO mutant, the expression of HY5 and GIS3 are both increased. Hence, NO was also involved in the regulation of the cGMP-HY5-root growth pathway. Moreover, exogenous cGMP recovered the expression of HY5 and GIS3 in Atnoa1 plant, indicating that cGMP is downstream of NO for inducing HY5 and GIS3 expression. By treating with hydrogen peroxide (H2O2) and diphenylene iodonium (DPI), an NADPH oxidase inhibitor, there was no effect on HY5 expression, but GIS3 expression was repressed. Moreover, the expression of GIS3 was still induced after ODQ treatment in HY5OE, while that was repressed after H2O2 and DPI treatment. These results indicated that NADPH oxidase mediated H2O2 production, which acted in the downstream of HY5. These results demonstrated that “NO-cGMP-HY5- GIS3-root growth” is a possible signal transduction pathway. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:52:53Z (GMT). No. of bitstreams: 1 ntu-108-D99b42001-1.pdf: 2748970 bytes, checksum: dc03597d08b03090a6f07b2e0c2502fc (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Content
誌謝 I 中文摘要 II Abstract IV Content i Introduction 1 1. Root growth in plants 1 2. cGMP is an important secondary messenger 2 3. ELONGATED HYPOCOTYL 5 (HY5) 5 4. Analysis of microarray data based on Arabidopsis roots treated with cGMP 6 5. The transgenic Arabidopsis overexpressing transcription factors 7 6. Research objectives 7 Materials and methods 8 1. Plant material and chemical treatments 8 2. Expression levels analysis 9 2-1. RNA isolation 9 2-2. RNA electrophoresis 10 2-3. RT-PCR 10 2-4. Quantitative real-time PCR 10 3. Plasmids construction 11 3-1. DNA gel electrophoresis 11 3-2. DNA gel extraction 12 3-3. DNA fragment ligation 12 3-4. E. coli transformation 13 3-5. Plasmid mini extraction 13 3-6. DNA sequencing 14 4. Arabidopsis transformation 14 4-1. Construction of transgenic plants 14 4-2. Preparation of Agrobacteria competent cells 15 4-3. Arabidopsis transformation 16 5. Statistical analysis 16 Results 18 1. Screening Arabidopsis transgenics from Dröge-Laser’s Lab in 1/2 MS medium containing ODQ 18 2. Genes downstream of the cGMP-HY5-root growth pathway 19 3. The expression of HY5 and GIS3 after treatment of cGMP and ODQ 21 4. Phenotype and genes expression in the HY5 overexpression transgenic plant created by ourselves 21 5. Phenotype of HY5OE, hy5, GIS3OE and GIS3RNAi plants under ODQ treatment 22 6. Relationship between NO and cGMP-HY5-root growth pathway 24 7. The relationship between H2O2 and cGMP-HY5-root growth pathway 25 Discussion 26 1. cGMP is essential of Arabidopsis rooting 26 2. Genes regulated by cGMP 26 3. Phytohormones may regulate cGMP-HY5-GIS3-dependent root growth 28 4. Nitric oxide participates in cGMP dependent root growth 30 5. GIS3 is repressed by both H2O2 and DPI 31 Conclusion 33 Table and figures 34 Table 1. Genes induced by both cGMP and HY5 pathway 34 Figure 1. Phenotypes of the Arabidopsis transgenics from Dröge-Laser’s Lab 35 Figure 2. Expression of genes regulated by HY5 36 Figure 3. Expression of HY5 and GIS3 in the presence of cGMP or ODQ 37 Figure 4. Genes expression of HY5 and GIS3 in HY5OE plants 38 Figure 5. Phenotypes of HY5OE and hy5 mutant plants under ODQ treatment 41 Figure 6. Phenotypes of GIS3OE and GIS3RNAi transgenic plants under ODQ treatment 43 Figure 7. Hypocotyl lengths of HY5OE, hy5 mutants, GIS3OE and GIS3RNAi plants 44 Figure 8. Effect of NO on the expression of HY5 and GIS3 46 Figure 9. Expression of GIS3 in HY5OE and nox1 transgenic plants under the presence of ODQ, DPI or H2O2 48 Figure 10. Proposed model of cGMP-HY5-GIS3-dependent root development pathway 50 Appendix 51 Table 1. Primers used in this study 51 Figure 1. Root and hypocotyl length of WT, HY5OE and hy5 plants 52 Figure 2. The expression of GIS3 in GIS3OE and HY5OE lines 53 Figure 3. Root length of WT in different concentration of SNP 54 References 55 | - |
| dc.language.iso | en | - |
| dc.subject | 一氧化氮 | zh_TW |
| dc.subject | 過氧化氫 | zh_TW |
| dc.subject | GIS3 | zh_TW |
| dc.subject | HY5 | zh_TW |
| dc.subject | 根生長發育 | zh_TW |
| dc.subject | cGMP | zh_TW |
| dc.subject | cGMP | en |
| dc.subject | HY5 | en |
| dc.subject | GIS3 | en |
| dc.subject | nitric oxide | en |
| dc.subject | hydrogen peroxide | en |
| dc.subject | root development | en |
| dc.title | cGMP參與阿拉伯芥根生長發育的角色之研究 | zh_TW |
| dc.title | The study of cGMP roles involved in root development of Arabidopsis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 黃麗芬;林信宏;林振祥;林盈仲 | zh_TW |
| dc.contributor.oralexamcommittee | Li-Fen Huang;Hsin-Hung Lin;Jeng-Shane Lin;Ying-Chung Lin | en |
| dc.subject.keyword | cGMP,HY5,GIS3,一氧化氮,過氧化氫,根生長發育, | zh_TW |
| dc.subject.keyword | cGMP,HY5,GIS3,nitric oxide,hydrogen peroxide,root development, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU201903234 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-14 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 植物科學研究所 | - |
| 顯示於系所單位： | 植物科學研究所 | |
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