- 學位論文
阿拉伯芥古氏基因啟動子研究分析與植物啟動子分析平台暨生物資料庫之建構
Promoter analysis of Ku genes from Arabidopsis and construction of novel web server and database for plant promoter analysis
摘要
啟動子在基因表現的調控上扮演極重要的角色。在本論文中，我們使用in vivo 和in silico的方法來研究植物的啟動子。除了用實驗的方法分析阿拉伯芥古氏基因(AtKu70 和AtKu80)啟動子外，也建立一以資料庫為輔的植物啟動子分析網路服務平台。 第一部份，我們以轉殖阿拉伯芥來研究阿拉伯芥古氏基因(AtKu70 和AtKu80)的啟動子活性。在高等植物中，阿拉伯芥古氏基因在DNA 修復的非同源末端結合(non-homologous endjoining)機制中扮演極重要的角色。阿拉伯芥古氏基因在植物系統與在哺乳類動物系統一樣都具有多重細胞功能，例如：參與維持染色體端粒(telomere)長度、參與轉錄過程、參與細胞凋亡(apotosis)等。然而，在哺乳動物中會持續性地表現大量的古氏基因，但相對之下，在高等植物中，古氏基因的表現量低。在本研究中，我們想要瞭解阿拉伯芥古氏基因在高等植物中是如何被調控的。因此我們選殖出阿拉伯芥古氏基因的啟動子區域，再接上GUS 報導基因，然後利用轉殖植物來研究該啟動子的活性。我們發現，在植物發育初期，阿拉伯芥古氏基因啟動子在下胚軸和子葉具有很高的活性，另外，在花與果莢發育初期，花的柱頭和果莢也都有較高的啟動子活性。另一方面，我們也發現吉貝素(gibberellic acid)、植物生長激素(auxins)和茉莉酸(jasmonic acid)會促進阿拉伯芥古氏基因啟動子活性；相反地，離酸(abscisic acid)、水楊酸(salicylic acid)、 熱逆境、乾旱逆境和冷逆境都會抑制阿拉伯芥古氏基因啟動子活性。除此之外，我們利用片段分析(deletion analysis)發現AtKu70 的最小功能啟動子區域約在轉錄起始點(transcription start site)上游約400 鹼基對的位置，而AtKu80 則在600 鹼基對的位置。綜合以上，我們發現阿拉伯芥古氏基因的調控與植物發育相關，同時，也受到植物賀爾蒙與環境逆境的影響。 第二部分，隨著許多植物基因體定序計畫的完成，例如：阿拉伯芥、稻米和玉米等，快速有效地研究植物基因轉錄的調控在植物科學中便是一非常重要的課題。目前已有許多關於植物啟動子的生物資訊服務平台和資料庫被開發出來，但他們多只著重於註解單一基因啟動子之轉錄因子結合位置，而忽略了啟動子上其他重要的調控因子，例如：串連的重複區域(tandem repeats) 和CpG/CpNpG 島 (CpG/CpNpG islands)。另一方面，組合的轉錄因子在調控一群表現情況類似的基因是非常重要的。因此，我們便想開發一工具用來分析調控一群基因啟動子的組合轉錄因子(combinatorial transcription factors)。在本研究中，我們建構一資料庫輔助的網路服務平台—PlantPAN (Plant Promoter Analysis Navigator)，本系統除可辨識出一群共同表現基因啟動子上之組合的近端調控因子(combinatorial cis-regulatory elements)外，同時還考慮組合轉錄因子之間的距離是否在合理範圍內。本系統所收集的植物轉錄因子主要是來自TRANSFAC、PLACE、AGRIS 和JASPER 四個資料庫，使用本系統分析組合轉錄因子時，使用者可輸入一群基因識別碼(Gene IDs)或啟動子序列，透過本系統的分析，在這一群基因啟動子上同時出現的組合轉錄因子結合位置便會被辨識出來，同時可限制組合的轉錄因子之間的距離在20 到200 鹼基對之間。除了轉錄因子結合位置的分析外，本系統也提供註解植物啟動子上串連重複區域和CpG/CpNpG 島。為使本系統更臻完善，同源基因啟動子保留區域(conserved regions)中的調控因子也可透過本系統被偵測及標示出來。另一方面，由於最近很多研究發現microRNA 在基因表現的調控上也扮演很重要的角色，因此在PlantPAN 中，我們特別發展一工具來辨識microRNA在傳訊RNA (mRNA)和5’端未轉譯區域 (5’UTR) 的標的結合位置。本新開發的系統目前已可在http://PlantPAN.mbc.nctu.edu.tw 免費使用。
並列摘要
Promoter is essential to gene expression regulation. In this study, in vivo and in silico methods were used to analyze plant promoters. In addition to experimental techniques used for investigating AtKu promoter, a database-assisted plant promoter analysis web server was developed. Firstly, transgenic Arabidopsis lines were obtained to investigate Arabidopsis Ku70 (AtKu70) and Ku80 (AtKu80) promoters activities. AtKu70 and AtKu80 have been found in higher plants and play a crucial role in non-homologous end joining (NHEJ) during DNA repair. Similar to that in mammalian cells, AtKu70 and AtKu80 protein are involved presumably in multiple cellular processes, such as telomere maintenance, transcription, and apoptosis. The expression of Ku gene is constitutively high in mammalian cells; nevertheless, it is relatively at low level in higher plants. In this study, we were thus prompted to elucidate the regulation