Molecular Cloning And Expression Analysis Of β-actin And TGF-β Recepter In Scylla Paramamosain

The mud crab Scylla paramamosain is an important marine aquaculture crab in China and it is mainly distributed along the Indo-West Pacific regions andthe southeast of China, including coastal region of Zhejiang, Fujian, Taiwan, Guangdong, Guangxi and Hainan. In recent years, more and more researchers begin to study Scylla paramamosain for its important economic value. During high-throughput sequencing work in our laboratory, wo found some functional genesassociated with growth traits—β-actin gene and TGF-β Recepter gene.1. β-actin gene is a member of the actin family, mainly involved in maintaining cell structure, movement and physical activity in cell division, and it plays an important role in quantitative genetic experiments or semi-quantitative research. β-actin gene has a potent promoter which can improve other genes’ expression. This advantage makesβ-actin play an impotant role in the reasearch of transgenic animals. And also β-actin is used to construct molecular evolutionary tree, to analyses the evolution of the relationship between species. In this study,RT-PCR and RACE techniques were used to amplify the cDNA full-length of β-actin in the mud crab (Scylla paramamosain). Sequence analysis showed that the full cDNA of β-actin gene was1434bp long, including a1131bp open reading frame (ORF), a83bp5’ untranslated region (5’ UTR) and a243bp3’ untranslated region (3’ UTR). The ORF encoded376amino acids residues with apredicted molecular mass and PI of approximately41.79KDa and5.205respectively. Multiple alignment with homologous amino acid residues was performed and it indicated a high similarity among the18species. Scylla paramamosain was more closely related to Gecarcinus lateralis than any other species according to the neighbor joining (NJ) phylogenetic tree. Real time-PCR results indicated that β-actin gene expressed in all eight different tissues including blood, heart, liver, stomach, gill, muscle, testis and connective tissue, with the highest expression in muscle and lowest expression in connective tissue. Owing to the significant differences of RNA expression among these tissues, this gene is not suitableused as an endogenous control. 2. TGF-β R genes are important receptors, to start the TGF-β super familymembers in signal transduction. These receptors start the different biological functions by binding to type II and type I receptors on the cell surface. In this study, TGF-β R is to be cloned, and the nucleotide and deduced amino acid sequences of each gene are analyzed. The expression level is analyzed by real-timeqRT-PCR.In this experiment, we get TGF-β R gene’s full length cDNA sequence forthe first time. Sequence analysis showed that the full cDNA is2035bp, including open reading frame1164bp, the5’UTR607bp, the3’UTR264bp, polyreal (A) tail16bp. Using SMART software to analysis TGF-βR gene’ secondary structure, we find that the gene has outside the membrane, intracellular and transmembrane region with characterized conserved domains of serine/threonine kinase receptors, and has obvious signal peptide sequence composed of22aminoacid residues. Sequence alignment analysis showed: the genes in the region ofthe GS is highly conservative, and the homology with type I receptor is higher.The gene is expressed in all organizations, with the highest expression in thegonads and muscle tissue. This indicates that the genes involved in the growthregulation in Scylla paramamosain.According to the amplification by the genetic sequence of a introns, wo get sequence of a intron of302bp. Alignment the sequences of the intron among17individuals, we found that sequences is completely consistent among17individuals. The result provides the basic condition for further studies SNPs ofTGF-βR genes correlation with growth traits analysis.