Association and Functional Analyses Revealed That PPP1R3B Plays an Important Role in the Regulation of Glycogen Content in the Pacific Oyster Crassostrea gigas.

Abstract

The Pacific oyster (Crassostrea gigas) is one of the most important aquaculture species worldwide. Glycogen contributes greatly to the special taste and creamy white color of oysters. Previous genome-wide association studies (GWAS) identified several single nucleotide polymorphism (SNP) sites that were strongly related to glycogen content. Genes within 100 kb upstream and downstream of the associated SNPs were screened. One gene annotated as protein phosphatase 1 regulatory subunit 3B (PPP1R3B), which can promote glycogen synthesis together with protein phosphatase 1 catalytic subunit (PPP1C) in mammals, was selected as a candidate gene in this study. First, full-length CgPPP1R3B was cloned and its function was characterized. The gene expression profiles of CgPPP1R3B in different tissues and seasons showed a close relationship to glycogen content. RNA interference (RNAi) experiments of this gene in vivo showed that decreased CgPPP1R3B levels resulted in lower glycogen contents in the experimental group than in the control group. Co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) assays indicated that CgPPP1R3B can interact with CgPPP1C, glycogen synthase (CgGS) and glycogen phosphorylase (CgGP), thus participating in glycogen metabolism. Co-sedimentation analysis in vitro demonstrated that the CgPPP1R3B protein can bind to glycogen molecules directly, and these results indicated the conserved function of the CgPPP1R3B protein compared to that of mammals. In addition, thirteen SNPs were precisely mapped in this gene. Ten of the thirteen SNPs were confirmed to be significantly (p < 0.05) related to glycogen content in an independent wild population (n = 288). The CgPPP1R3B levels in oysters with high glycogen content were significantly higher than those of oysters with low glycogen content, and gene expression levels were significantly associated with various genotypes of four associated SNPs (p < 0.05). The data indicated that the associated SNPs may control glycogen content by regulating CgPPP1R3B expression. These results suggest that CgPPP1R3B is an important gene for glycogen metabolic regulation and that the associated SNPs of this gene are potential markers for oyster molecular breeding for increased glycogen content.