of AtKu genes in higher plants. Promoters of the AtKu70 and AtKu80 were isolated from Arabidopsis and their activities characterized using GUS reporter-aided approach in transgenic plants. Ku promoter activities were determined relatively higher in hypocotyls and cotyledons upon germination and in stigma and siliques as well at their early developing stages. Furthermore, Ku promoter activities could be enhanced by gibberellic acid, auxins, and jasmonic acid, but repressed by abscisic acid, salicylic acid, heat, drought and cold. Deletion analysis demonstrates minimal lengths of about 400 bp and 600 bp upstream of transcription start site for functional promoters of AtKu70 and AtKu80, respectively. Taken together, expressions of Ku genes are regulated both by developmental programs as well as by plant hormones and environmental stresses. Secondly, it is well known that elucidating transcriptional regulation in plant genes is one of the most important and urgent areas of research for plant scientists, following the mapping of various plant genomes, such as A. thaliana, O. sativa and Z. mays. A variety of bioinformatics servers or databases of plant promoters have been established, although most of them have been aimed only at annotating transcription factor binding sites in a single gene and have neglected some important regulatory elements (tandem repeats and CpG/CpNpG islands) on promoter regions. Additionally, the combinatorial interaction of transcription factors (TFs) is important for regulating the gene group that is associated with same expression pattern. Therefore, we were thus prompted to develop a tool for detecting co-regulation of transcription factors in a group of gene promoters. In this study, a database-assisted system, PlantPAN (Plant Promoter Analysis Navigator, http://PlantPAN.mbc.nctu.edu.tw) was constructed, for recognizing combinatorial cis-regulatory elements with distance constraint in plant co-expressed genes. The system collects the plant transcription factor binding profiles from TRANSFAC, PLACE, AGRIS and JASPER databases, and allows users to input a group of gene IDs or promoter sequences, enabling the co-occurrence of combinatorial transcription factor binding sites (TFBSs) within a defined distance (20 bp to 200 bp) to be identified. Additionally, the new resource enables the annotation of other regulatory features in a plant promoter, such as CpG/CpNpG islands and tandem repeats. Moreover, the regulatory elements in the conserved regions of the promoters across homologous genes are detected and displayed. Furthermore, it is shown that microRNA is important in gene expression regulation, a tool for identified microRNA target sites in mRNA or 5’ un-translated region (5’ UTR) are also applied in PlantPAN. This novel analytical resource is now freely available at http://PlantPAN.mbc.nctu.edu.tw.