To characterize the regulatory function of Cg NacreinL1 in shell formation of Pacific oyster(Crassostrea gigas),Cg NacreinL1 was cloned,featured in structure characteristics and expressed in mantle,gills,blood lym-phocytes,adductor muscles,gonads,labial flaps,and hepatopancreas of Pacific oyster with shell length of 13 cm±1 cm exposed to normal aerated seawater(blank group),and injected with 100 μL of siRNA-NC(negative group)and 100 μLsiRNA-CgNacreinL1(CgNacreinL1 group)twice.The expression characteristics of Cg NacreinL1 were explored in Pacific oyster exposed to normal aerated seawater with pH of 8.10±0.05(control group),and 7.80±0.05(acidification stress group,prepared by mixture of air and CO2)and regulatory role of Cg NacreinL1 in CaCO3 deposition was verified in Pacific oyster.It was found that Cg NacreinL1 contained one conserved carbonic anhydrase(CA)domain and a region of acidic amino acid residues,with significantly higher expression levels in the adductor muscle(21.24-fold)and mantle(11.54-fold)(P<0.05)than that in the gonad.The maximal expression level was observed in the outer fold of the mantle,which was as high as 54.95-fold and 40.49-fold of that in the inner fold and middle fold,respectively(P<0.05).After incubating with Cg NacreinL1 protein,the average rate of pH de-crease was significantly lower in the NaHCO3-CaCl2composite solution(0.005/min)than that in the control group(0.01/min).After the mRNA expression of Cg NacreinL1 was interfered by RNAi,the prismatic layer structure of the Pacific oyster shell became more tightly arranged,with significantly fewer surface pores and cavities.During the 28-day acidification stress test,the mRNA expression level of Cg NacreinL1 was shown to be initially decreased and then increased across all parts of the mantle.Overall,Cg NacreinL1 was involved in shell formation by inhibiting CaCO3 deposition,with different response patterns in the early and late stages of acidification stress.The findings contribute to comprehensive understanding of the regulatory mechanism of Cg NacreinL1 in shell formation.
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