Isolation and characterization of differentially expressed genes during somatic embryogenesis in orchardgrass (Dactylis glomerata)

This dissertation describes the steps that were taken in pursuit of the identification of differentially expressed genes during initiation and early stages of somatic embryogenesis in orchardgrass, Dactylis glomerata L., Poaceae. The differential response of two genotypes, Embryogen-P and Nonembryogen, to the embryo inductive medium was used to compare gene expression in these genotypes and to isolate cDNAs that appeared only in leaf cultures induced for somatic embryogenesis. RT-PCR differential display performed with total RNA from basal leaf segments cultured for 10 d and from distal leaf segments cultured for 28 d resulted in the isolation of 17 cDNA fragments that appeared to be characteristic for somatic embryogenesis from leaf cultures. Thirteen of them were cloned and sequenced. They represented between 186 and 362 by from the 3' end of the transcripts, just before the poly(A) tail. Three of these cDNA fragments were used as probes for screening a cDNA library made from embryogenic leaf cultures.;Four clones were isolated from the cDNA library. One, designated as DGRPT6a was uniformly expressed in leaf segments and suspension cultures. It was 81% identical to the 26S proteasome AAA-ATPase subunit RPT6a from Arabidopsis thaliana L. The other three clones were confirmed to have differential expression in embryogenic tissues. The DGMPP sequence showed high homology with a putative mitochondrial processing peptidase alpha-II chain precursor from rice. It was expressed only in embryogenic leaf cultures. DGE1 and DGE2 showed no significant similarity with sequences from the GeneBank; therefore they encoded two novel proteins. The putative DGEs appeared to be nuclear proteins controlled by phosphorylation and might be involved in replication or transcription regulation. DGE1 was found to be expressed both in basal and distal leaf segments, while DGE2 was expressed only in distal leaf segments and in suspension cultures induced for embryogenesis. The isolation of three clones that were differentially expressed in embryogenic tissues of orchardgrass provides the opportunity for further detailed studies of the molecular events that underlay the process of somatic embryogenesis in grass species.