Tgf-β/Smad Signaling Pathway Genes: Molecular Cloning, Expression Analysis And Snps Associated With Growth Traits In Zhikong Scallop (Chlamys Farreri)

The transforming growth factor β (TGF-β) superfamily which comprises a largenumber of structurally related multifunctional cytokines, control a diverse set of cellularprocesses, including cell proliferation, cell differentiation and cell growth. These growthfactors transduce signals through an evolutionarily conserved mechanism. In most cases,TGF-β ligand dimers initiate the signaling cascade by binding to type II and type Ireceptors on the cell surface, and then transphosphorylase the Smad proteins totransduce the signal into the nucleus and regulate the transcription of target genes. Inthis study, there are11candidate genes in the TGF-β/Smad signaling pathway to becloned in Zhikong scallop (Chlamys farreri) through homology cloning and in silicocloning and the nucleotide and deduced amino acid sequences of each gene are analyzed.The expression levels of the11genes in embryos/larvae and adult tisues are analyzed byreal-time qRT-PCR. The single nucleotide polymorphisms (SNPs) in the11genes arescreened through resequencing and EST database screening and genotyped in a full-sibfamily and a wild population by high resolution melting (HRM) assays. The associationof these SNPs with growth traits and gene expression levels are analyzed. This studywill provide important information to our understanding of TGF-β/Smad signaling inscallops. The major results are as follows.1. The DNA and full-length cDNA sequences of two TGF-β ligands (Myostatin(MSTN) and Bone morphogenetic protein2(BMP2)) and an inhibitor (Follistatin) arecloned in Zhikong scallop. Both MSTN and BMP2consists of3exons and2introns, andthe encoded protein sequences share conserved structures such as signal domain,pro-domain, proteolytic processing site (RXXR) and mature domain with the reportedTGF-β members. Several muscle-specific regulatory elements including COMP、MEF2s、MTBFs and E-boxes are identified in the5’ flanking region of MSTN. Follistatin consists of5exons and4introns, encoding a polypeptide that contains signal domain,N-terminal domain and3Follistatin domains. Analysis of expression of these3genes atdifferent developmental stages and in different adult tissues reveal that higher expressionlevels of MSTN and BMP2are detected in gastrulae, and those of Follistatin are detectedin fertilized eggs and4-cell embryos. In the adult scallops, the3genes are expressed inall of the sampled tissues, and the highest expression levels of MSTN are detected in thestriated muscle, and those of BMP2and Follistatin are detected in the gill. The HRManalysis shows that5SNPs in Myostatin and1in Follistatin are polymorphic. Inscallops from a full-sib family, the SNP g.-1163G>T in the5’ flanking region of MSTNhas a significant association with growth traits such as shell length, shell height andstriated muscle weight and expression level of MSTN in striated muscles (P <0.05).2. The full-length cDNA sequences of Activin type II receptor (ActR2), Activin-likereceptor1(ALR1), TGF-β type I receptor (TGFBR1) and BMP type I receptor (BMPR1)are isolated, and all of them encode amino acids with characterized conserved domainsof serine/threonine kinase receptors. Their cDNA sequences are compared to thegenomic database of Zhikong scallop, and gene structures of ActR2and TGFBR1aredetermined, which consist of6and10exons, respectively. All the4receptor genes areexpressed at higher levels in the fertilized eggs,4-cell embryos and blastulae than inlarval stages. Transcripts of the4genes are detected in all the sampled adult tissues, andhigher expression levels are observed in the gonad and striated muscle. In scallops froma full-sib family, the SNP c.1815C>T in3’ untranslated region (UTR) of TGFBR1geneis significantly associated with growth traits and gene expression. Scallops withgenotype TT of c.1815C>T have higher shell length, shell height and striated muscleweight, and have lower TGFBR1expression level in striated muscle (P<0.05).3. The full-length cDNA of members of the Smad family including Smad1, Smad3,Smad4and Smad6are cloned, and genomic organizations of them are determined.Smad1, Smad3and Smad6have7,9and5exons, respectively, and all of them encodetwo conserved domains, MH1(mad homologous domain1) and MH2, as Smad proteinsin other species. While Smad4in Zhikong scallop consists of5exons and only encodeMH1, the MH2domain of which is missed. Real-time qRT-PCR reveals that all the4 Smad genes have high expression levels in gastrulae and lower expression levels inother embryo/larvae stages. Transcripts of Smad1, Smad3, Smad4and Smad6aredetected in all of the adult tissues sampled. Smad1and Smad3are expressed at higherlevels in the striated muscle, and Smad4and Smad6have higher expression levels in thegonad and gill, respectively. The SNP c.846G>T which is a synonymous mutation isidentified in the coding region of Smad3. The association of SD3c.846G>T with growthtraits are analyzed. Scallops with genotype GG have significantly higher values ofgrowth traits than those with genotype GT (P<0.05) in a wild population. The results ofreal-time qRT-PCR show that scallops with genotype GT have higher Smad3expressionlevel in striated muscle than those with genotype GG.