Genetics - Theses

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    Selection and characterisation of Arabidopsis mutants resistant to a glutathione biosynthesis inhibitor
    Maughan, Spencer C. ( 2003)
    This thesis describes the investigation of GSH regulation and signaling in Arabidopsis thaliana by a genetic approach. A procedure is described to select for mutants resistant to a chemical inhibitor of glutathione (GSH) biosynthesis, L-buthionine-SR-sulfoximine (BSO). Fifteen mutants were selected and preliminary genetic examinations indicated that BSO-resistance phenotypes are often highly variable. Consequently five mutants were chosen for further genetic, molecular and biochemical characterisation. All five of these mutants were shown to have altered GSH levels. Moreover, root growth among these mutants varied indicating that different mechanisms may be conferring BSO-resistance. Molecular mapping techniques were used to assign the BSO-resistance loci of four of the mutants to chromosome regions. Two mutants (a T-DNA insertion mutant and an EMS-derived mutant) were found to map to the same position between the markers CHS1 and R89998 on chromosome 5. Subsequently, complementation tests confirmed that these mutants define a single locus. The mutated locus in the T-DNA insertion mutant was determined using molecular techniques. The gene that contained the insert encodes a putative transport protein and was designated the BSO Resistance Transporter1 (BRT1). No transcript was detected in the T-DNA insertion mutant (brt1-1) which suggests that brt1-1 is likely to be a null allele. The EMS-derived mutant (brt1-2) contained an intron splice-site consensus sequence mutation which resulted in transcripts that included an unspliced intron. The function of the BRT1 gene was investigated by phenotype testing and despite the brt1 mutants having 2-fold GSH levels they were not resistant to known oxidising agents. Northern analysis of GCS and the use of a GCS-GUS reporter line indicated that the increase in GSH levels in these mutants was not accompanied by increases in GCS transcription and translation. This suggests that BRT1 is a novel component influencing GSH metabolism. BRT1 defines a novel family of three genes in Arabidopsis. Preliminary experiments indicate that these three genes are expressed in the same tissues providing for possible a level of redundancy between them. Supporting this is a newly identified insertion mutant in a second BRT gene (brt2-1) which is also resistant to BS0. Moreover, the double mutant, brt1-1;brt2-1, exhibits increased BSO-resistance suggesting an additive effect of the mutations. Database searches revealed that there were homologues in other organisms including one from the malaria parasite (PfCRT). Mutations in PfCRT confer resistance to a widely used antimalarial, chloroquine (CQ). However the function of PfCRT is not known. The BRT1 mutant was shown to have a level of functional conservation with a malaria parasite homologue, PfCRT, and therefore further characterisation of these proteins may provide insights into GSH metabolism and perhaps CQ-resistance in the malaria parasite.
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    The role of cytochrome P450s in pyrethroid resistance in the AN02 strain of Helicoverpa armigera
    Grubor, Vladimir ( 2003)
    Helicoverpa armigera is a lepidopteran moth species that is one of the largest agricultural pests in the world. In Australia, it is especially damaging to the cotton industry. Previous research on pyrethroid resistance in Helicoverpa armigera has suggested several resistance mechanisms present in the field. Research on a specific strain of H. armigera, AN02, suggested that resistance to pyrethroids in this particular strain is due to metabolism of the insecticide by cytochrome P450. This thesis describes work undertaken to gain further knowledge of the resistance mechanism present in this strain. Chapter 2 describes expression testing and elimination of the CYP6B cluster of P450s as candidates for resistance in the AN02, which were the most likely candidates available at the time. Chapter 3 describes construction, probing and sequence analysis of a midgut cDNA library of H.armigera to clone more P450 sequences and analyse the general transcriptome of one of the tissues where detoxification is likely to occur. Chapter 4 describes expression testing of new P450 sequences obtained from different sources to determine whether any of them have increased expression that is associated with the resistance genotype. Several new P450 genes were found to have increased expression and are candidates for further study.