| Shell physical barrier and non-specific immune system are the two main defense mechanisms of shellfish.It has been shown that phenoloxidase is not only a key catalytic enzyme of the prophenoloxidase activating system（proPO）,an important pathway of the non-specific immune system in shellfish,but it is also found to be widely present in shells as a matrix protein involved in the biomineralization process of shell formation.However,little is known about the types of phenol oxidase enzymes,their tissue distribution characteristics,and their biological functions in shellfish,especially in freshwater shellfish.In this study,we used the freshwater pearl mussel,Hyriopsis cumingii,as experimental material,and firstly screened the specific inhibitors of total phenol oxidase and its three types of tyrosinase,catecholase and laccase,and analyzed the tissue-specific distribution characteristics of the above enzymes.Secondly,we analyzed the characteristics and correlations of the changes in phenol oxidase enzyme activity,mRNA expression,oxidation level,and immune indicators such as SOD enzyme activity of H.cumingii with the three treatments of high-temperature stress,pearl-sac planting surgery and cerium treatment.Finally,a new tyrosinase gene HcTyr2 was isolated and cloned,and analysis of the correlation between the changes in the relative mRNA expression levels and the differences in the nacreous layer of newborn shells after shell notching with RNA interference,to explore the function of this gene in the shell biomineralization of H.cumingii.The results of the study are as follows.1.Identification of phenoloxidase species and their tissue-specific distributions in H.cumingiiFirstly,the enzyme activities of total phenol oxidase,laccase,and tyrosinase were used L-DOPA,hydroquinone,and L-tyrosine disodium salt as specific substrates respectively,which were determined in six tissues,including gill,hemolymph,and mantle pallial.Next,the phenol oxidase candidate inhibitors phenylthiocarbamide（PTU）,CTAB（cetyltrimethylammonium bromide）,PMFS（phenylmethylsulfonyl fluoride）,citric acid,kojic acid,and Na2EDTA were screened,and 4-hexylresorcinol（4-HR）was used as inhibitor of tyrosinase and laccase enzyme activities.Again,the tissue expression characteristics of laccase and tyrosinase gene mRNAs were analyzed by quantitative real-time PCR（qRT-PCR）.The results showed that the total phenol oxidase activity was highest in hemolymph supernatant,followed by gill,and lowest in mantle edge,mantle pallial and hepatopancreas;the laccase enzyme activity was significantly higher in hemolymph supernatant than in other tissues（P<0.05）;the tyrosinase enzyme activity was significantly higher in hemolymph cell than in gill,mantle edge,mantle pallial and hemolymph supernatant（P<0.05）.Finally,the relative mRNA expression levels of four genes,laccase1,Laccase2,HcTyr1,and HcTyr2,were significantly higher in mantle edge than in the other five tissues（P<0.05）.The above results showed that there were three types of phenol oxidase,laccase,tyrosinase and catecholase in H.cumingii with the highest enzyme activity in hemolymph supernatant,and laccase may be the main type of phenol oxidase.2.Changes in the response of phenol oxidase and functional analysis with three treatments of high-temperature stress,pearl-sac planting surgery,and cerium treatment in H.cumingiiIn this study,we investigated the role of phenol oxidase in the immune response of H.cumingii through the experiments of response of phenol oxidase to high temperature stress in mussels with different shell colors,the experiments of pearl-sac development of H.cumingii after planting surgery,and the experiments of phenol oxidase response change with different concentrations of cerium treatment.（1）Responses of phenol oxidase to high temperature stress in the mussels with different shell colorsIn this experiment,the mussels were subjected to high-temperature stress（34℃）,and the changes of phenol oxidase enzyme activity,relative expression levels of phenol oxidase-related genes and ROS levels in mantle pallial were measured at 0,6,12,24,48,and 96 h after treatment.The results showed that the phenol oxidase enzyme activity of both purple and white pearl-layered mussels showed an upward and then downward trend during high temperature stress,with the highest enzyme activity at 24 h（P<0.05）;the relative expression levels of HcTyr1,Laccase1,and Laccase2 mRNAs at 12 h were significantly higher than those at other time points（P<0.05）.The relative expression levels of SOD,CAT,T-AOC enzymes,and H2O2content increased significantly and then decreased（P<0.05）,and the relative expression levels of SOD and CAT mRNAs increased significantly and then decreased（P<0.05）.The results suggest that the mussel may increase the ROS level by activating the proPO system,increasing the activity of key enzyme phenol oxidase and mRNA expression level,and thus responding to the immune response induced by high temperature.