Preliminary Study On Air Exposure Response Mechanism Of Crassostrea Gigas Based On Transcriptome Data

The Pacific oyster Crassostrea gigas is a bivalve mollusc that lives in the intertidal zone and is also an important marine aquaculture species in China.The environment in the intertidal zone is complex and variable,especially the cyclical fluctuations of the tides that expose the C.gigas to the air for extended periods of time.The response mechanism against air exposure is extremely complex and involves multiple life processes such as energy metabolism,immune response,cell homeostasis and osmotic pressure balance.However,current research started with only a few molecules or signaling pathways and lack systemicity and comprehensiveness.Gene coexpression network analysis(WGCNA)constructs a correlation network based on gene expression profiles,and can reveal the gene expression patterns of each sample through modularization and systematic analysis.It is an ideal method to study complex life processes such as air exposure response.In this study,gene co-expression network analysis,protein interaction network and phylogenetic analysis were used to analyze the transcriptome data of oyster gills and adductor muscle tissue under air exposure.The molecular mechanism of stress response of C.gigas under air exposure was firstly elucidated from three aspects: energy metabolism,protein homeostasis and apoptosis.The transcriptome data of C.gigas and adductor muscle under air exposure were analyzed by WGCNA.Genes were clustered into 22 modules,among which red,cyan,darkgrey module had significant expression characteristics in the adductor muscle.Further analysis found that on the 5th day under air exposure,the differentially expressed genes in the adductor muscle were enriched as GO terms such as "respiratory chain" and "ribosomal biogenesis";on the 7th day,GO terms such as "TRAIL-activated apoptotic signals ","negative regulation of protein processing",and "negative regulation of protein phosphorylation" were enriched;on the 9th day,GO terms such as "galactolipid metabolism process" and "phospholipid catabolism process" were enriched.The above results indicate that C.gigas rely on glycogen metabolism to maintain energy supply in a short period of time(within 5 days)under air exposure,and the energy cost of maintaining basal metabolism of C.gigas under air exposure stress increases.With the continuation of stress,the negative regulation of protein processing and maturation and the activation of the apoptotic pathway suggest that C.gigas reduce metabolic activity in the body to maintain the basic energy expenditure required for survival.In the late stage of air exposure stress,C.gigas rely on metabolic lipids to sustain life and the glycogen and protein in the body are exhausted.The brown module is a module that significantly correlates the expression level of the gene in gills with the air exposure time.It was found that the enrichment of KEGG pathway is mainly related to “ER to Golgi transport vesicle membrane” and “Protein processing in endoplasmic reticulum”,which indicated that this gene cluster was mainly involved in the ERAD pathway and ER stress response.The construction of the hub gene co-expression network revealed that the network centered on STK17 A,a protein kinase that induces apoptosis.Protein-protein interaction network and KEGG pathway annotation analysis showed that endoplasmic reticulum-mediated Nterminal glycosylation,endocytosis and endoplasmic reticulum-related degradation pathways constitute the network,and the central gene LOC583350 is E3 ubiquitin ligase MIB2.Studies have shown that the brown module is a regulatory network related to ER stress in C.gigas cells.Some transcription factors containing zinc finger domains play an important role as the regulatory center of the network.The ER acts as a sensor of cellular stress and can regulate apoptosis,autophagy,and protein degradation to maintain cell homeostasis.Analysis about transcriptome data of gills tissue revealed that the expression level of IAP family genes increased significantly under air exposure.The phylogeny of the IAP family genes was analyzed and it was found that the IAP gene family is divided into two branches according to its domain and phylogenetic relationship,one of which is a unique evolutionary branch of molluscs,and the other is a ubiquitous branch of animals.Part of the IAPs in oyster herpes virus OsHV-1 is clustered with oyster IAPs,indicating that the IAP in the virus is derived from the gene level transfer of the host organism.The non-synonymous mutation rate of oyster IAPs is much higher than that of other species in this subtype,confirming that the presence of viral homologs of the host gene will result in selection pressures that favor amino acid substitution in the host molecule.The above results indicate that the response mechanism of C.gigas to air exposure stress has significant characteristics both in time and space.In time,different stages of air exposure have different energy utilization strategies.Rich glycogen provides sufficient energy to maintain damage caused by mild stress.And when stress increases,apoptotic pathways can be activated,and the overall energy strategy changes from a positive compensation strategy to a conservative energy-saving strategy.In space,different tissues respond differently to air exposure.As a sensitive sensor to the external environment,the ER in gill cells can adjust the homeostasis strategy according to the magnitude of the environmental stress,and rationally allocate energy to meet the needs of damage repair,transcription regulation and apoptosis.The relevant pathway induced by IAPs may be an important mechanism for C.gigas to adapt to the intertidal environment.The expansion of IAPs in arthropods and mollusks may be inextricably linked to virus infection and adaptation to complex environments.This study provides some guidance and reference for subsequent in-depth research.