Molecular biology of flax (Linum usitatissimum L) seed oleosin genes

Oil-bodies are intracellular spherical organelles that store oil in the form of triacylglycerols. They have a diameter of 0.6 to 2.0 mum and are surrounded by a half-unit membrane of phospholipids embedded with unique and abundant proteins called oleosins. Five different cDNA clones and four different genes encoding oleosin were isolated from flax (Linum usitatissimum L.) and their sequences determined. Sequencing of these genes and sequence similarity searches with other oleosin sequences in the data bank reveal that flax oleosins can be grouped into putative H- and L-oleosin isoforms. The H-isofonm oleosin genes are intronless. In contrast, the L-isoform genes have a single intron. The flax oleosin transcript accumulation was induced during embryo development. The protein accumulated to nearly equal levels in different embryo tissues, the radicle and cotyledons. Using genomic Southern hybridization, it was established that flax oleosins appear to be members of a multigene family, with two genes encoding the H-isoforms and four genes encoding the L-isoforms. Flax oleosin promoters are strong promoters driving expression levels of reporter beta-glucuronidase to comparable levels as compared to other seed storage gene promoters such as the beta-phaseolin from beans and the linin promoter derived from flax. In stable transgenic flax and Arabidopsis plants flax oleosin-GFP fusion protein can accumulate to 0.05--0.27% of total seed protein. The relative amounts of oil and oleosin deposited during seed development may control the size of oil-bodies (Tzen and Huang, 1992; Sarmiento et al., 1997). A heterologous oleosin was over-expressed in transgenic flax plants. The oil: oleosin ratio and the oil-body size was determined in the transgenic and wild type seed lines. High oil: oleosin ratio in wild type seeds resulted in bigger oil-bodies as compared to low oil: oleosin ratio in transgenic seeds. Olcosin heteromers are believed to impart stability to the proteins on the oil-bodies. However, in flax, expression of L- and H-oleosin isoforms do not seem to impart additional stability to the proteins accumulated on the oil-bodies.;The findings of this thesis have general implications on oleosin expression, evolution and function and for its use as carriers for recombinant proteins.