1. Materials & Methods
Figure 6. Experimental Design.
Figure 1. Plasmid pre-insertion of SESA1 and post-insertion of SESA1.
FUNCTION AND REGULATION OF SESA1 IN ARABIDOPSIS THALIANA
Anna Pleto, Kevin Sidoran, and Xiao-Ning Zhang, PhD
Biochemistry Program, St. Bonaventure University, St. Bonaventure, New York
Figure 7. Expression of SESA-1 across Genotypes relative to GAPDH.
Figure 8. Visualization of successful expression of SESA1 via SDS-
PAGE. L: P7712 Ladder. 1: Pre-protein extraction. 2: Post-exposure to
Lysozyme. 3: Completed protein extraction
Conclusion
• qPCR data shows downregulation of SESA1 by SR45
• SESA1 was successfully cloned using E. Coli
• Demonstrated successful expression of SESA1
Acknowledgments
Our team would like to acknowledge the National Science Foundation and
St. Bonaventure University.
References
(1) www.arabidopsis.org
(2) Meyer, K., Koester, T., & Staiger, D. (2015). Pre-mRNA Splicing in Plants: In Vivo
Functions of RNA-Binding Proteins Implicated in the Splicing Process. Biomolecules, 1717-
1740.
(3) Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., & Morimoto, T. (n.d.). Tertiary and
Quaternary Structures of 0.19 α-Amylase Inhibitor from Wheat Kernel Determined by X-ray
Analysis at 2.06 Å Resolution † , ‡. Biochemistry, 13503-13511.
SR45 is a splicing activator that has been shown to downregulate SESA1 with RNAseq data. We aim to assess the function of Seed Storage Albumin 1 (SESA1) in Arabidopsis thaliana given its bifunctional
trypsin/α-amylase inhibitory domain, and its involvement in lipid storage, as well as confirm the SR45 downregulation. We hypothesize that SR45 aids in the nutrient breakdown of amylose by downregulating SESA1 inhibiting
amylase activity thus promoting the breakdown of amylose. Total RNAs were extracted from wild type (Col-0), overexpression lines (OX1-1 and OX1-9) and mutant (sr45-1), and purified using DNase. Total RNAs were reverse
transcribed to provide cDNAs for Taq PCR amplification. Expression of SESA1 in Col-0, OX1-1, and OX1-9 were compared to the null mutant sr45-1 by qPCR. We report that ideal qPCR conditions for the SESA1 gene are a re-
annealing temperature of 56.3°C, and a concentration 1/50 of the amplified cDNA. We report that GXL Polymerase has proved to be the most effective at amplifying the full-length SESA1 cDNA. The goal of this study is to
express the SESA1 protein and assess its α-amylase inhibitory function with a reducing sugar assay, to support the involvement of SR45 in nutrient reserve activity.
Figure 4. Transformed E. Coli grown
on LB/Amp Agarose gel at 37°C.
Figure 5. Colony PCR (SESA1 amplification) on
12 different transformed E. Coli colonies.
(Starred wells were selected for expression.)
Figure 2. SESA1 Digestion with
XhoI and BamHI.
Figure 3. Restriction enzyme digestion. 1: SESA-
1. 2. Chimeric pET-16b/AFC2 (XhoI/BamHI-HF).
3: Undigested Vector.
0
5
10
15
20
25
30
35
40
45
50
Col-0 sr45-1 Ox1-1 Ox1-9
Log(ExpressionRatio)
Genotype
800 bp
400 bp
* *
10 kb
3 kb
0.5 kb
1 2 3
Digested
Vector
10 kb
0.5 kb
1 2
10 kb
0.5 kb
*
22 kb