Microalgae are a large and diverse group of photosynthetic organisms ranging from prokaryotic cyanobacteria to eukaryotic algae spread across many phyla. Traditional algal biotechnology approaches have focused on growing algae in monoculture, in contrast to nature, where algae live in association with many other organisms. One association of interest is between the bacterium Mesorhizobium loti (Rhizobiales) and the green alga, Lobomonas rostrata deficient in the production of vitamin B12. The alga provides fixed carbon to the bacterium whereas the bacterium supplies vitamin B12 to the alga. In the course of a screen for bacterial mutants altered in the interaction, a novel symbiosis was serendipitously identified involving the non-Rhizobiales bacterium Rhodococcus erythropolis and L. rostrata. This novel interaction, together with interaction of the more industrially relevant Chlamydomonas reinhardtii strain was characterized. Nitrogen is a major limiting nutrient for industrial scale algal production. An alternative option to the Haber-Bosch process of synthesising and supplementing fixed nitrogen into media is to utilise nitrogen fixing bacteria capable of secreting fixed nitrogen into the media otherwise known as biofertilisation. Anabaena sp. PCC 7120 is a filamentous cyanobacteria that can fix its own nitrogen and engineered strains capable of releasing fixed nitrogen in the form of amino acids and ammonium were cultured with the industrially relevant Chlamydomonas reinhardtii metE—+ M. loti consortium and Chlorella vulgaris in nitrogen-free media. There are relatively few published studies investigating and outlining the challenges involved in scaling algae production from the laboratory through to pilot scale. Furthermore, these studies have typically focused on growing axenic cultures. The B12-dependent strain of C. reinhardtii metE— was grown in the presence of supplemented B12 and B12 producing M. loti at lab scale (50 mL), pre-pilot scale (10 L) and pilot scale (60 L). The growth efficiency as determined by growth rate, was measured and compared for both cultures at all scales.

Yu, Wai Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / 摘要 --- p.vi / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- The importance of rice --- p.3 / Chapter 2.2 --- Limitation of essential amino acids in rice --- p.4 / Chapter 2.3 --- Lysine biosynthetic pathway --- p.6 / Chapter 2.3.1 --- The biosynthesis of aspartate --- p.6 / Chapter 2.3.2 --- Aspartate family pathway --- p.3 / Chapter 2.3.2.1 --- Aspartate kinase (AK) --- p.10 / Chapter 2.3.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.12 / Chapter 2.3.2.3 --- Other enzymes --- p.14 / Chapter 2.4 --- Regulation of lysine content in plant --- p.15 / Chapter 2.5 --- Enhancement of lysine content in plants --- p.16 / Chapter 2.5.1 --- "Breeding, selection and naturally occuring muatnts" --- p.17 / Chapter 2.5.2 --- Induced biochemical mutants --- p.18 / Chapter 2.5.3 --- Transgenic plants --- p.19 / Chapter 2.6 --- Hypothesis --- p.24 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.25 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Chemicals --- p.25 / Chapter 3.3 --- Bacterial strains --- p.25 / Chapter 3.4 --- Cloning of AK and DHPS cDNAs --- p.25 / Chapter 3.4.1 --- Plant materials --- p.25 / Chapter 3.4.2 --- RNA extraction --- p.26 / Chapter 3.4.3 --- RT-PCR amplification of AK and DHPS cDNAs --- p.26 / Chapter 3.4.4 --- Sequence modification of AK and DHPS cDNAs --- p.27 / Chapter 3.4.5 --- DNA sequencing of AK and DHPS cDNAs --- p.32 / Chapter 3.5 --- Chimeric gene construction for rice transformation --- p.32 / Chapter 3.5.1 --- Plasmid and genetic material --- p.32 / Chapter 3.5.2 --- Construction of chimeric genes with seed-specific promoter --- p.35 / Chapter 3.5.3 --- Sequence fidelity of chimeric genes --- p.37 / Chapter 3.6 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.37 / Chapter 3.7 --- Rice transformation --- p.38 / Chapter 3.7.1 --- Plant materials --- p.38 / Chapter 3.7.2 --- Preparation of agrobacterium --- p.33 / Chapter 3.7.3 --- Agrobacterium-mediated rice transformation --- p.39 / Chapter 3.7.3.1 --- Callus induction from mature rice seed embryos --- p.39 / Chapter 7.3.2 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.39 / Chapter 3.8 --- Analysis of transgenic expression --- p.41 / Chapter 3.8.1 --- Genomic DNA extraction --- p.41 / Chapter 3.8.2 --- Total RNA extraction --- p.41 / Chapter 3.8.3 --- Synthesis of DIG-labeled DNA probe --- p.42 / Chapter 3.8.4 --- Southern blot analysis --- p.43 / Chapter 3.8.5 --- Northern blot analysis --- p.43 / Chapter 3.8.6 --- Extraction of rice seed protein --- p.43 / Chapter 3.8.7 --- Tricine SDS-PAGE --- p.44 / Chapter 3.8.8 --- Raising AK and DHPS antibody --- p.44 / Chapter 3.8.9 --- Western blot analysis --- p.46 / Chapter 3.9 --- Free amino acid analysis --- p.46 / Chapter CHAPTER 4. --- RESULTS --- p.48 / Chapter 4.1 --- Cloning of AK and DHPS cDNAs from rice --- p.48 / Chapter 4.1.1 --- RNA extraction and cDNAs amplification --- p.43 / Chapter 4.1.2 --- Sequencing of AK and DHPS cDNAs --- p.50 / Chapter 4.2 --- Sequence modification of AK and DHPS cDNAs --- p.50 / Chapter 4.3 --- Construction of chimeric genes --- p.50 / Chapter 4.4 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.56 / Chapter 4.5 --- Rice transformation --- p.58 / Chapter 4.6 --- Detection of target genes in transgenic rice lines --- p.60 / Chapter 4.6.1 --- PCR of genomic DNA --- p.60 / Chapter 4.6.2 --- Southern blot analysis --- p.63 / Chapter 4.7 --- Northern blot analysis --- p.65 / Chapter 4.8 --- Western blot analysis of AK and DHPS proteins --- p.66 / Chapter 4.9 --- Free amino acid analysis --- p.68 / Chapter 4.9.1 --- Free lysine content --- p.68 / Chapter 4.9.2 --- Changes in other amino acids --- p.69 / Chapter CHAPTER 5. --- DISCUSSION --- p.82 / Chapter 5.1 --- Cloning and modification of AK and DHPS cDNAs --- p.82 / Chapter 5.2 --- Seed-specific expression of modified AK and DHPS in rice --- p.82 / Chapter 5.3 --- Free amino acid changes in transgenic rice lines --- p.83 / Chapter 5.4 --- Future perspectives --- p.85 / Chapter CHAPTER 6. --- CONCLUSION --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.102

