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The possible roles of soybean ASN genes in seed protein contents.

Wan Tai Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-111). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xi / Table of Contents --- p.xii / List of Figures --- p.xvi / List of Tables --- p.xvi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Soybeans --- p.1 / Chapter 1.1.1 --- Nutrient composition of soybean --- p.1 / Chapter 1.1.2 --- Nitrogen fixation and assimilation in soybean --- p.3 / Chapter 1.1.3 --- The role in nitrogen allocation and controlling the nitrogen sink-source relationship of asparagine --- p.3 / Chapter 1.1.4 --- Characterization of asparagine synthetase --- p.8 / Chapter 1.1.4.1 --- Biochemistry and molecular background of plant asparagine synthetase --- p.8 / Chapter 1.1.4.2 --- Asparagine synthetase in Arabadopsis thaliana --- p.9 / Chapter 1.1.4.3 --- "Asparagine synthesis in soybean, Glycine max" --- p.10 / Chapter 1.1.4.4 --- "Asparagine synthetase in rice, Oryza sativa" --- p.11 / Chapter 1.2 --- Seed protein quality and quantity improvement --- p.13 / Chapter 1.2.1 --- Nutrition composition of rice --- p.13 / Chapter 1.2.2 --- Molecular approaches for improving seed storage protein quality --- p.14 / Chapter 1.2.2.1 --- Protein sequence modification --- p.14 / Chapter 1.2.2.2 --- Synthetic genes --- p.16 / Chapter 1.2.2.3 --- Overexpression of homologous genes --- p.17 / Chapter 1.2.2.4 --- Transfer and expression of heterologous genes --- p.18 / Chapter 1.2.2.5 --- "Manipulation of pathway synthesizing essential amino acids, aspartate family amino acid" --- p.19 / Chapter 1.2.3 --- Research in improving rice seed protein quality and quantity --- p.22 / Chapter 1.3 --- Hypothesis and objective of this study --- p.23 / Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Plant materials --- p.25 / Chapter 2.1.2 --- Bacterial strains and vectors --- p.26 / Chapter 2.1.3 --- Growth conditions for soybean --- p.26 / Chapter 2.1.4 --- Chemicals and reagents --- p.26 / Chapter 2.1.5 --- "Buffer, solution and gel" --- p.26 / Chapter 2.1.6 --- Commercial kits --- p.27 / Chapter 2.1.7 --- Equipments and facilities used --- p.27 / Chapter 2.1.8 --- Primers --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Growth condition for plant materials --- p.28 / Chapter 2.2.1.1 --- General conditions for planting soybean --- p.28 / Chapter 2.2.1.2 --- Soybean seedlings for gene expression profile analysis --- p.28 / Chapter 2.2.1.3 --- Mature soybean for gene expression profile analysis --- p.29 / Chapter 2.2.1.4 --- Mature soybean for cloning of AS I and AS2 full length cDNA --- p.30 / Chapter 2.2.1.5 --- Mature soybean seed for amino acid profile analysis --- p.30 / Chapter 2.2.1.6 --- General conditions for planting transgenic rice in CUHK --- p.30 / Chapter 2.2.1.7 --- Transgenic rice seedling for PCR screening --- p.31 / Chapter 2.2.1.8 --- Transgenic rice for functional test and seed for biochemical analysis --- p.31 / Chapter 2.2.2 --- Molecular techniques --- p.32 / Chapter 2.2.2.1 --- Total RNA extraction --- p.32 / Chapter 2.2.2.2 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.2.2.3 --- Northern blot analysis --- p.33 / Chapter 2.2.2.3.1 --- Chemiluminescent detection --- p.33 / Chapter 2.2.2.3.2 --- Film development --- p.34 / Chapter 2.2.2.4 --- Preparation of single-stranded DIG-labeled PCR probes --- p.34 / Chapter 2.2.2.4.1 --- Primer design for the PCR probes of --- p.34 / Chapter 2.2.2.4.2 --- Amplification of AS1 and AS2 internal PCR fragments --- p.34 / Chapter 2.2.2.4.3 --- Quantitation of purified AS1 and AS2 PCR fragments --- p.35 / Chapter 2.2.2.4.4 --- Biased PCR to make single-stranded DNA probes --- p.35 / Chapter 2.2.2.4.5 --- Probe quantitation --- p.36 / Chapter 2.2.2.5 --- Probe specificity test --- p.37 / Chapter 2.2.2.6 --- Cloning of full length cDNA --- p.37 / Chapter 2.2.2.6.1 --- First strand cDNA synthesis from RNA of high protein content soybean leaf --- p.37 / Chapter 2.2.2.6.2 --- PCR for amplification of AS1 and AS2 full length cDNA --- p.38 / Chapter 2.2.2.6.3 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.38 / Chapter 2.2.2.6.4 --- Ligation of DNA inserts into pBluescript II KS(+) T-vector --- p.39 / Chapter 2.2.2.6.5 --- Preparation of E. coli DH5α CaCl2-mediaed competent cells --- p.39 / Chapter 2.2.2.6.6 --- Transformation of E. coli DH5α competent cell --- p.40 / Chapter 2.2.2.7 --- Screening of recombinant plasmids --- p.40 / Chapter 2.2.2.7.1 --- Isolation of recombinant plasimid DNA from bacterial cells --- p.41 / Chapter 2.2.2.7.2 --- PCR screening on recombinant plasmids --- p.41 / Chapter 2.2.2.7.3 --- DNA gel electrophoresis --- p.41 / Chapter 2.2.2.8 --- Sequencing and homology search --- p.42 / Chapter 2.2.2.9 --- Functional test using transgenic plant --- p.43 / Chapter 2.2.2.9.