Cloning And Expression Of Wnt3,Frizzled5/8 And ?-catenin Gene By Analysis Of Transcriptome Library From Rhopilema Esculentum

Based on the transcriptome library constructed from Rhopilema esculentum,through the bioinformatics analysis and in-depth mining,the cDNA and genomic structure of the key genes Wnt3?frizzled5/8 and ?-catenin in Wnt signaling pathway from R.esculentum was analyzed in this study.The expression of these genes in the asexual reproduction of R.esculentum was elucidate by the RT-PCR and Whole mount in situ hybridization technique,which provided reference for understanding the molecular regulation mechanism of asexual reproduction for R.esculentum.The main results obtained were showed below.1.Construction and bioinformatics analysis of transcriptome library of R.esculentum.More than 580000 sequences were obtained by 1/2 run 454 GS FLX sequencing in R.esculentum transcriptome library.The total length was about 240 Mb,the average length was 409.2 bp.490000 high quality of ESTs sequences were gained by removing primer and low quality of sequence(<100 bp).37628 functional genes gained by assembling,were classified as cellular localization,molecular functions and biological processes in R.esculentum.Functional genes involved in cellular localization,molecular functions and biological process accounted for 30.88%,31.45%and 37.67%of storage capacity respectively.Taking advantange of comparative transcriptomics,we found that R.esculentum contained the vast majority of Wnt signaling pathways related genes ever known.Except WntlO,R.esculentum contained 12 ESTs sequences of Wnt family,5 frizzed ESTs sequences and 2?-catenin ESTs sequences.2.Cloning,genome structure and expression of Wnt3 gene in R,esculentumThe full-length cDNA of ReWn3 firstly cloned was 2074 bp,containing an open reading frame(ORF)of 1080 bp encoding 360 amino acids,with 23 highly conservative cysteine.The full length of Re Wn3 genome was 4704 bp,including 5 exons and 4 introns,the structure of that was nearly similar to Hydra Wnt3.Multiple sequence alignment revealed that the amino acid sequence of ReWnt3 was highly consistent with that of other cnidaria,and the homology with C.hemisphaerica reached 32.8%.Phylogenetic analysis showed that members of the Wnt3 and Wnt3a family fiom vertebrates clustered into the two sisters groups respectively,then clustered together with Wnt3 groups of Hemichordata,finally clustered with cnidaria into a group,forming the pattern that Wnt3 evolved gradually from low to high animals,from simple to complex.Quantitative real-time PCR analysis revealed that the Re Wnt3 was widely expressed in four different processes of asexual reproduction of R.esculentum.varing with the development process.The mRNA expression level of ReWnt3 was the highest in strobila.3.Cloning,genome structure and expression of Frizzled 5/8 gene in R.esculentumThe full-length cDNA of Frizzled 5/8(ReFzd 5/8)firstly cloned was 2166 bp,containing an open reading frame(ORF)of 1701 bp encoding 567 amino acid.SMART analysis showed that ReFzd 5/8 comfomed to the structural characteristics of frizzled family,including a cysteine-rich domain with 10 conserved cysteine residues located in the N-terminal,a transmembrane domain with seven transmembrane segments,a putative signal peptide of 21 amino acids.The genome of ReFzd 5/8 contained no introns.Blast analysis showed that the deduced amino acid sequence of ReFzd5/8 had a high homology with that of Frizzled from Clytia hemisphaerica and Hydra magnipapillata.Phylogenetic analysis revealed that R.esculentum first clustered together with Fzd gene from sea anemone,then clustered together with Fzd5 and Fzd8 genes from different species.RT-PCR analysis revealed that ReFzd 5/8 gene expression was found at four different development stages,varing with different stages of development.The relative expression level of transverse was the highest in strobila,which was 6.90-fold,1.74-fold,and 4.35-fold of that in scyphistoma,ephyra and medusa,respectively.The results of whole-mount situ hybridization technique showed that the expression of ReFzd 5/8 gene was detected mainly in tentacles and base of scyphistoma.4.Cloning and expression of ?-catenin gene in R.esculentumThe full-length cDNA of R.esculentum ?-catenin(ReBcn)first cloned was 3035 bp,containing an open reading frame(ORF)of 2451 bp encoding 817 amino acid.SMART analysis showed that ReBcn involved coiled-coil domain and no putative signal peptide was found.BLAST analysis showed that the deduced amino acid sequence of ReBcn had a high homology with that from Clytia hemisphaerica,Hydra magnipapillata,Podocoryna carnea,Hydractinia echinata and Hydra vulgaris,with above 74%identity of amino acid sequence.A phylogenetic based on the neighbor-joining method revealed that ReBcn clustered together with cnidarians, forming independent branch of the cnidaria.R.T-PCR analysis revealed that the expression of ReBcn is expressed in four different stages of asexual reproduction.Compared with other developmental stages,the expression of ephyra was the highest.