No description available.

No description available.

Green microalgae are an important source of natural products such as β-carotene, and have recently become objects of intense study for producing biodiesel and valuable recombinant proteins. Application of chloroplast engineering in microalgae is limited by the availability of tools for genetically engineering the chloroplast of commercially important species. The phytoene desaturase gene of a previously isolated norflurazon tolerant mutant of Chlamydomonas reinhardtii was isolated and sequenced. A thymine to guanine transversion in exon 2 changes codon 131 resulting in a F131V mutation that is located in the NADP binding site domain on the primary structure. This mutation clusters with three conserved amino acids, whose substitution confers norflurazon tolerance in other species, in a pocket on a 3-D structure of the protein. The pocket identifies the target site of norflurazon. The side pocket is at the opening of a tunnel leading to the enzyme's NADP binding site. The mutant gene was cloned and used as marker for glass-bead mediated nuclear transformation of C. reinhardtii using direct selection with 5 μM norflurazon. Integration was by illegitimate recombination and transformants were able to grow in media containing 150 μM norflurazon. Transformants exhibited cross tolerance to fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wildtype. This allows mutant pds gene to act as a dual negative/positive selectable marker that is conditional on the herbicide used. The F131V mutation was introduced into a synthetic gene encoding a Dunaliella salina phytoene desaturase that contained codons used frequently in C. reinhardtii chloroplast genes. The 1.8 kbp CpPDS1 gene was assembled from 74 oligonucleotides by overlap PCR. The coding sequence was inserted into a Dunaliella tertiolecta chloroplast targeting vector that integrated the CpPDS1 sequence into the ycf3-trnL-rbcL region of the plastome. The resulting vector was transformed into D. salina and D. tertiolecta chloroplasts using particle bombardment with plasmid coated gold microprojectiles. Norflurazon tolerant colonies were isolated and the D. salina and D. tertiolecta clones were shown to contain a pds gene integrated in the plastome using PCR analyses. Transformation of the CpPDS1 gene into C. reinhardtii chloroplasts by rescue of an atpB mutation only gave rise to herbicide tolerant colonies if the presequence was removed. Industrial production of algae in large volumes is limited by the availability of light to drive algal growth. This problem was addressed by expressing fluorescent protein Katushka in the chloroplast of C. reinhardtii which converts yellow light to red light. The Katushka gene was transformed into chloroplasts using vector pB10, which was constructed to rescue a deletion in the chloroplast atpB gene in the mutant CC373 strain. The Katushka coding sequence was codon-optimised for expression in chloroplasts and expressed from three different promoter and 5' UTRs (atpA, atpB and psbD). C. reinhardtii wild type cells were able to grow under either blue or red LED lights but grew best when both were present. Wild type cell grew poorly in yellow LED lighting. Cells expressing Katushka in the chloroplast exhibited enhanced autotrophic growth in yellow light and under conditions where yellow light was present and red light was limiting. The improvement in growth was related to the levels of Katushka fluorescence detected in chloroplast transformants, which was highest for the atpA promoter and UTR.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on March 18, 2009. Major professor: Ilhami Yildiz, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture, with specialization in: Agricultural Engineering Technology." "February 2009." Includes bibliographical references (p. 115-122). Also available on microfiche.

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Changes in the nutrient loading to the Ythan estuary over the past twenty-five years were examined by analysing available long-term data sets to attempt to correlate nutrient changes with reported increases in the growth of opportunistic green macroalgae in the estuary. Analysis revealed a three-fold increase in nitrogen and a two-fold increase in phosphorus as a result of increases in agricultural run-off and sewage effluent respectively. Concurrent with these nutrient increases, the peak summer biomass of Enteromorpha on Newburgh South Quay Mudflat increased from around 1kg FW./m2 in the mid 1960's to 3kgFW./m2 in 1985/86, with evidence for further increases in the biomass and distribution of other opportunistic species such as Ulva and Chaetomorpha elsewhere in the estuary. Much of the primary production was found to become incorporated into the sediment, through burial of macroalgae in autumn. The effects of dense accumulations of algae on the underlying macrofauna were investigated by means of a field experiment. Corphium was shown to be intolerant of algal cover, but the abundance of Macoma, Nereis and Capitella increased. Algal cover was also shown to cause sediment anoxia and lead to accretion of silt. A further field experiment was carried out to investigate the processes by which algal cover affected macrofaunal abundance. Both the physical and organic (biological) effects of algal mats were shown to be important in determining invertebrate abundance, mostly as a result of changes in sediment chemistry. A tidal multi-microcosm system was designed and constructed to conduct laboratory based studies, but treatment effects were found to be enhanced compared to field conditions, such that results could not easily be extrapolated to the natural environment. It was recommended that monitoring of water nutrient, concentrations together with algal biomass and distribution, be continued, as changes in macrofaunal abundance under algal mats may create food shortages for certain shorebirds and fish.