Poster abstracts
Poster number 56 submitted by Claire Schaef
: Discovering the role of SAP18 in transcriptional regulation by creating a SAP18 knockdown line.
Claire Schaef (Biochemistry Program, Department of Biology, St. Bonaventure University), Xiao-Ning Zhang (Biochemistry Program, Department of Biology, St. Bonaventure University)
Abstract:
SAP18 is a highly conserved gene in eukaryotic systems, shown to control stress-related responses through transcriptional regulation. The highly conserved nature of SAP18 suggests that this gene is important for proper life functioning. As one of the three subunits of the apoptosis and splicing associated complex (ASAP), SAP18functions in regulating RNA metabolism in various ways, recruiting histone deacetylases and controlling polyadenylation for example. While known to function in transcriptional silencing, it is largely unknown which specific genes are regulated by SAP18 as part of the ASAP complex. To study the processes that SAP18 regulates, a SAP18-specific amiRNA was cloned under the control of the native SAP18 promoter. To create SAP18 knockdown lines, this construct was introduced into the flowering pSAP18::SAP18-GFP qrt homozygote plants through transformation with Agrobacteria. The first generation was screened with kanamycin. This gave 42 candidate transformants. Then the GFP signal strength was visualized in their pollen tetrads with confocal imaging to determine the knockdown effect. 19 plants which tetrads had two dim and two bright pollen grains were kept and allowed to seed. They were reexamined in second generation, looking for plants homozygous for the SAP18knockdown. In both rounds of screening, several abnormal pollen phenotypes were repeatedly observed, including multiple plants with closed anthers or shriveled pollen. Expression levels of SAP18 in 7 promising lines were then analyzed with RT-qPCR, comparing SAP18 expression to Col-0 wild type. Preliminary results suggest potential success in several silencing lines, with the best knockdown of SAP18 expression to 0.46 and 0.53 of wild type. Once these results are confirmed, the plant lines with the least SAP18 expression will be used to explore how a decrease in SAP18 alters the expression of other genes. If SAP18 is as important as its highly conserved nature indicates, we expect to see abnormalities in the knockdown lines, such as in abiotic and biotic stress responses, and fertility. Based on the prevalence of shriveled pollen seen in mutant lines, a hypothesis is that SAP18 may regulate gamete formation.
References:
Chen, S. L., Rooney, T. J., Hu, A. R., Beard, H. S., Garrett, W. M., Mangalath, L. M., . . . Zhang, X. (2019). Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana. Frontiers in Plant Science, 10. doi:10.3389/fpls.2019.01116
Song, C., & Galbraith, D. W. (2006). AtSAP18, An Orthologue of Human SAP18, is Involved in the Regulation of Salt Stress and Mediates Transcriptional Repression in Arabidopsis. Plant Molecular Biology, 60(2), 241-257. doi:10.1007/s11103-005-3880-9
Zhang, Y., Iratni, R., Erdjument-Bromage, H., Tempst, P., & Reinberg, D. (1997). Histone Deacetylases and SAP18, a Novel Polypeptide, Are Components of a Human Sin3 Complex. Cell, 89(3), 357-364. doi:10.1016/s0092-8674(00)80216-0
Keywords: SAP18, artificial microRNA, transcriptional regulation