Promoter analysis of Ku genes from Arabidopsis and construction of novel web server and database for plant promoter analysis

• Main Authors: Wen-Chi Chang, 張文綺
• Other Authors: Rong-Long Pan
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/03772650732153391323

博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 96 === 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.