A leaf-specific phage T7 RNA polymerase-based system for transgene expression in tobacco chloroplasts
Citation:
Alan Magee, 'A leaf-specific phage T7 RNA polymerase-based system for transgene expression in tobacco chloroplasts', [thesis], Trinity College (Dublin, Ireland). School of Genetics and Microbiology, 2001, pp 201Download Item:
Abstract:
This thesis describes the development of a chloroplast-localised gene expression
system based on the phage T7 RNA polymerase. In order to direct T7 RNAP
expression in a leaf-specific manner and to target it to the chloroplast, we constructed
three chimeric genes (st8, st14 and st25) in which tobacco rbcS sequences encoding
the Rubisco small subunit (SSU) transit peptide and either the first 8, 14 or 25 amino
acids of mature SSU were fused in-frame with the coding sequence of T7 RNAP.
Transgenic tobacco plants expressing the STB, STM and ST25 chloroplast-targeted
fusion proteins in their leaves were generated using the Agrobacterium system for
nuclear transformation. These transgenic plants were shown to express a chloroplastlocalised
transcriptional activity which was specific for the phage T7 DNA template.
All transgenic lines grew normally, the transgenes were inherited in a Mendelian
fashion in the progeny, and homozygous lines expressing the STM and ST25 fusion
proteins were identified. We further determined that a large proportion of all three ST
fusion proteins was localised within the chloroplast. In addition we determined that
the ST25 fusion protein had an in vivo half-life of at least 16 days in the dark and that
the transcription of eight plastid genes in the ST25 line appeared to be unaltered by the
presence of the T7 RNAP activity.
Author: Magee, Alan
Advisor:
Kavanagh, TonyQualification name:
Doctor of Philosophy (Ph.D.)Publisher:
Trinity College (Dublin, Ireland). School of Genetics and MicrobiologyNote:
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Full text availableKeywords:
Genetics, Ph.D., Ph.D. Trinity College DublinMetadata
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