Tsai Sau-Na. / Thesis submitted in: 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 157-170). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xii / Table of contents --- p.xiv / List of figures --- p.xx / List of tables --- p.xxii / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Salinization is a global problem --- p.1 / Chapter 1.2 --- Causes of soil salinization in agricultural lands --- p.2 / Chapter 1.3 --- Toxicity of salinity in plants --- p.4 / Chapter 1.3.1. --- Physiological drought --- p.4 / Chapter 1.3.2. --- Nutritional imbalance --- p.5 / Chapter 1.3.3 --- Specific ion toxicity --- p.6 / Chapter 1.4 --- Plant adaptation to salinity --- p.7 / Chapter 1.5 --- Ion transport proteins in plant --- p.10 / Chapter 1.5.1 --- "Pump, channel and carrier" --- p.10 / Chapter 1.5.2 --- Pumps --- p.11 / Chapter 1.5.2.1 --- P-ATPase --- p.11 / Chapter 1.5.2.2 --- V-ATPase --- p.12 / Chapter 1.5.2.3 --- PPiase --- p.12 / Chapter 1.5.3 --- Cation channels --- p.13 / Chapter 1.5.3.1 --- K+ channels --- p.13 / Chapter 1.5.3.1.1 --- Shaker family --- p.14 / Chapter 1.5.3.1.1.1 --- KIRCs --- p.16 / Chapter 1.5.3.1.1.2 --- KORCs --- p.17 / Chapter 1.5.3.1.1.3 --- VICs --- p.18 / Chapter 1.5.3.1.2 --- Kir family --- p.18 / Chapter 1.5.3.1.2 --- KCO family --- p.19 / Chapter 1.5.3.2 --- Ca2+ channels --- p.20 / Chapter 1.5.3.2.1 --- TPC family --- p.20 / Chapter 1.5.3.2.2 --- CNGC family --- p.21 / Chapter 1.5.4 --- Anion Channels --- p.22 / Chapter 1.5.5 --- Carriers --- p.23 / Chapter 1.5.5.1 --- High affinity K+ carriers --- p.23 / Chapter 1.5.5.1.1 --- HKT transporter --- p.24 / Chapter 1.5.5.1.2 --- HAK/KUP transporter --- p.25 / Chapter 1.5.5.2 --- Cation/H+ antiporters --- p.26 / Chapter 1.5.5.2.1 --- Na+/H+ antiporter --- p.27 / Chapter 1.5.5.2.2 --- Ca2+/H+ antiporters --- p.30 / Chapter 1.6 --- Ion homeostasis and salt tolerance --- p.31 / Chapter 1.6.1 --- Ion transporters involved in ion homeostasis during salt stress --- p.31 / Chapter 1.6.2 --- Sodium uptake under salt stress --- p.32 / Chapter 1.6.4 --- Sodium extrusion --- p.36 / Chapter 1.6.5 --- Intracellular compartmentation --- p.37 / Chapter 1.6.6 --- Genetic engineering of ion transporter for improvement of salt tolerance --- p.40 / Chapter 1.7 --- Soybean as a target for studies of salt tolerance --- p.41 / Chapter 1.7.1 --- Economic importance of soybean --- p.41 / Chapter 1.7.2 --- Salt tolerant soybean in China --- p.43 / Chapter 1.7.3 --- Previous studies of Wenfeng7 and Union in our laboratory --- p.43 / Chapter 1.7.4 --- Hypothesis and research strategy of my project --- p.46 / Chapter 2. --- Materials and methods --- p.49 / Chapter 2.1 --- Materials --- p.49 / Chapter 2.1.1. --- Plant materials --- p.49 / Chapter 2.1.2. --- Bacteria strains and plasmid vectors --- p.50 / Chapter 2.1.3. --- Growth media for soybeans and A. thaliana --- p.50 / Chapter 2.1.4. --- Chemicals and reagents used --- p.50 / Chapter 2.1.5. --- Solutions used --- p.51 / Chapter 2.1.6. --- Commercial kits used --- p.51 / Chapter 2.1.7. --- Equipment and facilities used --- p.51 / Chapter 2.1.8. --- Primers used --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- Cloning of ion transporters --- p.52 / Chapter 2.2.1.1. --- Sample preparation --- p.52 / Chapter 2.2.1.2 --- Total RNA extraction --- p.52 / Chapter 2.2.1.3 --- Primer design for RACE --- p.53 / Chapter 2.2.1.4 --- 5´ة& 3´ة RACE of ion transporters --- p.54 / Chapter 2.2.1.5 --- Subcloning of RACE cDNA fragments --- p.56 / Chapter 2.2.1.6 --- PCR screening of white colonies --- p.57 / Chapter 2.2.1.8 --- Preparation of recombinant plasmid for sequencing --- p.57 / Chapter 2.2.1.9 --- Sequencing and homology search --- p.58 / Chapter 2.2.1.10 --- Cloning of full length coding regions of ion transporters --- p.58 / Chapter 2.2.1.11 --- "Sequence comparison, analysis and multialignment" --- p.62 / Chapter 2.2.2 --- Gene expression profiles --- p.62 / Chapter 2.2.2.1 --- Sample stepwise treatment with different concentration of NaCl --- p.62 / Chapter 2.2.2.2 --- Sample treatment with different Hoagland's solution supplement with 1.2% NaCl --- p.63 / Chapter 2.2.2.