The organization and structure of the leghemoglobin (Lb) genes in soybean have been investigated. Using molecular cloning techniques, a Lb-cDNA recombinant molecule, pLb1, was prepared and characterized. Hybridization of this clone of genomic DNA revealed the presence of at least seven Lb genes in the soybean genome. The arrangement of these genes appeared to be the same in DNA isolated from infected or uninfected tissues. The plasmid pLb1 was also used to isolate three Lb sequences from a genomic library constructed in (lamda) Charon4 vector. Nucleotide sequence analysis showed that one Lb gene, present on an 11.5 kilobases (kb) Eco RI genomic fragment, spans about 1 200 nucleotides and appears to code for Lbc(,3). Its coding sequence is interrupted at amino acid positions 32 to 33, 68 to 69 and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain some consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the recombinant phage containing the 11.5 kb Eco RI fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequences of the second gene showed that this gene is incomplete, containing only two exons. The deduced amino acid sequence of this gene, although showing 78% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present in a second genomic clone containing a 4.2 kb Eco RI fragment revealed a truncated Lb gene showing homology with the last exon and the non-coding region at the 3' end of the two other Lb genes. A very high homology is found among the nucleotide sequence of the Lbc(,3), Lba and Lbc(,1) genes. Comparison of the intervening sequences of these genes indicated that they diverged mainly through the creatio

The nodulin-23 gene is an abundantly transcribed soybean gene induced in nodules during symbiosis with Rhizobium. Sequencing of the cDNA and genomic clones revealed one intron within an open reading frame. A 24,275 dalton protein was predicted. The transcription of nodulin-23 gene occurs concomitantly with Lbc$ sb3$ and nodulin-24 genes. The 5$ sp prime$-regions of nodulin-23 and Lbc$ sb3$ genes were sequenced and compared with that of nodulin-24. Three potential cis-regulatory sequences were identified. The presence of trans-acting molecule(s), possibly regulating the expression of these genes, was tested for in vitro by preincubating nuclei from embryonic axes with nodule extract and assaying for gene activation. Nodulin-23, nodulin-24, and Lbc$ sb3$ genes were specifically activated and demonstrated similar kinetics. Several genes used as controls were not stimulated. A nodule factor(s) was shown to bind the 5$ sp prime$-region of nodulin-23 gene. The corresponding DNA regions from the other two coordinately expressed nodulin genes specifically competed for this binding, whereas other genes did not bind this factor at all.

