Molecular Regulation Of Rhopilema Esculentum Asexual Reproduction Process

On the basis of constructing Rhopilema Esculentum 454 transcriptome library, the reseach screened and identified three signaling pathways which are Notch, Hedgehog and TGF-β / Smads, and we cloned full-length c DNA of Notch, TβR-I, Hedgehog and analyzed theirs m RNA expression in different developmental stages of R.Esculentum by expressed sequence tag(EST), rapid amplification of c DNA ends(RACE) techniques and RT-PCR. This research includes the following contents:The full-length c DNA of R.Esculentum Notch was 6671 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,which includes a putative signal peptide of 21 amino acid residues, its mature peptide includes 2000 amino acid residues and consists of 37 structure domain, including 26 EGF-like domains,6 ANK domains, and a transmembrane domain except 3 Notch / Lin-12( NL) structure domain,a NOD and a NODP domain which were only exsising Notch family; The homology and phylogenetic analysis showed that the amino acid sequence similarity of R.Esculentum Notch with Cnidaria Nematostella vectensis amino acid was 39%, and with invertebrates and vertebrates amino acid similarity respectively were 35% ~ 37%, 34% ~ 38%. Phylogenetic tree of Notch based on neighbor-joining method show that R.Esculentum was clustered with Cnidaria Nematostella vectensis. Quantitative real-time PCR analysis revealed that the expression of Notch transcript was detected in all four developmental stages, The m RNA expression level of Notch was the highest in scyphistoma and the lowest in strobili. Scyphistoma is 1.85 times of that strobili, Along with the development of strobili to the ephyra, its expression quantity rising again. Podocyst generation is the most important way of asexual reproduction to produce young R.Esculentum, namely scyphistoma after two closely related development of segmental and abnormal condition product ephyra.it indicates that Notch gene involved in regulation of asexual reproduction of R.Esculentum.The full-length c DNA of R.Esculentum Hedgehog was 1506 bp,containing 5’-untranslated region(UTR) of 13 bp,an open reading frame(ORF) of 1371 bp and 3’-untranslated region(UTR) of 119 bp which contains a polyadenylation signals of AATAAA.and Predict Hedgehog protein Molecular weight and theoretical isoelectric point(p I) 50.71 k Da and 6.57 respectively. SMART analysis showed that: R.Esculentum Hedgehog, as a secretory protein,which includes a putative signal peptide of 20 amino acid residues, its mature peptide includes 457 amino acid residues and consists of 4 structure domain, including a SVWC domain, a transmembrane domain,a Hint N domain and a Hint C domain. Also includes three potential N- glycosylation sites: NPTT,NLSP and NESA which is identified in the 50-53, 63-66, 391-394 site amino acids respectively. The homology and phylogenetic analysis showed that the amino acid sequence similarity of R.Esculentum Hedgehog with nematode oesophagostomum dentatum amino acid was 25%, and with invertebrates and vertebrates amino acid similarity respectively were 20%~24%, 21% ~ 24%. Phylogenetic tree of Hedgehog based on neighbor-joining method show that R.Esculentum was clustered with nematode oesophagostomum dentatum.Quantitative real-time PCR analysis revealed that the expression of Hedgehog transcript was detected in all four developmental stages, The m RNA expression level of Hedgehog was the highest in strobila and the lowest in Scyphistoma. strobila is 5.37,2.31, 2.47 times of that Scyphistoma, ephyra, medusa, Along with the development of Scyphistoma to the strobili, its expression quantity is significantly rising,and from strobili to ephyra on the contrary. Podocyst generation is the most important way of asexual reproduction to produce young R.Esculentum, namely scyphistoma after two closely related development of segmental and abnormal condition product ephyra. it indicates that Hedgehog gene involved in regulation of asexual reproduction of R.Esculentum.The full-length c DNA of R.Esculentum TβR-I was 1977 bp,containing 5’- untranslated region(UTR) of 90 bp,an open reading frame(ORF) of 1509 bp and 3’-untranslated region(UTR) of 376 bp which contains a polyadenylation signals of AATAAA.and Predict TβR-I protein Molecular weight and theoretical isoelectric point(p I) 56.54 k Da and 8.63 respectively. SMART analysis showed that: R. Esculentum TβR-I, as a secretory protein,which includes a putative signal peptide of 19 amino acid residues, its mature peptide includes 503 amino acid residues and consists of 3 structure domain, including extracellular domain, a transmembrane domain and intracellular domain which is mainly concaining glycine/serine enrichment area sequence(GS) and serine/threonine kinase area(S- TKc). Also includes 4 potential N- glycosylation sites: NNTC, NVSC, NLSA, NGSL which is identified in the 533-36, 80-83, 113-116, 284-287 site amino acids respectively. The homology and phylogenetic analysis showed that the amino acid sequence similarity of R.Esculentum TβR-I with cnidaria hydra amino acid was 39%, and with invertebrates and vertebrates amino acid similarity were 44%~47%, 47%~54% respectively. Phylogenetic tree of TβR-I based on neighbor-joining method show that R.Esculentum was clustered with cnidaria hydra.Quantitative real-time PCR analysis revealed that the expression of TβR-I transcript was detected in all four developmental stages, The m RNA expression level of TβR-I was the highest in strobila and the lowest in medusa. strobila is 1.56,1.31 å'Œ 1.81 times of that Scyphistoma, ephyra, medusa, Along with the development of Scyphistoma to the strobili, its expression quantity is significantly rising,and from strobili to ephyra on the contrary. it indicates that TβR-I gene involved in regulation of asexual reproduction of R.Esculentum.