La caracterització funcional de dos gens en la peridermis, la ω hidroxilasa d'àcids grassos CYP86A33 -candidata per la funcionalització del carboni ω-terminal dels monòmers alifàtics de la suberina- i la ketoacyl-CoA sintasa StKCS6 -candidata per elongar àcids grassos o derivats llargs de suberina i ceres- es realitza per silenciament per RNA d'interferència en patata. La deficiència de CYP86A33 comporta una gran reducció dels monòmers principals de la suberina, l'àcid gras ω-hidroxilat i l'α,ω-diàcid C18:1, juntament amb una reducció total de la quantitat de suberina del 60%. Aquesta deficiència altera l'estructura lamel·lar típica de la suberina, així com també la funció barrera de la peridermis. La deficiència en StKCS6 comporta que els monòmers de la suberina de 28 carbonis o més llargs es redueixin i que els de 26 carbonis o més curts s'incrementin. Aquesta deficiència suggereix que la llargada dels compostos alifàtics pot contribuir a les propietats impermeabilitzants de la peridermis. / The functional characterization of two genes in the periderm, the ω-hydroxylase CYP86A33 -candidate for the functionalization of the ω-terminal carbon of suberin aliphatic compounds- and the putative ketoacyl-CoA synthase StKCS6 -candidate for the elongation of VLCFA and derivatives of suberin and waxes of periderm- is performed by RNA interference-mediated silencing in potato The CYP86A33 deficiency leads to a great reduction of the main suberin monomers, the C18:1 ω-hydroxyacid and α,ω-diacid, together with an overall decrease of the suberin total amount by 60%. The deficiency in these ω-oxidized fatty acids alters the typical suberin lamellar structure as well as the periderm water barrier function.StKCS6 deficiency leads to a decrease of suberin and wax compounds with chain-length C28 and higher and an increase of those with chain-length C26 and lower. This deficiency suggests that the aliphatics chain-length can contribute to the sealing properties of periderm.

Il existe chez D. melanogaster et D. simulans un polymorphisme des hydrocarbures mâles qui pourrait jouer un rôle dans la mise en place d’un isolement sexuel. Le 7-tricosène (7-T; C23:1) et le 7-pentacosène (7-P; C25:1) sont les phéromones principales des mâles. Elles interviennent dans le comportement de cour et varient en fonction des paramètres géoclimatiques. Cette thèse a pour objectif d'étudier le rôle des phéromones mâles sur l’isolement sexuel ainsi que les changements génétiques à l’origine de leur variation et de leur évolution. Un des volets de cette thèse porte sur des aspects physiologiques et comportementaux. Nous avons étudié l'impact de la température sur la synthèse des hydrocarbures mâles et l'influence du rapport 7-T/7-P sur la résistance à la dessiccation et sur la réceptivité des femelles de différentes populations de D. melanogaster et D. simulans. Nous avons observé un isolement sexuel significatif entre des souches 7-T et 7-P de ces deux espèces présentes au laboratoire depuis des décennies, ainsi qu'entre des lignées issues d'une même population soumises à une sélection artificielle. Il apparaît également que les souches synthétisant de grandes quantités de 7-P s’adaptent plus rapidement aux modifications importantes de température. La seconde partie de cette thèse consiste en la détermination et l'étude des gènes d’élongase pouvant être impliqués dans la synthèse du 7-T et du 7-P chez les mâles de D. melanogaster et dans une moindre mesure chez ceux de D. simulans. Les travaux réalisés nous ont permis de mettre en évidence qu’un gène, situé sur le chromosome II, joue un rôle majoritaire dans la synthèse du 7-P. / In D. melanogaster and D. simulans there is a male hydrocarbon polymorphism that may play a role in sexual isolation. The main male pheromones are the 7-tricosene (7-T C23: 1) and the 7-pentacosene (7-P, C25: 1). They are involved in courtship behavior and depend on geo-climatic parameters. The aim of this thesis is to study the role of male pheromones on sexual isolation and to better understand the genetic changes responsible for the variation and the evolution of male pheromones. The first part investigates the plasticity of CHCs in response to temperature and