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by Li Wing Yen Francisca. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 94-105). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgemnets --- p.vii / Abbreviations --- p.ix / Table of contents --- p.xii / List of figures --- p.xvi / List of tables --- p.xvii / Chapter 1. --- General Introduction / Chapter 1.1 --- Introduction to oxidative stress / Chapter 1.1.1 --- Introduction to Reactive Oxygen Species --- p.1 / Chapter 1.1.2 --- Major sites of ROS production / Chapter 1.1.2.1 --- Chloroplast --- p.4 / Chapter 1.1.2.2 --- Mitochondria --- p.4 / Chapter 1.2 --- Regulation of intercellular ROS content in plant cells / Chapter 1.2.1 --- Enzymatic defense ofROS --- p.6 / Chapter 1.2.1.1 --- Superoxide dismutases --- p.6 / Chapter 1.2.1.2 --- "Ascorbate peroxidase, Glutathione reductase and the Ascorbate-Glutathione cycle" --- p.7 / Chapter 1.2.1.3 --- Catalase --- p.11 / Chapter 1.2.1.4 --- Alternative oxidase --- p.11 / Chapter 1.2.2 --- Non-enzymatic / Chapter 1.2.2.1 --- Ascorbate and Glutathione --- p.12 / Chapter 1.2.2.2 --- α-tocopherol --- p.12 / Chapter 1.3 --- "Salt, dehydration and oxidative stress" / Chapter 1.3.1 --- Oxidative stress is induced when plants were under salt stress --- p.13 / Chapter 1.3.2 --- Oxidative stress is induced when plants were under dehydration stress --- p.14 / Chapter 1.4 --- ROS scavenging: the road to achieve multiple-stress tolerance? --- p.16 / Chapter 1.5 --- Purple acid phosphatase and its relationship with oxidative stress in plants / Chapter 1.5.1 --- General introduction to plants purple acid phosphatase (PAP) --- p.20 / Chapter 1.5.2 --- Purple acid phosphatases that found to be involved in ROS scavenging in plants --- p.21 / Chapter 1.6 --- Previous studies in GmPAP3 --- p.23 / Chapter 1.7 --- Hypothesis and significance of this project --- p.25 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- "Plants, bacterial strains and vectors." --- p.26 / Chapter 2.1.2 --- Chemicals and reagents --- p.27 / Chapter 2.1.3 --- Commercial kits --- p.28 / Chapter 2.1.4 --- Primers and adaptors --- p.29 / Chapter 2.1.5 --- Equipments and facilities used --- p.31 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.31 / Chapter 2.1.7 --- Software --- p.31 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Molecular techniques / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.32 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.32 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5α, DE3 and Agrobacterium competent cells" --- p.33 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.36 / Chapter 2.2.1.5 --- DNA and RNA extraction --- p.37 / Chapter 2.2.1.6 --- Generation of single-stranded DIG-labeled PCR probes --- p.38 / Chapter 2.2.1.7 --- Testing the concentration of DIG-labeled probes --- p.40 / Chapter 2.2.1.8 --- Northern blot analysis --- p.40 / Chapter 2.2.1.9 --- PCR techniques --- p.41 / Chapter 2.2.1.10 --- Sequencing --- p.42 / Chapter 2.2.2 --- Plant cell culture and transformation / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.43 / Chapter 2.2.2.2 --- Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells --- p.44 / Chapter 2.2.3 --- Growth and treatment conditions for plants / Chapter 2.2.3.1 --- Growth and salt treatment condition of soybean samples for gene expression studies of GmPAPS --- p.45 / Chapter 2.2.3.2 --- Root assay of GmPAP3 transgenic Arabidopsis thaliana --- p.46 / Chapter 2.2.4 --- "Immunolabeling, mitochondria integrity, ROS detection and confocal microscopy" / Chapter 2.2.4.1 --- Immunolabeling of GmPAP3-T7 transgenic cell lines --- p.47 / Chapter 2.2.4.2 --- Mitochondria integrity --- p.48 / Chapter 2.2.4.3 --- Detection of Reactive oxygen species (ROS) --- p.48 / Chapter 2.2.4.4 --- Confocal microscopy --- p.49 / Chapter 2.2.4.5 --- Images processing and analysis --- p.49 / Chapter 2.2.5 --- Statistical analysis --- p.50 / Chapter 3. --- Results / Chapter 3.1 --- "Expression of GmPAP3 was induced by NaCl stress, oxidative stress, and dehydration stress" --- p.51 / Chapter 3.2 --- Establishment of GmPAP3-T7 fusion transgenic cell lines / Chapter 3.2.1 --- Subcloning of GmPAP3-T7 into the binary vector system W104 --- p.53 / Chapter 3.2.2 --- Transformation of W104-GmPAP3-T7 into tobacco BY-2 cells --- p.56 / Chapter 3.3 --- Establishment of GmPAP3 trangenic cell lines / Chapter 3.3.1 --- Subcloning of GmPAP3 into the binary vector system W104 --- p.58 / Chapter 3.3.2 --- Transformation of W104-GmPAP3 into tobacco BY-2 cells --- p.58 / Chapter 3.4 --- Establishment of GmPAP3 transgenic Arabidopsis thaliana / Chapter 3.4.1 --- Transformation of W104-GmPAP3 into Arabidopsis thaliana --- p.61 / Chapter 3.5 --- Colocalization of GmPAP3 with MitoTracker-orange --- p.66 / Chapter 3.6 --- Effect of expressing GmPAP 3 on mitochondria integrity of BY-2 cells under NaCl and dehydration stress. --- p.71 / Chapter 3.7 --- Effect of expressing GmPAP3 on ROS production in BY-2 cells under salt and PEG treatment --- p.75 / Chapter 3.8 --- Effect of expressing GmPAP3 in Arabidopsis thaliana under salt stress --- p.81 / Chapter 4. --- Discussion / Chapter 4.1 --- Gene expression profile of GmPAP3 --- p.83 / Chapter 4.2 --- Subcellular localization of GmPAP3 --- p.84 / Chapter 4.3 --- Functional tests of GmPAP 3 transgenic BY-2 cells / Chapter 4.3.1 --- GmPAP3 could protect the plant cells' mitochondria integrity when under salt and dehydration stress --- p.86 / Chapter 4.3.2 --- Expressing GmPAPS in tobacco BY-2 cells were able to reduce the production ofROS under salt and dehydration stresses --- p.88 / Chapter 4.4 --- Functional tests of GmPAP3 transgenic Arabidopsis --- p.91 / Chapter 5. --- Conclusion and perspectives --- p.92 / References --- p.94 / Appendix I: Restriction and modifying enzymes --- p.106 / Appendix II: Chemicals --- p.107 / Appendix III: Commercial kits --- p.111 / Appendix IV: Equipments and facilities used --- p.112 / "Appendix V: Buffer, solution, gel and medium formulation" --- p.113