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.43 / Chapter 2.2.2.9.2 --- Agrobacterium mediated transformation into rice calli to regenerate transgenic AS1/ AS2 rice --- p.44 / Chapter 2.2.2.10 --- PCR Screenig of homozygous and heterozygous transgenic plants --- p.44 / Chapter 2.2.2.10.1 --- Isolation of genomic DNA from transgenic plants --- p.45 / Chapter 2.2.2.10.2 --- PCR screening using genomic DNA --- p.46 / Chapter 2.2.2.11 --- Quantitative PCR analysis on transgenic plants --- p.48 / Chapter 2.2.3 --- Biochemical Analysis --- p.49 / Chapter 2.2.3.1 --- Quantitative amino acid analysis in mature soybean seeds --- p.49 / Chapter 2.2.3.2 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.49 / Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Amino acid analysis on mature soybean seeds --- p.50 / Chapter 3.2 --- Expression pattern analysis of AS genes by Northern Blot analysis --- p.54 / Chapter 3.2.1 --- Making of single strand digoxigenin (DIG)-labeled probe --- p.54 / Chapter 3.2.2 --- Probe specificity --- p.57 / Chapter 3.2.3 --- AS expression level under light/dark treatments by Northern Blot analysis --- p.58 / Chapter 3.2.4 --- AS expression level in young seedlings by Northern Blot analysis --- p.62 / Chapter 3.2.5 --- AS expression level in podding soybean by Northern Blot analysis --- p.64 / Chapter 3.3 --- Cloning of AS genes from high protein content soybeans --- p.66 / Chapter 3.3.1 --- "PCR amplification of AS1 and AS2 full length cDNA from the first-strand cDNA of high portein content cultivar soybean, YuDoul2" --- p.66 / Chapter 3.3.2 --- Nucleotide sequences analysis of AS1 and AS2 full-length cDNA clones --- p.68 / Chapter 3.4 --- Construction of AS1 and AS2 transgenic rice --- p.75 / Chapter 3.4.1 --- Construction of AS1 and AS2 constructs --- p.75 / Chapter 3.4.2 --- Transformation of chimeric gene constructs into Agrobacterium tumefaciens --- p.75 / Chapter 3.4.3 --- Agrobacterium mediated transformation into Oryza sativa calli to regenerate transgenic rice --- p.76 / Chapter 3.4.4 --- PCR screening of transgene from transgenic AS1 and AS2 rice --- p.76 / Chapter 3.4.5 --- Quantitative PCR analysis of the transgene expression --- p.81 / Chapter 3.4.6 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.83 / Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- The role of asparagine and asparagine synthetase in nitrogen assimilation and sink-source relationship in soybean --- p.89 / Chapter 4.2 --- Comparative study of AS between different high seed protein content crops --- p.92 / Chapter 4.3 --- The attempt to find out the reason for the strong AS1 expression detected in high protein soybean cultivars --- p.92 / Chapter 4.4 --- Other factors affecting seed protein contents --- p.93 / Chapter 4.5 --- Rice seed quality improvement by nitrogen assimilation enhancement --- p.94 / Chapter 4.6 --- Comparative study of amino acid profile and seed total protein in other transgenic rice --- p.95 / Chapter 4.7 --- Possible reason of higher seed protein content in AS2 transgenic rice --- p.96 / Chapter 4.8 --- Selectable marker --- p.97 / Chapter 5 --- Conclusion and Prespectives --- p.99 / Chapter 6 --- References --- p.102 / Chapter 7 --- Appendix --- p.112 / Appendix I: Major chemicals and reagents used in this research --- p.112 / "Appendix II: Major buffer, solution and gel used in this research" --- p.114 / Appendix III: Commercial kits used in this research --- p.117 / Appendix IV: Major equipments and facilities used in this research --- p.118 / Appendix V: Primer list --- p.119

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325880
Date January 2006
ContributorsWan, Tai Fung., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 120 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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