AN INVESTIGATION OF MECHANISMS OF AN INTRON-MEDIATED GENE SILENCING
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Introns, non-coding regions between exons, exist in most eukaryotic genes. Many studies have shown that introns regulate gene expression through both transcriptional and posttranscriptional mechanisms. I revealed that in Arabidopsis thaliana, over-expression of the first and longest intron of CAULIFLOWER (CAL) gene led to the silence of the endogenous CAL gene through DNA methylation, which is mediated by the intron-derived 24 nt siRNAs. I investigated mechanisms of this intron-mediated gene silencing phenomenon through several different approaches, including northern blot, qRT-PCR, small RNA sequencing, bisulfite sequencing, McrBC-PCR, and bioinformatics. The CAL first intron does not show evolutionary conversation among different species in Rosid family. A distinctly folded stable secondary structure was found in the CAL first intron but its relevance to the silencing remains to be determined. Further, the CAL first intron likely possesses regulatory sequences demonstrated by the intron's ability to induce GUS reporter gene expression when fused upstream of a TATA box and the GUS gene. Antisense long non-coding RNAs (lncRNAs) from the intron was detected by qRT-PCR, which may pair with the over-expressed sense CAL first intron transcript from the transgene to from double stranded RNAs and subsequently generate 24 nt siRNAs. Therefore, this study provides a potentially novel and simple method to silence target genes by over-expressing cis regulatory elements either in introns or in promoters. I investigated how the intron-mediated silencing is inherited and showed that the silencing of CAL occurs in seeds and the silencing efficiency is dependent on the length of seed storage time. The extent of methylation in the CAL first intron increases when the seed age increases. This work has important biotechnology implications. Combined, my research not only describes a novel phenomenon of intron-mediated silencing but also sheds light in the mechanism of intron- or cis regulatory element-mediated gene silencing. Hence my research work will have broad implications both in basic research and in biomedical and agricultural applications.