（2）Responses of phenoloxidase to implantation surgeryThe phenoloxidase enzyme activity and ROS levels in hemolymph of H.cumingii were measured at 6 h,12 h,24 h,48 h,96 h,8 d,and 12 d after implantation surgery.The results showed that the phenol oxidase enzyme activity increased significantly（P<0.05）at 12 h,48 h,and 8 d,laccase enzyme activity increased significantly（P<0.05）at 48 h.ROS levels increased significantly（P<0.05）at 48 h.The results suggest that the pearl sac of H.cumingii may respond to the immune response by activating the proPO system,increasing the enzymatic activity of the key enzyme phenol oxidase,and producing large amounts of ROS after implantation surgery.（3）Responses of phenol oxidase to different concentrations of rare-earth cerium treatment in H.cumingiiIn this experiment,we used different concentrations of rare earth cerium(10-4,10-3,10-2 M)as treatmen with implantation surgery,measured the phenol oxidase enzyme activity in the pearl sac tissue,screened the phenol oxidase related differential genes,measured the oxidative stress-related indexes such as ROS level,and analyzed the changes and correlation in the chemical characteristics of the pearl sac tissue on the 6th day after treatment.The results showed that the phenol oxidase enzyme activity in the pearl sacs was significantly higher（P<0.05）in the group treated with high concentration(10-2 M)than in the control group.Comparative transcriptome analysis of the different concentration treatment groups showed that the mRNA expression levels of three Tyrosinase unigenes and two Laccase unigenes were significantly different among the twenty-six phenoloxidase-related genes.Compared with the control group,ROS level in pearl sac increased significantly in the 10-2 M rare earth cerium-treated group and decreased significantly in the 10-3 M rare earth cerium-treated group（P<0.05）;meanwhile,SOD and CAT enzyme activities increased significantly with low concentration of rare earth cerium treatment（P<0.05）.The MDA and H2O2 contents increased significantly（P<0.05）with treatment of high concentration cerium.17 SOD genes related unigenes obtained in comparative transcriptomes,three of which were significantly differentially expressed.The results of histochemical analysis in pearl sac showed that the formation of pearl sacs was accelerated with the increase of rare earth cerium treatment concentration,which promoted the aggregation of hemolymph in the lumen of pearl sacs and the morphological transformation of pearl sac epithelial cells.The results suggest that the rare-earth cerium treatment of H.cumingii may participate in the immune response during pearl sac development by activating the proPO system and increasing the enzymatic activity of the key enzyme phenol oxidase,which in turn regulates the developmental process of pearl sacs.The above results suggest that the mussel may regulate the autoimmune response by activating the proPO system and increasing the phenol oxidase activity and ROS level in vivo with exogenous stimulation.3.Molecular characterization of the tyrosinase HcTyr2 gene and its function in the biomineralization of the mussel shellIn this study,the changes in the relative expression levels of HcTyr2 mRNA were detected by qRT-PCR at different developmental stages and after shell notching treatment,and the changes in the relative expression levels of HcTyr2 mRNA and newborn seashell crystal structure difference in the mussel treated with ds HcTyr2after shell notching treatment by qRT-PCR and scanning electron microscopy.The results showed that the relative expression levels of HcTyr2 mRNA increased significantly（P<0.05）in the gastrulae stage and unmatured glochidia stage,and the relative expression levels of HcTyr2 mRNA increased significantly（P<0.05）at 24 h and 9 d during the shell regeneration stage after shell notching.The inhibition of tyrosinase and RNA interference not only inhibited periostracum growth but also led to structural disorders of aragonite tablets in the pearl layer.The above results suggest that HcTyr2 may be involved in the development of mussel shells and may regulate the growth of shell crystals by cross-linking quinone proteins from soluble matrix proteins to insoluble matrix proteins through oxidation.In summary,three phenol oxidase types are present in H.cumingii:laccase,catecholase,and tyrosinase.Laccase may be mainly involved in the non-specific immune response,while tyrosinase may be mainly involved in the regulation of biomineralization.The above results provide a basis for resolving the biological functions of phenol oxidases in H.cumingii. |
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