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.64 / Chapter 2.2.2.4 --- Testing the concentration of DIG-labeled probes --- p.65 / Chapter 2.2.2.5 --- Northern blot technique --- p.66 / Chapter 2.2.2.6 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.67 / Chapter 2.2.3 --- Functional test using transgenic plants --- p.68 / Chapter 2.2.3.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.68 / Chapter 2.2.3.2 --- "Eletroporation of Agrobacterium, tumefaciens" --- p.69 / Chapter 2.2.3.3 --- Seed sterilization and plant growth --- p.70 / Chapter 2.2.3.4 --- Vacuum infiltration transformation of Arabidopsis thaliana --- p.71 / Chapter 2.2.3.5 --- Selection of hemizygous and homozygous transgenic plants --- p.72 / Chapter 2.2.3.6 --- Genomic DNA extraction and PCR screening --- p.72 / Chapter 2.2.3.7 --- RT-PCR and Northern Blot of transgenic plants --- p.73 / Chapter 2.2.3.8 --- Functional test on MS plate supplemented with NaCl --- p.73 / Chapter 2.2.3.9 --- Functional test on sand supplemented with Hoagland's solution and NaCl --- p.74 / Chapter 3. --- Results --- p.76 / Chapter 3.1 --- "Cloning of Nhx, AKT1 and CLC from Wenfeng7 and Union" --- p.76 / Chapter 3.1.1 --- "Cloning of 5'- & 3'- RACE cDNA fragments of Nhx, AKT1 and CLC" --- p.76 / Chapter 3.1.2 --- "Cloning of full length coding regions of Nhx, AKT1 and CLC from Wenfeng7 and Union" --- p.77 / Chapter 3.1.3 --- "Sequence comparison, analysis and multialignment" --- p.82 / Chapter 3.1.3.1 --- Sequence analysis and multialignment of GmNhx1 and GmNhx2 --- p.82 / Chapter 3.1.3.2 --- Sequence analysis and multialignment of GmAKTl --- p.92 / Chapter 3.1.3.3 --- Sequence analysis and multialignment of GmCLC --- p.101 / Chapter 3.2 --- "Gene expression profiles of GmNhx, GmCLC and GmAKTl" --- p.111 / Chapter 3.2.1 --- Induction of GmNhx and GmCLC gene expression by NaCl in different Hoagland's solution --- p.111 / Chapter 3.2.2 --- RT-PCR using gene specific primers to distinguish the gene expression of GmNhx1 and GmNhx2 --- p.116 / Chapter 3.2.3 --- RT-PCR analysis of the transcripts of GmAKTl in Wenfeng7 and Union --- p.118 / Chapter 3.3 --- Functional analysis of transgenic plants in salt tress --- p.120 / Chapter 3.3.1 --- "Construction of chimeric gene of GmNhx1´ة GmNhx2, GmCLC and GmAKT1 into V7 vector" --- p.120 / Chapter 3.3.2 --- Transformation of chimeric gene constructs into A. tumefaciens --- p.122 / Chapter 3.3.3 --- Vacuum infiltration transformation of Arabidopsis thaliana and selection of transgenic plants --- p.123 / Chapter 3.3.4 --- PCR screening of transgene from transgenic plants --- p.130 / Chapter 3.3.5 --- PT-PCR and Northern blot analysis of the transgene transcripts --- p.133 / Chapter 3.3.6 --- Functional test of transgenic plants under salt stress --- p.135 / Chapter 4. --- Discussion --- p.139 / Chapter 4.1 --- "Isolation of GmNhx, GmAKTl and GmCLC from Wenfeng7 and Union" --- p.139 / Chapter 4.1.1. --- GmNhx1 and GmNhx2 are putative vacuolar Na+/H+ antiporters from Wenfeng7 and Union --- p.139 / Chapter 4.1.2. --- GmAKT1 is an inward-rectifying K+ channel from Wenfeng7 and Union --- p.141 / Chapter 4.1.3 --- GmCLC is a putative vacuolar voltage-dependent chloride channel from Wenfeng7 and Union --- p.144 / Chapter 4.2 --- "Gene expression profiles of GmNhx, GmAKT1 and GmCLC from Wenfeng7 and Union" --- p.146 / Chapter 4.2.1 --- Differential expression between GmNhx1 and GmNhx2 in Wenfeng7 and Union --- p.146 / Chapter 4.2.2 --- Coordinated expression of GmNhx and GmCLC in wenfeng7 and Union --- p.147 / Chapter 4.2.3 --- GmAKT1 is preferentially expressed in roots of wenfeng7 and Union and presented in low abundance --- p.148 / Chapter 4.3 --- Functional tests of transgenic Arabidopsis plants --- p.150 / Chapter 4.3.1 --- Screening of heterozygous and homozygous transgenic plant --- p.150 / Chapter 4.3.2 --- Function tests of heterozygous and homozygous transgenic plants under salt stress --- p.151 / Chapter 4.3.3 --- Gene silencing in transgenic plants --- p.152 / Chapter 5. --- Conclusion and perspectives --- p.155 / References --- p.157 / "Appendix I: Buffer, restriction and modifying enzymes" --- p.171 / Appendix II: Major chemicals and reagents used in this research --- p.171 / Appendix III: Major common solutions used in this research --- p.174 / Appendix IV: Commercial kits used in this research --- p.177 / Appendix V: Major equipment and facilities used --- p.177