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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Cloning and characterization of ion transporter genes from a salt-tolerant soybean variety.

January 2004 (has links)
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

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