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標題: | 探討NRT1.13在開花調控中的下游機制 The downstream mechanisms of NRT1.13 in flowering regulation |
作者: | Jeng-Jung Wu 吳任容 |
指導教授: | 蔡宜芳 |
關鍵字: | 硝酸鹽,NRT1.13,CIPK8,開花, Nitrate,NRT1.13,CIPK8,flowering, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 硝酸鹽不僅是植物必需的氮源,同時也是植物重要的信號分子。硝酸鹽對植物從營養期到繁殖期的轉變扮演著重要的一環。但植物如何偵測硝酸鹽來調節開花還所知甚少。先前研究顯示,NRT1家族中第10到第11個跨膜區域的脯胺酸(在NRT1.1序列中為第492個)在NRT1家族中為高度保守,且對於硝酸鹽轉運能力而非硝酸鹽的感受能力是極重要的。NRT1.13是一個膜蛋白且表達在木質部旁的薄壁組織細胞,在NRT1家族內是一個不具有此高度保守性脯胺酸的例外。相較於全營養環境,生長在氮限制環境下,nrt1.13-1單突變株表現出更延遲開花的性狀。因此本實驗室推測,NRT1.13可能是一個植物內部的硝酸鹽感受器,且能透過偵測木質部中硝酸鹽含量,調控植物開花。本篇研究的目的是想了解NRT1.13調控開花的分子機制。前人研究顯示,主要抑制開花基因FLC的表現量在nrt1.13-1突變株中上昇。我透過研究nrt1.13-1、flc-3及nrt1.13-1 flc-3雙突變株的開花情形來瞭解NRT1.13與FLC的關聯性。nrt1.13-1在低硝酸鹽的情況下會延遲開花,但在nrt1.13-1 flc-3雙突變株中這個性狀消失了,推測NRT1.13可能透過FLC調控開花。為了進一步找其他的下游基因,我們用酵母菌雙雜合研究尋找。結果顯示,蛋白激酶CIPK8與NRT1.13有結合能力,且CIPK8的調控區域和激酶區域都與NRT1.13有結合能力。植株的表現型分析發現,nrt1.13-1和cipk8-1雙突變株的開花性狀與野生型相似而非與nrt1.13-1相似,因此推測CIPK8可能是NRT1.13調控開花機制中的下游傳訊分子之一。第三部分,我們還觀察到轉錄因子NLP1、2、7和9的PB1區域對NRT1.13有結合能力,但全長NLP卻沒有。因此在開花調控中,NLPs是否同樣參與在NRT1.13的下游傳導並調控開花,將是非常有趣的問題。最後,我們觀察到NRT1.13並沒有表現在頂端分生組織中,顯示NRT1.13是在蓮座葉中而非頂端分生組織調控開花時間。綜合所有結果,我們找到了可能是位於NRT1.13下游的傳導信號分子,它們參與在NRT1.13應對氮源限制環境時調控開花的機制內。 Nitrate is not only an essential nutrient but also a signaling molecule for plants. Efficient utilization of nitrate is important for the conversion from vegetative stage to reproductive stage. Little is known about how plants sense nitrate to regulate flowering. Proline residue (492 in CHL1) between 10th and 11th transmembrane domains is highly conserved in NRT1 family and important for nitrate transportation but not for the nitrate sensing. NRT1.13, expressed in the xylem parenchyma cell, was an exception in NRT1 family without the highly conserved proline residue. In low nitrate condition, nrt1.13-1 showed delayed flowering. Previous results in our group suggested that NRT1.13 might be an internal sensor to monitor nitrate content in xylem and then regulate flowering. The aim of this study is to understand how NRT1.13 regulate flowering. Previous study showed that FLC, repressor of flowering, is up-regulated in nrt1.13-1. In this study, phenotype analysis showed that the nitrate-dependent late flowering phenotype of nrt1.13-1 is lost in the double mutant of nrt1.13 and flc-3, suggesting that NRT1.13 might regulate the flowering time through FLC. In the search for additional downstream candidates, yeast two-hybrid study showed that protein kinase CIPK8 interacts with NRT1.13. Both regulatory domain and kinase domain of CIPK8 can interact with NRT1.13. Phenotype analysis showed that the disruption of CIPK8 lead to early flowering while double mutant of nrt1.13-1 and cipk8-1 behave like as wild type instead of nrt1.13-1, suggesting that CIPK8 is required for the late flowering phenotype of nrt1.13-1, and it may act in the downstream of NRT1.13 to regulate flowering. In the third part, we also observed that the PB1 domain of transcription factor NLP1, 2, 7 and 9, can interact with NRT1.13, but the full-length of NLP cannot. In the last, it was found that NRT1.13 is not expressed in the shoot apical meristem, suggesting that NRT1.13 regulates the flowering time in the leaves instead of shoot apical meristem. Taken together, through these studies, we have identified several potential signal components in the downstream of NRT1.13 to regulate flowering in an N-dependent manner. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67634 |
DOI: | 10.6342/NTU201702083 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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