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Title: Genetic modifiers of hairpin-induced gene silencing in Arabidopsis thaliana
Author: Hunter, B. C.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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The Chalcone Synthase (CHS) gene codes for the first step of biosynthesis of the purple pigment anthocyanin. In this study, transgenic Arabidopsis plants with a hairpin construct homologous to part of the CHS coding sequence were used in a genetic screen to identify modifiers of hairpin silencing. Some putative genetic modifiers were identified, but these appear to be the result of transgene:transgene interaction based on shared promoter homology rather than being due to second site suppressors in the Arabidopsis genome. New transgenic Arabidopsis lines were made with hairpin constructs directed to either the promoter or the reading frame of the CHS gene and both types of transgenic showed an anthocyanin-deficient phenotype. For each construct a representative single-insert homozygous transgenic line was selected for subsequent work. Both transgenic lines showed increased methylation of their respective target site and contained short interfering RNA (siRNA) homologous to their target site. A collection of 59 putative modifiers of CHS silencing were tested with both transgenic lines using segregation analysis of the anthocyanin-deficient phenotype in the F2 generation of appropriate crosses. Mutants in AGO4, AGO6, DRD1, DRM2, NRPD2a and NRPE1 act as recessive second site modifiers of the CHS promoter hairpin line phenotype. These mutants are associated with a decrease in DNA methylation at the hairpin target site. Mutant alleles of the DCL4 and HEN1 genes reduce silencing of the CHS coding sequence hairpin. The reduction in silencing is greater in mutants that are hemizygous for the hairpin construct than in those that are homozygous for it. The increase in siRNA in the construct homozygotes, compared to that in the hemizygotes, is perhaps sufficient to overcome any modification of the phenotype by mutants of DCL4 and HEN1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available