Identification and Expression Characterization of the Smad3 Gene and SNPs Associated with Growth Traits in the Hard Clam (Meretrix meretrix)

Lulu Fu,Qiudie Chi,Yinghui Dong,Zhihua Lin,Yongbo Bao,Hanhan Yao

doi:10.3390/fishes6040083

Abstract

It has been demonstrated that the sekelsky mothers against decapentaplegic homolog 3 (Smad3) plays an important role in the growth and development of vertebrates. However, little is known about the association between the Smad3 gene and the growth traits of mollusks. In this study, Smad3 from the hard clam Meretrix meretrix (Mm-Smad3) was cloned, characterized, and screened for growth-related single nucleotide polymorphisms (SNPs) in its exons. The full-length cDNA of Mm-Smad3 was 1938 bp, encoding a protein with 428 amino acid residues. The protein sequence included an MH1 (27–135 aa) and MH2 domain (233–404 aa). Promoter analysis showed that the promoter sequence of Mm-Smad3 was 2548 bp, and the binding sites of Pit-1a, Antp, Hb, and other transcription factors are related to the growth and development of hard clams. The phylogenetic tree was divided into two major clusters, including mollusks and vertebrate. The expression level of Mm-Smad3 was predominantly detected in the mantle and foot, while extremely less expression was observed in the digestive gland. The low expression level of Mm-Smad3 was detected at the stages of unfertilized mature eggs, fertilized eggs, four-cell embryos, blastula, gastrulae, trochophore, and D-shaped larvae, whereas an opposite trend was observed regarding the highest expression at the umbo larvae stage (p < 0.05). In the mantle repair experiment, the time-course expression profiles showed that compared to the expression level at 0 h, Mm-Smad3 significantly decreased at 6 h (p < 0.05) but increased at 12 and 48 h. Further, the association analysis identified 11 SNPs in the exons of Mm-Smad3, of which three loci (c.597 C > T, c.660 C > T, c.792 A > T) were significantly related to the growth traits of clam (p < 0.05). Overall, our findings indicated that Mm-Smad3 is a growth-related gene and the detected SNP sites provide growth-related markers for molecular marker-assisted breeding of this species.

Highlights

Drosophila mothers against decapentaplegic proteins (Smads) are pivotal intracellular molecules that transfer signals of transforming growth factor-β (TGF-β) super-family members from the cell surface to the nucleus [1]
The full-length cDNA sequence of Mm-sekelsky mothers against decapentaplegic homolog 3 (Smad3) showed high identity in sequence size to that from other species, such as mammals and shellfish, in which the MH1 and MH2 domain and the C-terminal SSXS phosphorylation sites were more than 90% identical
The domains MH1 and MH2 of the Smad3 play an irreplaceable role in transmitting TGF-β signals and acting on target genes

Summary

Introduction

Drosophila mothers against decapentaplegic proteins (Smads) are pivotal intracellular molecules that transfer signals of transforming growth factor-β (TGF-β) super-family members from the cell surface to the nucleus [1]. The TGF-β superfamily members, including TGF-βs, activins, bone morphogenetic proteins, and growth/differentiation factors, transduce signals via serine/threonine kinase receptors on the cell surface, forming heteromeric complexes, and propagating through phosphorylation of Smad proteins [2]. One of R-Smads, is an important intracellular signaling effector for the TGF/activin signaling pathway It has been identified and characterized in a wide range of species ranging from worms to mammals [4,5,6,7,8], and plays a critical role in cell proliferation and differentiation [9], embryonic development [10], tissue homeostasis and growth, fibrosis [11], bone formation [12] and rebuilding [13], disease treatment [14,15], and wound healing [16] in vertebrates. The mantle is the main organ for shell formation and regulates the extracellular growth of crystals, and secretes matrix proteins [20]

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Conclusion