High external Pi could reduce the fold of induction of GmSOS1 and GmCNGC by salinity stress, while posses no effect on other gene candidates. The possible effects on the repression of GmSOS1 and GmCNGC by high external Pi were discussed according to the current understandings on their roles in the salt stress responses. / In this study, phenotypical, physiological, cellular and molecular investigations were carried out to delineate the interactive effects of salinity and external Pi in "Pi tolerant" soybeans. The ultimate goals are to provide essential scientific background for practicing soybean cultivation in saline lands and to explore the possibility to improve the salt tolerance together with P-deficiency tolerance of soybeans. / It was found that high external Pi could reduce the salt tolerance capability of 15 "Pi tolerant" soybean germplasms. Such detrimental effect was common among soybeans, regardless of the type (cultivated versus wild), the salt tolerant capability in optimum Pi level, and the sensitivity to Pi level (Pi tolerant versus Pi sensitive). / Salinity is a major abiotic stress significantly reducing crop yield. Moreover, high salinity in soil is usually accompanied with deficiency of available phosphorus (P). Supplementation of inorganic phosphate (Pi) could be an agricultural strategy to enhance crop production on saline lands. However, ionic components in soil often interact to each other to affect the final growth performance of plants. / Soybean is an important crop that is sensitive to both high salinity and P deficiency in soil. Based mainly on the studies using "Pi sensitive" soybean cultivars, physiological investigations concluded that high external Pi could reduce the salt tolerance via excessive accumulation of P and chloride in the foliar tissues. "Pi tolerant" and "Pi sensitive" are relative terms to describe the response of a soybean cultivar to 1.6mM Pi when grown in non-saline nutrient solutions. The "Pi sensitive" cultivars developed a reddish-brown discoloration on their leaves and exhibited retarded growth. By contrast, the "Pi tolerant" cultivars thrived under high Pi supplements. / The physiological mechanism underlining such interaction in "Pi tolerant" soybeans was distinct from that in "Pi sensitive" cultivars. At the in planta level, high level of external Pi external Pi diminished when de-rooted plants were used, suggesting that the root is the primary organ interacting with Pi in the growth medium. Two cell models, including soybean suspension cells and the tobacco Bright-Yellow-2 cell line, were also employed to study the effects of high external Pi at the cellular level. Consistent to the results using the whole plant, high external Pi uplifted cellular sodium ion uptake and reduced cell viability under salinity stress. / To identify the possible molecular targets of high external Pi, the expression of 12 gene candidates in roots of "Pi tolerant" soybean was investigated in response to NaCl stress supplemented with 0.2mM Pi or 2mM Pi. The putative functions of these gene candidates are involved in: (a) Na+ and/or K+ transportation (GmSOS1, GmNHX; GmGLR3, GmCNGC, GmNKCC and GmAKT1); (b) regulation of ion homeostasis (GmSAL1, GmCIPK1 and GmSCA1); and (c) energetic system for the operation of ion transporters (GmAHA1, GmVHA-C and GmVP1). / Phang, Tsui Hung. / "June 2008." / Adviser: Lam Hon Ming. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1525. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 132-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2003. / South Africa produces 190 000 tonnes of soybean per annum. Seed producing companies require knowledge of the diversity of the germplasm to produce hybrids that will be competitive in local and overseas markets. Furthermore, they need to ascertain the presence/absence of the anti-nutritional factor, Kunitz trypsin inhibitor protein. Currently, seed producing companies plant the seed and wait for the grow-out in order to select desirable traits. This process is time-consuming, tedious and does not necessarily ensure the selection of the best genetic stability as it is based on phenotypic expression alone. This study was undertaken to evaluate a molecular method to determine the genetic diversity among soybean parent lines and optimize a method which can be used to evaluate seeds for the Kunitz trypsin inhibitor protein / M

Call number: LD2668 .T4 1978 K55 / Master of Science

In effort to breed for daylength-neutral (DNP) soybean germplasms (Glycine max (L.) Merrill), selected longday cultivars (LDP) from Maturity Group 00 were crossed to local adapted shortday cultivars (DP) from Maturity Group VI. In the segregating populations there were many new hybrid combinations, some of which flowered and set pod as early as the Group 00 parents, however, they were larger in size and matured normally. These lines were considered daylength-neutral plants (DNP), and were evaluated in the field nursery in biweekly date of planting experiments from early May to late July during 1978 and 1979. Selected LDP and local adapted SDP cultivars were utilized as check lines. It was observed that LDP cultivars flowered and set pods normally. However, they did not mature normally; the pods ripened and shattered while the stem and leaves remained green and these plants were short and unproductive. Local SDP were the most sensitive plants in response to the change in planting date and daylength. Number of days to flowering, pod setting, and maturity as well as plant heights had decreased sharply in response to the decrease in daylength of later planting dates. In contrast, DNP lines flowered, set pod and matured normally on large vigorous plants in approximately the same period of time regardless of planting date or the daylength during the growing season. To study the inheritance of the daylength-neutral trait in soybeans, crosses were made between DNP lines and local SDP cultivars. These were extremely wide crosses. Segregating populations from these crosses were tested under three light treatments of 12, 18 and 24 hours. Only DNP plants flowered and set pod normally under the long photoperiod treatments of 18 and 24 hours. The magnitude and continuous nature of the frequency distribution of the segregating populations as well as the low heritability estimates of each trait imply that this response is under polygenic control.