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

This research investigates the protein interactions that occur when soy protein is added to milk and subjected to renneting or heating. Milk was fortified with 20% soy protein and enzymic coagulation studied at 35°C at various pH's and CaCl2 levels. The first part deals with the interaction between milk and soy proteins during rennet-induced milk coagulation. The first goal was to determine how soy proteins affected milk coagulation. The effects of native versus heat-denatured soy proteins on rennet coagulation time and curd firmness were compared. lmmunogold labeling along with transmission electron microscopy was used to identify and localfze soy proteins in coagulated milk. Partitioning of ß-conglycinin and glycinin, the two main soy protein fractions, between cheese and whey was determined by electrophoresis. Soy proteins affected milk coagulation to the greatest extent at pH 6.6. Both heat-denatured and native soy proteins increased rennet coagulation time. Only heat-denatured soy proteins affected final curd firmness. Most of ß-conglycinin was lost in whey, whereas glycinin was retained in curd. Soy proteins existed in the curd as aggregates that were less electron dense than casein micelles. At pH 6.6, heat-denatured soy proteins were fibrous and adhered to the surfaces of casein micelle, preventing direct micelle-micelle contact. This would delay aggregation rate and decrease curd firmness by decreasing the number and strength of links between casein micelles. Native soy proteins did not bind to the casein micelles but rather were physically trapped within curd. Their effect of delaying aggregation is thought to be a function of their binding of calcium. Adding CaCl2 or lowering the pH to 6.3 or 6.0 helped restore coagulation properties. The second goal was to determine what heat-induced interaction occurs between milk and soy proteins, specifically between κ-casein and glycinin. Both κ-casein and glycinin are heat labile and form insoluble aggregates when heated. When glycinin and κ-casein were heated together, some acidic polypeptides of glycinin crosslinked with κ-casein via disulfide linkages. However, when disulfide linkage was prevented by adding ß-mercaptoethanol , non-covalent interactions between κ-casein and both acidic and basic polypeptides of glycinin occurred that prevented the heat precipitation of glycinin. This non-covalent interaction between glycinin polypeptides and κ-casein may explain why the heat-treated soy proteins became attached to the surfaces of casein micelles during rennet coagulation of milk.

No description available.

A novel approach for the production of soy protein isolates was investigated integrating electroacidification and membrane ultrafiltration. The effect of electroacidification on the ultrafiltration performance and physicochemical properties of the soy protein extracts was obtained by comparing an electroacidified (pH 6) and a non-electroacidified (pH 9) soy protein extract. The effect of membrane fouling on the permeate flux decline was studied in a hollow fiber and a dead end ultrafiltration system. Due to more significant membrane fouling, the permeate flux was always lower for the electroacidified extract, resulting in at least 1.5-fold increase in the total fouling resistance compared to the non-electroacidified extract. The total amount of protein deposited on the membrane surface during unstirred dead-end ultrafiltration was comparable (about 7 mg/cm2) for both soy protein extracts. The discrepancy between the total fouling resistance and the protein deposition estimates was attributed to the formation of denser (less permeable) fouling deposit for the electroacidified extract, which was supported by scanning electron microscopy studies of fouled membranes. The removal of carbohydrates and minerals was evaluated for direct ultrafiltration and two-stage discontinuous diafiltration using a hollow fiber system. The carbohydrate removal results were always consistent with the theoretical predictions, indicating that the carbohydrates were freely permeable across the membrane. In contrast, the minerals were partially retained by the membrane, but to a higher extent for the non-electroacidified extract, which demonstrated that the electroacidification pretreatment enhanced the mineral removal during the ultrafiltration. Incorporation of the diafiltration step improved the ash (mineral) and carbohydrate removal. Stronger electrostatic interactions between soy proteins, calcium/magnesium, and phytic acid (antinutrient) at alkaline pH resulted in less efficient removal of calcium, magnesium, and phytic acid during the ultrafiltration of the non-electroacidified extract compared to the electroacidified extract. Consequently, the soy protein isolates produced by electroacidification and the hollow fiber ultrafiltration had a lower mineral and phytic acid content. The protein content was at least 88 % (dry basis), with or without the electroacidification pretreatment. The study of the viscosity revealed that the electroacidification pretreatment reduced the viscosity of the soy protein extract, which resulted in a lower axial pressure drop increase during the ultrafiltration of the electroacidified extract compared to the non-electroacidified extract. Adjusting the pH of the electroacidified extract to 9 and the pH of the non-electroacidified extract to 6 had a great impact on the particle size distribution but only a marginal effect on the viscosity of the pH adjusted extracts. This indicated that the pH and the particle size distribution were not responsible for the viscosity difference between the electroacidified and the non-electroacidified soy protein extracts. It was proposed that the electroacidification pretreatment had some impact on the water hydration capacity of the soy proteins, which consequently affected the viscosity.

