| Oyster is an important marine fishery resource cultivated globally and the largest sector of bivalve in global trades.Oysters become the most popular shellfish because of its soft flavor and its rich nutritional value with high quality protein,taurine,amino acids,vitamins and other nutrients.There are more than 30 oysters along the coast of China,while the Pacific oyster,Crassostrea gigas is the major cultivated oysters distributed in the coast of Bohai Sea and Yellow Sea.Glycogen,which contributes to the flavor of oysters,is not only one of the important evaluation index of oyster nutritional value,but also closely associated with physiological activities such as growth,reproduction and stress tolerance.Oysters have great glycogen variation between individuals,but little is known about the molecular and chemical mechanisms underlying glycogen content differences in Pacific oysters.In this study,the molecular basis of glycogen content difference in Pacific oysters were revealed based on regulatory network via multiple-omics analysis and functional study of pivotal genes.The mechanism research of the oyster glycogen regulation can provide new insights for high-quality oyster molecular breeding.We selected the top 10–15 high and low glycogen content individuals from 185half--sibling oyster families raised under a consistent environmental condition which were used to conduct transcriptome and metabolome analysis.Homogeneous culture helps minimize the impact of environment factors.We identified 1888differentially-expressed genes,seventy-five differentially-abundant metabolites,which are part of twenty-seven signaling pathways that were enriched using an integrated analysis of the interaction between the differentially-expressed genes and the differentially-abundant metabolites.Accordingly,the following findings were based on the oyster glycogen regulation network constructed by transcriptome and metabolome analysis.(1).Oyster glycogen content is close related to the fatty acid degradation capacity.A correlation analysis showed oyster glycogen content had a positive correlation with the free fatty acids.Simultaneously,in high-glycogen content oysters,high expression of OLAH and PPT,which participate in the release of fatty acids from the fatty acid synthase complex,together with abundant levels of myristic acid and palmitic acid implicate the higher fatty acid accumulation ability in high glycogen oysters.While in low-glycogen oysters,a high level of CPT2 expression increases flux through the fatty acid degradation pathway,causes a lowering of the free fatty acid levels and a reciprocal increase in long chain fatty acyl-Co A levels that then suppress glycogen accumulation.(2).Amino acid content and conversion efficiency to glucose play crucial role in glycogen content.We observed an increase in the abundance of glucogenic amino acids,as well as increase in the level of pyruvic acid and PEPCK expression which catalyzes the first committed step in gluconeogenesis.Taken together,we speculate that this increase in abundance of glucogenic amino acids(i.e.gluconeogenic substrates)coupled with an increase in gluconeogenic capacity leads to increased de novo production of glucose and ultimately to increased glycogen synthesis.(3).Oysters with higher glycogen content exhibited increased energy metabolism.The high level of expression of the HK(Hexokinase)and PK(Pyruvate kinase)genes involved in glycolysis and Malate dehydrogenase(MDH)and pyruvate carboxylase(PYC)that participate in the TCA cycle in high-glycogen oysters reveals that these oysters have a higher energy metabolism compared to low-glycogen content oysters and implies that they have a greater resistance to stress.(4).Higher glycogen content oyster manifests as a higher anti-adversity ability.Although it has been widely recognized that glycogen participates in stress resistance in the oyster,the molecular mechanism behind this has not been identified.Making use of integrated analysis of transcriptome and metabolome,we suggest that GPX5 may be an important enzyme in the mechanism by which glycogen contributes to stress resistance in oysters.We selected glycogenin as candidate gene through transcriptome analysis.Although the glycogen synthase and glycogen phosphorylase involved in glycogen metabolism in Pacific oysters have been basically studied,the study of glycogenin as the initiator of glycogen synthesis is absent.Therefore,we identified and cloned C.gigas glycogenin(CgGN)to investigate its role in the initiation of glycogen synthesis and the correlation with glycogen content.Alignment of CgGN c DNA with the genomic sequence revealed that CgGN have three isoforms containing alternative exon regions.The phylogenetic tree showed that vertebrate and invertebrate glycogenin clustered separately in two distinct groups and CgGN belongs to glycogenin-1.With the aid of q-RT-PCR,we demonstrate that CgGN expression varied seasonally in the adductor muscle and gonadal area and was the highest in the adductor muscle.CgGN expression was significantly related with glycogen content in oysters.Furthermore,a site-directed mutagenesis experiment was performed to investigate the glycosylation site of CgGN.Sequence alignment indicates that Tyr-200 in CgGN corresponding to Tyr-195 of mammalian glycogenin-1,known to be the site of carbohydrate attachment.Differences in molecular weight among the Western blotting bands revealed that the Tyr200 Phe and Tyr202 Phe mutations could affect CgGN autoglycosylation.After autoglycosylation,CgGN can interact with glycogen synthase(CgGS)to complete glycogen synthesis.Additionally,subcellular localization analysis showed that CgGN isoforms and CgGS were located in the cytoplasm.In conclusion,CgGN has a glycosylation site corresponding to mammalian glycogenin-1 and can interact with CgGS to complete glycogen synthesis.We used q-RT-PCR to detect the expression patterns of CgFFAR4 in different developmental stages and tissues of Pacific oysters.CgFFAR4 expression increased sharply at the D-larval stage which is the critical moment of velum development.CgFFAR4 express in various tissues while was highly expressed in hepatopancreas,stomach,and intestine,which are associated with the oyster digestion tract.Additionally,relative expression of FFAR4 to EF(elongation factor)in C.gigas hepatopancreas and nerves significantly declined after fasting.All these data demonstrated that CgFFAR4 have a critical role in oyster ingestion and digestion.To further elucidate CgFFAR4 function,we performed CgFFAR4 knock down in oysters.Reduced glycogen content after CgFFAR4 knockdown revealed that it is involved in regulation of fatty acid and glycogen content.Decreased gene expression of insulin receptor,insulin receptor substrate and glycogen synthase in oysters indicates that FFAR4 is involved in the regulation of glycogen and FFA content via insulin pathway.Additionally,FFAs were not only nutrients but also contributes to the anti-inflammatory response.In Pacific oysters,we found that LPS stimulation decreased the expression of CgFFAR4,which may contribute to the animal's immune response.The immune function of FFAR4 is conserved in both invertebrates and mammals.In conclusion,this is the first time to map the regulation networks of oyster glycogen integrating transcriptome and metabolome analysis.Furthermore,we screened the candidate gene CgGN and comprehensively explain its function of glycogen synthesis initiation.Meanwhile,FFAR4 function was firstly studied on nutrition regulation and immune response in invertebrate.Our study has not only revealed the molecular basis of the relationship between amino acids,fatty acids,and glycogen,but also provides a molecular explanation for stress resistance in oysters.These findings provide new insights into the study of quality traits and could promote research into the molecular breeding of oysters. |
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