Controlling transpositon in the male gametophyte of transgenic plants.
thesisposted on 19.11.2015, 09:09 by Michael Richard Roberts
Investigations were carried out to determine the feasibility of a transposon tagging experiment in flax, Linum usitatissimum. The excision of the maize transposable element Activator (Ac) from the genome of transgenic flax callus was demonstrated, whilst a Dissociation element (Ds) was found to be stable. However, reintegration of excised Ac elements was not detected, and this barrier to gene tagging led to an examination of procedures which might improve the general applicability of transposon tagging. A recombinant Ac transposase gene was constructed in order to achieve a high germinal transposition frequency in transgenic plants; this feature is an essential component of an efficient transposon tagging strategy. The Ac construct was produced by fusing the promoter of an anther-specific gene to the transposase coding region. The anther-specific gene, APG, was cloned from Arabidopsis thaliana, following the identification of four putative microspore-specific mRNAs from Brassica napus. Of these mRNAs, one, termed 13, was analysed in detail and found to encode a novel oleosin protein, and was apparently confined to developing pollen. The I3 cDNA was used as a molecular probe to clone the APG gene, which encodes a proline-rich protein of unknown function. A small gene family encoding proteins with high sequence similarity to the APG protein was identified in B. napus via the isolation of three cDNAs termed CEX1, CEX2 and CEX6. Promoter fragments of the APG gene were demonstrated to drive expression of a ?-glucuronidase reporter gene in the male gametophyte, tapetum, stomium and anther wall of Nicotiana tabacum and Arahidopsis during the microspore development stage of gametogenesis. The restriction of transposition to these cells would permit the production of a seed population containing a wide range of unique transposon inserts which would be stable in during vegetative growth. Such applications of the APG/Ac fusion are discussed.