Tissue and organelle targeted transgene expression in plants

Abstract

This research develops systems for tissue and organelle targeted transgene expression in commercial strawberry. Two approaches were taken to achieve this. I) the development of a root-specific expression system for nuclear transgenes and II) the development of plastid transformation methodologies for strawberry. FavRB7, a root-specific, RB7 tonoplast intrinsic protein homologue was isolated from strawberry root tissue using reverse transcnptase-polymerase chain reaction (RT-PCR). A 2.8 kb region of promoter sequence associated with the FavRB7 gene was isolated and cloned upstream of the β-glucuronidase (gusA) reporter gene. Transgenic strawberry and tobacco were produced harbouring the gusA gene under control of the FavRB7 gene promoter. Promoter activity in these lines was characterised using histochemical, fluorometric and RNA molecular assays. FavRB7 promoter activity was shown to be strong and near root-specific in strawberry. However in contrast, in the heterologous species tobacco, FavRB7 promoter activity was shown to be constitutive. A series of novel plastid transformation vectors, harbouring selectable and scorable marker genes under control of strawberry plastome regulatory elements, were created to enable plastid transformation in strawberry and tobacco. Plastid transformation methodologies were developed for strawberry utilising the aminoglycoside 3'-adenyltransferase (aadA) gene and spectinomycin selection. Additionally, plastid transformation using a non-antibiotic selection system was investigated utilising the xylose isomerase (xylA) gene and D-xylose selection. Heteroplasmic plastid transformed strawberry lines were recovered using both selection systems and expression of the soluble modified green fluorescent protein (smGFP) scorable marker gene visualised specifically in strawberry chloroplasts of three individual lines. Additionally, heteroplasmic plastid transformed tobacco lines harbouring transgenes under control of strawberry plastome regulatory elements were generated using spectinomycin selection.