Cloning And Expression Of The Key Gene In Wnt Signaling Pathway From Rhopilema Esculentum

In this study, by454GS-FLX sequencing technique and RACEtechnique, the cDNA and genomic organization of the key gene in Wntsignaling pathway from R. esculentum was selected and cloned. Real-timePCR and Whole mount in situ hybridization technique was used to anlysethe expression and distribution of these genes in asexual reproductionstage of R. esculentum, and discussed the regulation of these genes inasexual reproduction process of R. esculentum.The cDNA and genome of Wnt4from R. esculentum was the first timecloned. The full-length cDNA of Re-Wnt4was1212bp, containing anopen reading frame (ORF) of1032bp encoding a poly-peptide with343amino acid residues. SMART analysis showed that Re-Wnt4shared thecommon features of Wnt family, including a putative signal peptide of28amino acid residues the24conserved cysteine residues involved in theformation of the internal disulfide bridges. The deduced amino acidsequence of Re-Wnt4had a high homology with that of Wnt4fromStrongylocentrotus purpuratus, Saccoglossus kowalevskii and H. sapiens.A phylogenetic tree of Wnt amino acid sequences were constructed with the neighbor-joining method and it revealed that all the Re-Wnt4wasclustered together with Wnt4from H. Sapiens and D. rerio. The genomeof Re-Wnt4contained three exons and two introns. Quantitative real-timePCR analysis revealed that the expression of Re-Wnt4transcript wasdetected in all four developmental stages. The mRNA expression level ofRe-Wnt4was the highest in strobila, which was5.75-fold,3.06-fold, and2.76-fold of that in scyphistoma, ephyra and medusa, respectively.The cDNA and genome of Wnt5from R. esculentum was the first timecloned. The full-length cDNA of Re-Wnt5was1646bp, containing anopen reading frame (ORF) of1059bp encoding a poly-peptide with353amino acid residues. SMART analysis showed that Re-Wnt5shared thecommon features of Wnt family, including a putative signal peptide of20amino acid residues, two N-glycosylation sites and the24conservedcysteine residues involved in the formation of the internal disulfidebridges. The deduced amino acid sequence of Re-Wnt5had a highhomology with that of Wnt5from Cnidaria, invertebrate and Chordata, aswell that of Wnt5and Wnt5b from vertebrate by the multiple sequencealignment. A phylogenetic tree of Wnt5amino acid sequences wereconstructed with the neighbor-joining method and it revealed that all theWnt5amino acid sequence from Cnidaria clustered together. After theBranchiostoma floridae, the Wnt5divergence occurred and formed into two evolutionary branches. The origin of Wnt5gene may be accompaniedby the formation of multicellular animal and the divergence of Wnt5a andWnt5b cluster occured after the divergence of vertebrates (Branchiostomafloridae). The genome of Re-Wnt5contained four exons and three introns,which was similar to that of Wnt5from Cnidaria and wnt5a and wnt5bfrom vertebrate in structure. In length, the genome of Re-Wnt5was like tothat of wnt5from Cnidaria, but smaller than that of wnt5a and wnt5b fromvertebrate. Quantitative real-time PCR analysis revealed that theexpression of Re-Wnt5transcript was detected in all four developmentalstages. The mRNA expression level of Re-Wnt5was the highest in strobila,which was12.38-fold,9.99-fold, and13.01-fold of that in scyphistoma,ephyra and medusa, respectively.The cDNA and genome of ReFzd-1from R. esculentum was the firsttime cloned. The full length cDNA of ReFzd-1was2488bp, and itcontained a complete open reading frame of1761bp encoding apoly-peptide with353amino acid residues. Nucleotide sequence analysisshowed that the predicted amino acid sequence contained a typicalFrizzled protein architecture, including：a putative signal peptide of1–23amino acids (aa); a cysteine-rich domain (CRD); a conserved region withseven transmembrane segments. Blast analysis showed that the deducedamino acid sequence of ReFzd-1had a high homology with that of Frizzled from H. echinata, H. vulgaris, C. hemisphaerica and N. vectensis,as well as that of Frizzled1, Frizzled2and Frizzled7from H. sapiens, M.musculus, X. laevis and D. rerio. A neighbour-joining phylogenetic tree ofFrizzled amino acid sequences was constructed. All the amino acidsequences were divided into11main distinct branches, includingfrizzled1-frizzled10of H. sapiens, M. musculus, X. laevis and D. rerio,frizzled of Cnidaria. The ten frizzled genes fall into four main clusters.The ReFzd-1was firstly clustered together with the other Cnidarias, andthen clustered together with Frizzled1, Frizzled2and Frizzled7from H.sapiens, M. musculus, X. laevis and D. rerio. Real-time PCR analysis wasemployed to examine the expression of ReFzd-1in the four growingperiod (scyphistoma, strobila, ephyra and medusa). The results indicatedthat ReFzd-1expression was gradually increased in stages of scyphistoma,strobila and ephyra, but dropped in medusa stage. The expression inendoderm from mature R. esculentum was higher than that in ectoderm,about5fold. The whole mount in situ hybridization showed that ReFzd-1not only expressed in the tentacles and the foot region of strobila but alsothe region where the strobilation formed.All the results indicated that three key genes (Wnt4, Wnt5, ReFzd-1)in Wnt signaling pathway from R. esculentum were participated in asexualreproduction process of R. esculentum and played important regulatory roles in growing period of strobila.