Undergraduate Student Hendrix College Little Rock, Arkansas, United States
J. Johnson (Hendrix College)| Andrew Schurko (Hendrix College)
Bdelloid rotifers are unique asexual invertebrates that possess a remarkable DNA repair system that allows them to recover from extensive DNA damage sustained through desiccation. While the mechanism of this DNA repair is unknown, we have used RNA-seq to identify several candidate genes that are upregulated following DNA damage. The goal of this project was to use RNA interference (RNAi) to silence expression of two of these candidate genes, RAD51 and DNA ligase K (LIGK) and then determine recovery from DNA damage caused by desiccation. To induce RNAi, the bdelloid Adineta vaga would be fed an E. coli “feeder strain” that could be induced with IPTG to express a double stranded RNA (dsRNA) copy of the target gene. To make a feeder strain for each gene, a plasmid (with the pL4440 backbone) was constructed by inserting a section of the gene between two inverted T7 RNA polymerase promoters. The plasmids were then transformed E. coli HT115(DE3) containing an IPTG-inducible T7 RNA polymerase. RNA was isolated from feeder strains grown in the presence of IPTG and analyzed by agarose gel electrophoresis to ensure that target gene dsRNA was expressed. Next, bdelloids were fed 108 cells of an induced (+IPTG, + dsRNA) and non-induced (-IPTG, -dsRNA) E. coli feeder strain for different lengths of time (6 and 24 hours). Real-time PCR was used to determine if gene expression was decreased in bdelloids fed the +IPTG feeder strain. For several replicates, we found that the Log2FC for RAD51 and LIGK was < -1 after 6 and 24 hours of being fed the +IPTG feeder strain, indicating that RNAi was induced. Optimized RNAi conditions could then be used to silence RAD51 and LIGK expression during recovery from desiccation. Induction of RNAi during desiccation or during rehydration following desiccation would then be done and the average recovery rate (defined as a rotifer being able to produce at least on offspring) for each treatment would be determined. A significantly lower recovery rate for rotifers fed the +IPTG strain would be consistent with a role for the target gene in DNA repair. Understanding the remarkable abilities of bdelloid rotifers to repair massive DNA damage will have the potential to understand DNA repair in other organisms on a deeper level.
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