Abstract
In the giant ciliate Stentor coeruleus, oral apparatus (OA) regeneration is an experimentally tractable regeneration paradigm that occurs via a series of morphological steps. OA regeneration is thought to be driven by a complex regulatory system that orchestrates the temporal expression of conserved and specific genes. We previously identified a S. coeruleus-specific gene (named SCSG1) that was significantly upregulated during the ciliogenesis stages of OA regeneration, with an expression peak at the stage of the first OA cilia appearance. We established a novel RNA interference (RNAi) method through cyanobacteria Synechocystis sp. PCC6803 feeding in S. coeruleus. The expression of SCSG1 gene was significantly knocked down by using this method and induced abnormal ciliogenesis of OA regeneration in S. coeruleus, suggesting that SCSG1 is essential for OA regeneration in S. coeruleus. This novel RNAi method by cyanobacterial feeding has potential utility for studying other ciliates.
Highlights
Regeneration of the oral apparatus (OA) and holdfast, and normal shape reconstitution have all been described in S. coeruleus [1]
After inducing double-stranded RNA expression from the L4440 plasmid, E. coli HT115 cells are fed to the ciliate to elicit RNA interference (RNAi) till the appearance of phenotypes [6]
Successful replication of the pSCT3C plasmid in E. coli DH10B was confirmed by PCR and sequencing
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. After inducing double-stranded RNA (dsRNA) expression from the L4440 plasmid, E. coli HT115 cells are fed to the ciliate to elicit RNAi till the appearance of phenotypes [6]. Using this method, RNAi against the kinase regulator Mob has been successfully induced in S. coeruleus [11]. We established a novel RNAi method for the constitutive expression of exogenous gene dsRNA in Synechocystis 6803 and successfully applied this method to knock down a previous identified S. coeruleus-specific gene (named SCSG1), known to be associated with. OA regeneration [5], and verified its function in ciliogenesis of OA regeneration
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