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標題: | cGMP參與阿拉伯芥根生長發育的角色之研究 The study of cGMP roles involved in root development of Arabidopsis |
作者: | 金禹圻 Yu-Chi King |
指導教授: | 鄭石通 Shih-Tong Jeng |
關鍵字: | cGMP,HY5,GIS3,一氧化氮,過氧化氫,根生長發育, cGMP,HY5,GIS3,nitric oxide,hydrogen peroxide,root development, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77254 |
DOI: | 10.6342/NTU201903234 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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