Abstract:Notch Pathway is a very conservative signaling pathway in evolution, through the interaction between Notch receptor and the ligand DSL protein which conveys signals to regulate cell proliferation, differentiation, apoptosis, and determine the fate of cells. Rhopilema esculentum belongs to cnidaria, it is an ideal model organism to study cnidaria asexual propagation because of the mature artificial breeding and aquaculture technology. The cDNA cloning and expression analysis of Notch were first performed in the R. esculentum based on 454 GS-FLX sequencing technique by RT-PCR and RACE method. The results showed that the full-length cDNA of Notch was 6768 bp, containing 5¢- untranslated region (UTR) of 90 bp, an open reading frame (ORF) of 6066 bp and 3¢- untranslated region (UTR) of 612 bp which contains a polyadenylation signals of AATAAA. SMART analysis showed that R. esculentum Notch, as a secretory protein, included a putative signal peptide of 21 amino acid residues; its mature peptide included 2000 amino acid residues and consisted of 37 structure domains, which includes 26 EGF-like domains, 6 ANK domains, and a transmembrane domain except 3 Notch / Lin-12(NL) structure domain, a NOD and a NODP domains which only exist in Notch family. The homology and phylogenetic analysis showed that the amino acid sequence similarity between R.esculentum Notch and Cnidaria Nematostella vectensis amino acid was 39%; by contrast, compared with invertebrates and vertebrates, R. esculentum shares 35%-37% and 34%-38% respectively, in the similarity of the amino acid. Phylogenetic tree of the Notch based on neighbor-joining method showed that R.esculentum was clustered with Cnidaria N. vectensis. Quantitative real-time PCR analysis revealed that the expression of Notch transcript was detected in all four developmental stages, with the highest level in scyphistoma stage and the lowest in strobili stage. The expression level of scyphistoma stage was 1.85 -fold of that in strobili. The results indicate that Notch gene is involved in the regulation of asexual reproduction of R.esculentum.
