Promoter analysis and expression of the tomato purple acid phosphatase (TPAP1) in tobacco.

Suen Pui Kit.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 154-168).Abstracts in English and Chinese.Acknowledgements --- p.iAbstract --- p.iiiList of Figures --- p.viiList of Tables --- p.ixList of Abbreviations --- p.xChapter Chapter 1: --- Introduction --- p.1Chapter Chapter 2: --- Literature Review --- p.3Chapter 2.1 --- Phosphorus and Plants --- p.3Chapter 2.1.1 --- Importance of phosphorus --- p.3Chapter 2.1.2 --- Phosphorus is a limiting nutrient --- p.3Chapter 2.2 --- Responses of Plants to Phosphate Deficiency --- p.4Chapter 2.2.1 --- Morphological changes of plants during phosphate deficiency --- p.5Chapter 2.2.1.1 --- Modification of the root system --- p.5Chapter 2.2.1.2 --- Symbiotic association of roots with mycorrhiza --- p.6Chapter 2.2.2 --- Maintenance of phosphate levels in plants during phosphate deficiency --- p.7Chapter 2.2.2.1 --- Phosphate homeostasis in plants --- p.7Chapter 2.2.2.2 --- "Enhancement of Pi scavenging, recycling and uptake" --- p.9Chapter 2.2.2.3 --- Pi-limited metabolism --- p.11Chapter 2.2.3 --- Hormones and phosphate starvation responses --- p.12Chapter 2.2.4 --- Regulation of gene expression during phosphate starvation --- p.14Chapter 2.2.4.1 --- The pho regulon in bacteria and yeast --- p.14Chapter 2.2.4.2 --- The coordination of phosphate starvation induced genes in plants --- p.19Chapter 2.2.4.3 --- Signaling phosphate starvation --- p.19Chapter 2.2.4.4 --- Phosphite and phosphate starvation --- p.21Chapter 2.2.4.5 --- Transcriptional regulation during phosphate starvation --- p.22Chapter 2.3 --- Acid Phosphatases in Higher Plants --- p.26Chapter 2.3.1 --- Enzymatic properties of acid phosphatases --- p.26Chapter 2.3.2 --- Localization and function of acid phosphatases --- p.27Chapter 2.3.3 --- Expression of acid phosphatases --- p.28Chapter 2.4 --- Purple Acid Phosphatases --- p.29Chapter 2.4.1 --- Properties of purple acid phosphatases --- p.29Chapter 2.4.2 --- Regulation and expression of plant purple acid phosphatase --- p.32Chapter 2.5 --- Tomato Purple Acid Phosphatases --- p.33Chapter 2.6 --- Promoter Analysis --- p.35Chapter 2.6.1 --- Structure of an eukaryotic promoter --- p.35Chapter 2.6.2 --- Promoter analysis by deletion mapping --- p.37Chapter 2.6.3 --- The computational approaches in promoter analysis --- p.38Chapter 2.6.4 --- Transient expression assay and transgenic expression assay --- p.39Chapter 2.7 --- Transcriptional Regulation of Tomato Purple Acid Phosphatase Expression --- p.40Chapter 2.8 --- Hypothesis --- p.41Chapter Chapter 3: --- Materials and Methods --- p.43Chapter 3.1 --- Introduction --- p.43Chapter 3.2 --- Materials --- p.44Chapter 3.2.1 --- Chemicals --- p.44Chapter 3.2.2 --- Plant materials --- p.44Chapter 3.2.3 --- Plasmid vectors and bacterial strains --- p.44Chapter 3.2.4 --- Primers design --- p.45Chapter 3.2.5 --- Confirmation of sequence fidelity --- p.46Chapter 3.3 --- Cloning of the TPAP1 Promoter Fragments --- p.46Chapter 3.3.1 --- Genomic DNA extraction --- p.46Chapter 3.3.1.1 --- Materials --- p.46Chapter 3.3.1.2 --- Procedures --- p.47Chapter 3.3.2 --- Cloning strategy of TPAP1 promoter --- p.47Chapter 3.3.3 --- TPAP1 promoter cloning --- p.48Chapter 3.3.3.1 --- Long-distance PCR --- p.48Chapter 3.3.4 --- Chimeric gene constructs --- p.48Chapter 3.3.4.1 --- Chimeric gene construction for particle bombardment --- p.51Chapter 3.3.4.2 --- Chimeric gene construction for tobacco transformation --- p.51Chapter 3.4 --- Transient Expression Assay of the TPAP1 Promoter Fragments --- p.54Chapter 3.4.1 --- TPAP1 promoter activity assay --- p.54Chapter 3.4.2 --- Preparation of MS culture medium --- p.54Chapter 3.4.3 --- Growing tomato seedlings in MS liquid medium --- p.56Chapter 3.4.4 --- Biolistic bombardment --- p.56Chapter 3.4.5 --- GUS histochemcial staining --- p.57Chapter 3.4.5.1 --- Materials --- p.57Chapter 3.4.5.2 --- Procedures --- p.57Chapter 3.5 --- Transgenic Assay of the TPAP1 Promoter Fragments --- p.58Chapter 3.5.1 --- Materials for tobacco transformation --- p.58Chapter 3.5.2 --- Agrobacterium tumefaciens preparation --- p.58Chapter 3.5.3 --- Tobacco transformation and regeneration --- p.59Chapter 3.5.4 --- Promoter activity analysis --- p.60Chapter 3.5.4.1 --- Materials --- p.60Chapter 3.5.4.2 --- Procedures --- p.60Chapter 3.5.5 --- Southern blot analysis --- p.61Chapter 3.5.6 --- RNA isolation --- p.61Chapter 3.5.6.1 --- Materials --- p.61Chapter 3.5.6.2 --- Procedures --- p.61Chapter 3.5.7 --- Northern blot analysis --- p.62Chapter 3.6 --- Biochemical Analysis of Acid Phosphatase Activities --- p.63Chapter 3.6.1 --- Excretion of acid phosphatase into the environment --- p.63Chapter 3.6.2 --- Growing tomato seedlings in MS medium --- p.63Chapter 3.6.3 --- Acid phosphatase activity assay by p-nitrophenyl phosphate --- p.64Chapter 3.6.4 --- Activity-gel detection --- p.65Chapter 3.6.4.1 --- Materials --- p.65Chapter 3.6.4.2 --- Procedures --- p.65Chapter 3.7 --- "Sequence Analysis of the TPAP1 gene, cDNA and promoter" --- p.66Chapter 3.7.1 --- Isolation of TPAPl cDNA --- p.66Chapter 3.7.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.66Chapter 3.7.1.2 --- RT-PCR --- p.67Chapter 3.7.2 --- Isolation of TPAP1 gene --- p.67Chapter 3.7.2.1 --- PCR amplification of the TPAP1 gene --- p.67Chapter 3.7.2.2 --- TPAP1 gene sequence determination --- p.68Chapter 3.7.3 --- Sequence analysis --- p.69Chapter 3.8 --- Statistical analysis --- p.70Chapter Chapter 4: --- Results --- p.72Chapter 4.1 --- "Cloning of the TPAP1 Promoter Fragments, Gene and cDNA" --- p.72Chapter 4.1.1 --- TPAP1 promoter fragment constructs --- p.72Chapter 4.1.2 --- TPAP1 cDNA cloning --- p.72Chapter 4.1.3 --- TPAP1 gene cloning --- p.72Chapter 4.2 --- "Sequence analysis of the TPAP1 promoter, gene, cDNA and predicted amino acid sequence" --- p.76Chapter 4.2.1 --- "The DNA sequence of the TPAP1 promoter, gene and cDNA" --- p.76Chapter 4.2.2 --- Properties of TPAP1 cDNA and protein --- p.83Chapter 4.2.3 --- Identification of potential metal ligating residues on TPAP1 --- p.85Chapter 4.2.4 --- Phylogenetic relationship of TPAPl to other plant PAPs --- p.86Chapter 4.2.5 --- Sequence comparison of 5'UTR ofTPAPl and NtPAP12 --- p.89Chapter 4.3 --- APase Activity Assay --- p.90Chapter 4.3.1 --- p-NPP APase activity assay --- p.90Chapter 4.3.2 --- Activity-gel detection --- p.90Chapter 4.4 --- "Comparison of TPAP 1, IAP，SAP 1 and SAP2" --- p.96Chapter 4.5 --- Potential Cis-acting Regulatory Elements (CAREs) on the TPAP1 Promoter --- p.100Chapter 4.5.1 --- Search for potential CAREs --- p.100Chapter 4.5.2 --- Functions of CAREs --- p.100Chapter 4.6 --- Transient Expression Analysis --- p.102Chapter 4.6.1 --- Biolistic bombardment of TPAP1 promoter fragments into tomato roots --- p.102Chapter 4.7 --- Transgenic Expression Analysis --- p.104Chapter 4.7.1 --- Transformation of tobacco --- p.104Chapter 4.7.2 --- Northern and RT-PCR analysis of GUS expression --- p.110Chapter 4.7.3 --- GUS activity analysis --- p.114Chapter 4.7.4 --- Histochemical staining of GUS --- p.123Chapter Chapter 5: --- Discussions --- p.135Chapter 5.1 --- Properties ofTPAPl --- p.135Chapter 5.1.1 --- "Structure of the TPAP1 promoter, gene and cDNA" --- p.135Chapter 5.1.2 --- Potential flmction(s) ofTPAPl --- p.135Chapter 5.1.3 --- The potential relationship between TPAP1 and NtPAP12 --- p.137Chapter 5.2 --- Induction of Secretory APases during Pi Starvation --- p.137Chapter 5.3 --- Putative Protein Encode by theTPAP 1 cDNA --- p.138Chapter 5.4 --- Promoter Analysis of TPAP1 --- p.140Chapter 5.4.1 --- Construct preparation --- p.140Chapter 5.4.2 --- Potential CAREs located on the TPAP1 promoter --- p.141Chapter 5.4.3 --- Transient expression analysis --- p.142Chapter 5.4.4 --- Transgenic expression analysis --- p.143Chapter 5.4.4.1 --- Northern analysis and RT-PCR analysis of GUS expression --- p.143Chapter 5.4.4.2 --- GUS activity analysis --- p.143Chapter 5.4.4.3 --- Histochemical staining of GUS --- p.145Chapter 5.5 --- Hypothetical Model for TPAP1 Promoter Activities --- p.146Chapter 5.5.1 --- Model for expression level --- p.146Chapter 5.5.2 --- Models for spatial expressions --- p.148Chapter 5.6 --- Future Perspectives --- p.150Chapter Chapter 6: --- Conclusions --- p.152References --- p.15