The zebrafish is rapidly becoming a vital tool in studies of genetic disease. Use of the zebrafish embryo as an experimental model combines the efficiency of techniques specific to invertebrates with the human applicability of vertebrate studies, along with a number of other advantages such as optical transparency and high spawn number. Sequencing maps and mutant screen data are available, and gene ontology annotation is progressing. Furthermore, a number of highly important projects are underway to expand the utility of the zebrafish still further (eg. Mutant screens and TILLING projects; see (Lieschke and Currie, 2007) for review). As such the zebrafish has become a vital model organism for study of a variety of genetic defects, toxicology and pharmacological screens etc. These papers trace the development of zebrafish embryos as a model organism for both genetic disease and, as part of this, the development of a relatively high throughput approach to analysing relative levels of apoptosis. The first paper describes the fmr1 gene family in zebrafish (fmr1, and its orthologs fxr1 and fxr2). This paper includes a phylogenetic analysis of the gene family that demonstrates the high conservation between human and zebrafish, in the context of Drosophila. We then describe expression of the genes in the embryo (using in situ hybridisation) and adult (using real time pcr). The conclusions are that the zebrafish is an appropriate model in which to study Fragile X Mental Retardation genetic disease. The second paper builds upon this conclusion and further establishes the appropriateness of the model by recapitulating elements of the disease that had already been modelled in other model organisms. The research is validated using a number of controls. We describe a number of original findings that extended the body of knowledge regarding pharmacological rescue of the FMRP loss phenotypes. A craniofacial phenotype is identified, the first such discovery in a model of Fragile X syndrome. These findings are a vital step toward understanding the pathway from gene, to molecular phenotype, to cellular morphology, to gross morphology. As part of these studies, we found it necessary to analyse apoptosis. The technique developed to facilitate this analysis is described in our third paper. Given the highly stochastic nature of the apoptotic patterns we developed a method to take full advantage of the characteristics of zebrafish embryos, primarily their transparency and availability in large numbers. As the zebrafish becomes more widely accepted as a model for a diverse range of scientific questions, the development of such a technique is doubly important given the necessity of a cheap, reliable and simple generalizable method of analysing processes affecting cell viability in fish. This has clear importance for pharmacological studies, but is also a long overdue addition to the battery of controls available for highly invasive techniques such as microinjection, in which apoptosis is regularly found among its non specific effects. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311173 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

<p>Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study.</p>

The production of barley (Hordeum vulgare L.) haploids by crossing with H. bulbosum is a widely used tool in breeding and genetics. Certain barley genotypes have low seed set in this interspecific cross, a phenomenon known as partial incompatibility. Haploid production efficiency and gamete sampling are important issues with the bulbosum technique, particularly when partially incompatible genotypes are used. An in vitro floret culture system was developed that substantially increases haploid production efficiency by optimizing caryopsis growth, haploid embryo development, and plant regeneration. The individual and combined effects of three plant growth regulators (2,4-D, GA₃ and kinetin) on haploid production efficiency and its determinants were compared in the floret culture system. 2,4-D alone was superior to GA₃ alone in haploid production efficiency. 2,4-D alone or kinetin + 2,4-D are recommended for the purpose of haploid production in floret culture using the bulbosum method. Partial incompatibility between H. vulgare and H. bulbosum was studied by doubled haploid progeny analysis. Two different loci were hypothesized to account for the inheritance of partial incompatibility in the crosses of Vada x Klages, Harrington x Klages, and Vada x Harrington. The partial incompatibility gene in Harrington was found to be recessive. The dominant nature of the partial incompatibility gene (Inc) in Vada was confirmed. An association between the (Inc) gene and a deficiency in a stigma/stylodium-specific high pl protein was found in the cosegregation analysis of doubled haploid progeny. The Inc gene may be linked to the gene coding for the stigma/stylodium-specific protein, or the Inc gene may regulate expression of the protein-encoding locus. Segregation analysis of Mendelian markers in doubled haploid progeny showed that there is no evidence that the partial incompatibility status of the parents has an effect on gamete sampling by the bulbosum technique. / Graduation date: 1991

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 89-102).

published_or_final_version / Molecular Biology / Master / Master of Philosophy

Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry. / The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.

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