Post-transcriptional regulation of the pea plastocyanin gene (PetE)

• Main Author: Brown, Naomi Jane
• Published: University of Cambridge 2004
• Subjects: 581.35
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597002

Expression of the pea plastocyanin gene (<i>PetE</i>) is regulated both by light and by signals from the chloroplast. Previous work has indicated that the light and chloroplast-controlled regulation operates post-transcriptionally in transgenic tobacco, requiring the correct 5’ terminus of the transcript and elements within the plastocyanin-coding region. The post-transcriptional light and chloroplast-controlled regulation of pea <i>PetE</i> has now been demonstrated to operate in transgenic <i>Arabidopsis</i> plants, indicating that the regulation is conserved in an additional plant species. The overall aim of the research described in this dissertation was to investigate the mechanisms by which light and plastid signals influence the stability of <i>PetE </i>transcripts. <i>PetE</i> constructs containing premature stop codons in the coding region were generated to investigate whether translation has a role in the light or chloroplast-controlled regulation. RNA-gel-blot analysis of transgenic plants containing these constructs was used to examine the effects of light and plastid inhibitors on pea <i>PetE</i> transcript accumulation in 7-day-old tobacco seedlings. The results obtained suggested that translation of the start of the <i>PetE</i> coding region is required for both light and plastid-regulated transcript stability. Constructs containing progressive 3’ deletions of the <i>PetE</i> coding region, fused to the <i>Luc </i>reporter gene, were generated to examine how much of the coding sequence is necessary for the regulation. Luciferase assays and RNA-gel-blot analysis were carried out on transgenic tobacco seedlings containing the constructs, to examine the effects of light and plastid inhibitors on the regulation. The results indicated that an element important in the light and chloroplast-controlled regulation is located in the first 12% of the coding region, corresponding to the first 60 nucleotides. The start of the plastocyanin-coding region therefore appears to contain sequences important in the regulation by light and plastid signals, and these sequences may need to be translated in order for the regulation to operate.