Studies On The Mechanism Of Copper And Zinc Accumulation In Differential Metal Accumulation Breeding Lines Of Fujian Oyster Crassostrea Angulata

Oysters have very high nutritional value and are cultivated all over the world.The production of oysters in China reached 5.226 million tons in 2019,ranking first in the world.Oysters have a strong ability to accumulate and tolerate metals,especially copper and zinc,which often leads to excessive metal concentrations in oysters,affecting food safety.Therefore,it is of great significance to understand the molecular genetic mechanism of metal accumulation in oysters and to cultivate oyster varieties with low metal accumulation for the healthy and sustainable development of oyster culture.The current researches on metal accumulation in oysters mainly focused on exploring the toxic effects of metals on oysters and the development of biomarkers.The molecular genetic mechanism of metal accumulation is still not deep enough.In this study,Crassostrea angulata,the main species of oysters cultured in Fujian,were used as the research object.The differences in tolerance to copper/zinc were analyzed between the breeding lines with high and low copper and zinc accumulation.Kinetics,proteomics,metabolomics,RNAi and other methods were used to analyze the mechanism of copper and zinc accumulation between different breeding lines.Key functional genes and molecular mechanism of copper and zinc accumulation in oysters were preliminarily explored.The main results are as follows:1.Analysis of tolerance to copper and zinc of oystersThe D-type larvae and 6-month-old adult oysters from different accumulation lines were subjected to copper/zinc acute challenge experiments,and the lethal concentrations(LC50)were calculated.The results showed that in the D-type stage,the oysters from high accumulation line were more tolerant to zinc than oysters from low accumulation line,but there was no significant difference in copper tolerance between the two breeding lines.In adult stage,the oysters from high accumulation line were more resistant to both copper and zinc than oysters from low accumulation line,indicating that the metal accumulation and detoxification mechanisms of the two breeding lines might be different,and would change with the development stage.2.Analysis of zinc accumulation mechanism in oystersThrough kinetic experiments,it was found that the absorption rate and discharge rate of zinc in the oysters from high accumulation line were higher,indicating that the oysters from high accumulation line had stronger zinc absorption and efflux capacity.In addition,the difference in zinc absorption rate constants between the two accumulation lines was much higher than the difference in discharge rate constants,indicating that the difference in zinc accumulation between the two lines might mainly due to the different absorption capacity of zinc.The Zn-binding proteins in the mantles of oysters from different zinc accumulation lines were isolated and purified by improved IMAC technique at 0 and 10 days after zinc exposure.The results showed that oysters with high and low zinc accumulation had different coping mechanisms under zinc exposure.When exposed to zinc,ZIP12,ZIP 14 and ZnT2 were up-regulated in the oysters from high accumulation line,which may promote the process of zinc absorption and transportation.Sulfatase,alcohol dehydrogenase class-3,S-formylglutathione hydrolase and other proteins may play an important role in the storage and detoxification of zinc in oysters.The oysters from low accumulation line might reduce zinc intake by expressing double-stranded RNAspecific editase 1 protein,and increase the expression of ZnT1 during zinc exposure to promote zinc efflux.Glutathione gamma glutamylcystainyl transferase and laccase-2 might play important roles in the detoxification.The expression patterns of snapin,YKT6,PI3K,guanine nucleoside-binding protein related to membrane fusion were different in the two lines,which might indicate that the two lines had different forms of membrane fusion,affecting the accumulation or efflux of zinc.These results indicate that oysters from high accumulation line are more likely to absorb zinc,and store zinc in vesicles,lysosomes and other parts than to excrete zinc.Oysters from low accumulation line absorbed less zinc and tended to excrete more zinc.3.Analysis of copper accumulation mechanism in oystersLC-MS analysis of the metabolites in the mantles of oysters from two accumulation lines exposed to copper for 0 d,3 d and 15 d showed that 125 metabolites were significantly different,mainly involved in amino acid metabolism,purine metabolism and energy metabolism.It was found that oysters from high accumulation line tended to accumulate copper in response to copper exposure.Amino acids and 3aminopropanesulphonic acid might play important regulatory roles in this process.The content of L-cysteic acid in oysters from high accumulation line was always higher than that of oysters from low accumulation line,which indicated that oysters from high accumulation line might have stronger ability to regulate the content of taurine.Taurine might play an important role in alleviating the toxicity of copper,which partly explained the reason why oyster from high accumulation line had stronger ability of copper accumulation and detoxification.The content of guanosine and inosine in oysters from high accumulation line was significantly lower than that of oysters from low accumulation line,and the content of xanthine in oysters of two accumulation lines decreased significantly after 15 days of copper exposure,indicating that copper accumulation or copper exposure may affect the purine metabolism of oysters.The variation trends of glucose,phosphoenolpyruvate,palmitic acid and other energy substances in oysters from two accumulation lines were different,indicating that copper exposure affected the energy metabolism process of oysters,and the energy supply modes of oysters from high and low accumulation lines might be different.The mechanism of copper enrichment related gene ATP7A was studied by RNA interference.Through feeding RNAi experiments,it was found that the ATP7A gene interference group had a higher amount of copper accumulation compared to the control group,indicating the ATP7A gene played an important role in the excretion of copper.Individuals with high ATP7A expression accumulated lower copper,which may be one of the important reasons for the difference in copper accumulation among oysters.