A novel approach for the production of soy protein isolates was investigated integrating electroacidification and membrane ultrafiltration. The effect of electroacidification on the ultrafiltration performance and physicochemical properties of the soy protein extracts was obtained by comparing an electroacidified (pH 6) and a non-electroacidified (pH 9) soy protein extract. The effect of membrane fouling on the permeate flux decline was studied in a hollow fiber and a dead end ultrafiltration system. Due to more significant membrane fouling, the permeate flux was always lower for the electroacidified extract, resulting in at least 1.5-fold increase in the total fouling resistance compared to the non-electroacidified extract. The total amount of protein deposited on the membrane surface during unstirred dead-end ultrafiltration was comparable (about 7 mg/cm2) for both soy protein extracts. The discrepancy between the total fouling resistance and the protein deposition estimates was attributed to the formation of denser (less permeable) fouling deposit for the electroacidified extract, which was supported by scanning electron microscopy studies of fouled membranes. The removal of carbohydrates and minerals was evaluated for direct ultrafiltration and two-stage discontinuous diafiltration using a hollow fiber system. The carbohydrate removal results were always consistent with the theoretical predictions, indicating that the carbohydrates were freely permeable across the membrane. In contrast, the minerals were partially retained by the membrane, but to a higher extent for the non-electroacidified extract, which demonstrated that the electroacidification pretreatment enhanced the mineral removal during the ultrafiltration. Incorporation of the diafiltration step improved the ash (mineral) and carbohydrate removal. Stronger electrostatic interactions between soy proteins, calcium/magnesium, and phytic acid (antinutrient) at alkaline pH resulted in less efficient removal of calcium, magnesium, and phytic acid during the ultrafiltration of the non-electroacidified extract compared to the electroacidified extract. Consequently, the soy protein isolates produced by electroacidification and the hollow fiber ultrafiltration had a lower mineral and phytic acid content. The protein content was at least 88 % (dry basis), with or without the electroacidification pretreatment. The study of the viscosity revealed that the electroacidification pretreatment reduced the viscosity of the soy protein extract, which resulted in a lower axial pressure drop increase during the ultrafiltration of the electroacidified extract compared to the non-electroacidified extract. Adjusting the pH of the electroacidified extract to 9 and the pH of the non-electroacidified extract to 6 had a great impact on the particle size distribution but only a marginal effect on the viscosity of the pH adjusted extracts. This indicated that the pH and the particle size distribution were not responsible for the viscosity difference between the electroacidified and the non-electroacidified soy protein extracts. It was proposed that the electroacidification pretreatment had some impact on the water hydration capacity of the soy proteins, which consequently affected the viscosity.

The effects of feeding soy protein (SP) or SP plus isoflavones (IF) (150 and 400 mg IF/kg diet) on bone metabolism were assessed in female weanling and retired breeder (RB) rats fed low calcium (Ca) for five weeks. Young rats fed SP-based diets had significantly smaller reductions in bone mineral density (BMD) and bone mineral content (BMC) as a result of the low Ca diet compared to those fed casein-based diets. Added IFs had no further benefits. Soy protein also affected bone metabolism in both the young and RB rats as indicated by markers of bone resorption. Neither the SP nor the added IFs had any effects on BMD or BMC in the RB rats. Feeding SP to young rats resulted in beneficial changes in BMD, BMC, and biochemical markers of bone metabolism. This study indicates that SP positively affects bone metabolism and minimizes the negative effects associated with low Ca intakes in young rats.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 151-159). Also available on the Internet.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Aug 16.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 29, 2007) Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 151-159). Also available on the Internet.