focuses on the role of 7-T and 7-P in resistance to desiccation and in sexual selection in D. melanogaster and D. simulans. In both species there was partial sexual isolation between 7-T and 7-P flies from wild-type laboratory strains and also from lines that have been artificially selected. Males with high levels of 7-P seemed to modify their CHCs profiles more quickly in response to temperature changes. The second part is the identification of elongase genes that might be involved in the synthesis of 7-T and 7-P in D. melanogaster and D. simulans males. A gene, located on chromosome II, could play a major role in the synthesis of 7-P in D. melanogaster.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Made available in DSpace on 2014-08-20T13:44:45Z (GMT). No. of bitstreams: 1 tese_helen_miranda.pdf: 927475 bytes, checksum: d105f02684c8c60f51894561bc5a61c7 (MD5) Previous issue date: 2008-07-11 / Soybean is one of the main world commodities and the seed is the key for high yields in this culture. Nevertheless, the soybean seed coat is the main modulator of seed quality, so, in this structure we could find answers for high seed quality production. The study goal is to identify structural contrasting traits, to obtain cDNA and to draw primers related to lignin synthesis and wax deposition in seed coat from two soybean genotypes. Plants were grown in greenhouse, flowers were identified and dated to follow the development after flowering until legumes were harvested (25, 30, 35, 40, 45, 50, 55 and 60 days). Seed coats were then analyzed in electronic microscopy and used to build a soybean seed coat cDNA library for permeability traits (lignin synthesis and wax deposition). In silico, analyzes were performed to obtain specific primers to detect soybean seed coat lignin and wax biosynthesis genes. According to the seed coat traits, we identified differences in the palisade cell thickness between genotypes. The protocol based on litio chloride was efficient to extract total RNA from both genotypes in all phases. The unidirectional cDNA multiplication was efficient for the generation of enough cDNA quantity for future projects. In silico analyzes allowed the development of 16 primers related to lignin and wax deposition biosynthesis in soybean seed coat. / A soja é uma das principais commodities mundiais e a semente o principal insumo para sua produção. O tegumento das sementes é o principal modulador da qualidade, podendo ser encontrado, nessa estrutura a resposta para a produção de sementes com caracteres superiores para vigor e viabilidade. O estudo teve como objetivo identificar características estruturais contrastantes, obter cDNA e desenhar primers relacionados à síntese de lignina e deposição de cera em tegumentos de sementes de soja de genótipos contrastantes em relação à permeabilidade CD 202 (permeável) e IAC Santa Maria 702 (semipermeável). As plantas foram cultivadas em casa de vegetação e a partir da primeira floração as flores foram marcadas para que o tempo de desenvolvimento de cada legume fosse identificado. Legumes com 25, 30, 35, 40, 45, 50, 55 e 60 dias de desenvolvimento foram coletados e os tegumentos extraídos e armazenados para extração de RNA. Tegumentos com 25, 40, 55 e 70 dias de desenvolvimento dos genótipos de soja CD 202 e IAC Santa Maria 702 também foram submetidos a Microscopia Eletrônica. Após a extração do RNA total, foram obtidos os RNAm, seguido do cDNA. Em paralelo foi realizada análise in silico para desenho de primers específicos de genes envolvidos na rota metabólica da lignina e na deposição de cera nas sementes. Foram detectadas diferenças na espessura da camada paliçádica dos tegumentos de sementes de soja dos genótipos CD 202 e IAC Santa Maria 702. O protocolo a base de cloreto de lítio foi eficiente para extrair RNA total de ambos os genótipos em todas as fases de coleta. A estratégia de multiplicação unidirecional do cDNA foi eficiente para geração de um volume considerável para o desenvolvimento de estudos futuros de expressão diferencial de genes. A análise in silico permitiu o desenvolvimento de 16 primers relacionados aos genes da lignina e da elongase.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.