| Variability of transgene expression levels resulting from gene silencing is considered as a hindrance to the successful application of plant genetic engineering. Towards alleviating gene silencing, I decided to screen for novel genes involved in transgene silencing and to investigate how these genes regulate plant development. Genes encoding putative chromatin remodeling factors, especially those including a SET domain, were selected as candidate targets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed and these genes were classified into 6 groups based on the domain architecture.; RNA interference (RNAi) vectors were constructed for ∼ 20 AtSET genes and were introduced into both wild type lines and transgenic lines silenced for a GFP reporter gene. Surprisingly, altered developmental phenotypes were only observed for three constructs, raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsis system. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the same RNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereas the RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in various tissues and among different plants can vary dramatically. Contradictory to previous reports, I found that there is correlation between transcript level and silencing phenotype. Possible reasons for this discrepancy are discussed. No apparent correlation between transgene copy number and RNAi phenotypes was observed.; Among the three RNAi constructs that caused an abnormal development in Arabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate a silenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screened for morphological phenotypes. Abnormal number of flower organs, loss of viability of male gametophytes, and decreased seedling germination percentage were found in SuvR3 RNAi lines. A progressive increase in both severity and frequency of abnormal phenotypes were seen in subsequent generations, suggesting an epigenetic regulatory mechanism involved with SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues. One of the protein isoforms, SuvR3alpha, lacks 16 amino acids within the highly conserved SET domain. Possible effects of isoform interaction are proposed. |
