Molecular Mechanism Of The Tolerance Under Air Exposure And Response Of Pinctada Fucata Martensii

Air exposure is one of the environmental stress factors often faced by intertidal organisms.Although bivalves living in the intertidal zone have certain air exposed tolerance after long-term selective evolution,there are still great differences in air exposed tolerance among different species or populations,which have an important impact on their survival,physiology,metabolism.P.f.martensii is the only industrialized sea pearl oyster in China.In the process of pearl production,nucleus insertion operation and monthly fixtures cleaning often result in long-term exposure to air,which causes stress response.This study explores the P.f.martensii of the black shell color selection of dry tolerance ability,build a air exposed sensitive group(S)and tolerance(T),the use of the transcriptome and analyses the difference expression of miRNAs group,the preliminary analysed P.f.martensii revealing the molecular mechanism of stress response,in order to meet P.f.martensii art mechanism and variety to accumulate basic data and theoretical guidance.The results are as follows:(1)Under the conditions of 22℃-34℃,the air exposed tolerance of P.f.martensii decreased with the increase of temperature and the extension of air exposed time;At22 ℃,the expression levels of proline hydroxylase(PHD),glucose transporter(GLUT),myeloid differentiation factor(MYD88),heat shock protein(HSP70,HSP20)and apoptosis inhibitor(c IAP)in groups S and T showed an increasing trend during air exposed,and the differences between groups were significant(P <0.05);The results showed that the survival rate of the tolerant group was significantly higher than that of the sensitive group(P <0.05).(2)The transcriptome library of S and T groups before air exposure,2h air exposed(large opening time of S group)and 14 h air exposed(near death time of S group)was constructed.The transcriptome data were analyzed,and the results showed that:Under initial air exposed stress,small GTP protein,anti-apoptotic system,HSP family genes,cell matrix and antioxidant defense system cooperated to protect the body cells from oxidative damage in T group.T group significantly upregulated a large number of genes related to energy metabolism,oxidative phosphorylation,anti-apoptotic gene BIRC and hypoxia-inducible factor HIF-α in response to adverse environment under long-term air exposed stress.In group S,the opening phase was mainly mediated by the adjustment of extracellular matrix and early stress genes(HSP and BIRC).P.f.martensii oysters,transcriptome expression of hyperactive,heat shock protein,antiapoptotic gene,solute carrier family,still increase amount of gene expression,such as cytoplasmic matrix through to cut gene function analysis found that energy metabolism and electronic chain enzymes related to express a cut,may be the important cause of mother P.f.martensii overall failure.These results indicate that HSP family genes,cytoplasmic matrix regulation,timely anti-apoptotic events,signal transduction mediated by small molecule GTP-protein,energy release mediated by oxidative phosphorylation and regulation of metabolic rate play important roles in the response to air exposed stress of P.f.martensii.Comparison between T group and S group showed that signal transduction mediated by small GTP-protein,high expression of HSP family genes and anti-apoptotic gene BIRC,and enrichment of HIF-1 signal pathway in T group might be the reasons for the strong air exposure tolerance in T group.(3)In this study,197 differential miRNAs were obtained by Small RNA sequencing,including 129 unknown miRNAs and 68 known miRNAs.The following miRNAs were obtained through further analysis and screening:miR-7-5p＿4、miR-7-5p＿6、miR-31＿3、miR-33＿3、miR-34＿2、miR-34＿4、miR-34-5p＿4、let-7-5p＿6、miR-278-3p＿6、novel＿mir14、novel＿mir25、 novel＿mir54、novel＿mir149.These different miRNAs mainly target genes related to redex,material transport ABC and GLUT,cytoskeleton regulation MUC,lipid metabolism SCD and PARP1,heat shock protein HSPA5,small GTP-protein GPCRs,anti-apoptotic XIAP,ubiquitin-protease TRIM,etc.The temporal sequence expression results showed that these differences were significantly correlated with the air exposed stress of P.f.martensii,and there were significant differences between the S group and the T group.The above results indicated that in the regulation network of miRNA-m RNA,The 7 differential miRNAs screened may be an important part of their adaptive stress response by regulating the target genes screened by transcriptome under air exposed stress.(4)Analysis of the transcriptome data obtained in some key genes(Pfm-ALDOB,Pfm-ABC,Pfm-GLUT,Pfm-XIAP,Pfm-PHD)gene cloning and the total length of the transcriptome are used to get the total length of the sequence,obtained the full length of gene ORF box,the physical and chemical properties,functional site,secondary structure,the structure of the protein domains,the transmembrane domain structure,more species homology comparison,three-dimensional structure of protein bioinformatics analysis,and examined these genes in different tissues and the expression differences under air exposed stress significantly(P<0.05),and the expression levels of Pfm-ALDOB,Pfm-GLUT,Pfm-XIAP and Pfm-PHD were the highest in gills,while the expression levels of Pfm-ABC were the highest in gonads.The expression of Pfm-HIF,Pfm-PHD,Pfm-GLUT,Pfm-ALDOB,Pfm-PEPCK,Pfm-ABC,and Pfm-XIAP increased significantly with the extension of air exposure time,and the expression of S group was higher than that of T group except Pfm-XIAP.Pfm-Hsp20 and Pfm-Hsp70 showed an upward trend in the S group and 0-16 h in the T group,and were significantly higher than those in the S group at 16 h(P<0.05),and then showed a downward trend at 24 h.The expression of Pfm-DLL fluctuated up and down in the T group,but there was no significant change in the S group at 16 h(P<0.05),and there was a downward trend at 24 h.Pfm-RAS decreased in both groups.Pfm-STRAP showed a downward trend in group S and a downward trend in group T.In addition,dual luciferase results show that there is a targeted regulatory relationship between novel＿miR54 and Pfm-GLUT,and between novel＿miR149 and Pfm-XIAP.These results indicated that the genes related to hypoxia,immunity and metabolism might play an important role in the air exposed stress of P.f.martensii,and there were significant differences in the expression of these related genes between the tolerant group and the sensitive group.It was speculated that the different expression patterns in the S and T groups reflected the different tolerance of the phenotypes to air exposure.