Li, Man Wah.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 109-119).Abstracts in English and Chinese.Thesis Committee --- p.IStatement --- p.IIAbstract --- p.III摘要 --- p.VAcknowledgements --- p.VIAbbreviations --- p.VIIIAbbreviations of Chemicals --- p.XList of Tables --- p.XIList of Figures --- p.XIITable of Contents --- p.XIIIChapter Chapter 1 --- Literature Review --- p.1Chapter 1.1 --- General amino acid control in yeast --- p.1Chapter 1.2 --- Mammalian eIF2α kinases --- p.7Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11Chapter 1.3 --- Plant General Amino Acid Control --- p.12Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14Chapter 1.4 --- Previous works in our lab --- p.15Chapter 1.5 --- Hypothesis and Objectives --- p.17Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- Materials --- p.18Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18Chapter 2.1.2 --- Primers --- p.21Chapter 2.1.3 --- Commercial kits --- p.25Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25Chapter 2.1.6 --- Enzymes --- p.25Chapter 2.1.7 --- Antibodies --- p.25Chapter 2.1.8 --- Equipments and facilities --- p.25Chapter 2.2 --- Methods --- p.26Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26Chapter 2.2.2 --- Basic molecular techniques --- p.27Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27Chapter 2.2.2.3 --- Restriction digestion --- p.27Chapter 2.2.2.4 --- DNA purification --- p.28Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28Chapter 2.2.2.6 --- DNA ligation --- p.29Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30Chapter 2.2.2.10 --- DNA sequencing --- p.30Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31Chapter 2.2.2.13 --- cDNA synthesis --- p.32Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33Chapter 2.2.2.15 --- Western blotting --- p.33Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47Chapter 2.2.6 --- Gel mobility shift assay --- p.47Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50Chapter 2.2.8.2 --- In vitro translation --- p.51Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53Chapter Chapter 3 --- ResultsChapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83Chapter Chapter 4 --- Discussion --- p.85Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97AppendicesAppendix I Commercial kits used in this project --- p.98"Appendix II Buffer, solution, gel and medium" --- p.99"Appendix III Chemicals, reagents and consumables" --- p.102Appendix IV Enzymes --- p.103Appendix V Antibodies --- p.104Appendix VI Equipments and facilities --- p.105Appendix VII Supplementary Data --- p.106Appendix VIII Amplification efficiency of real time primers --- p.108References --- p.10
