Mechanism Of Ceruloplasmin In Cadmium Tolerance Of Dunaliella

Ceruloplasmin（CP）is a glycoprotein found in various organisms.The known physiological functions include copper transport,organic amine oxidation,iron oxidase activity,regulation of cellular iron level,glutathione peroxidase and ascorbic acid oxidase activity and antioxidant activity.In this study,transcriptome analysis was performed on Dunaliella Dp815 exposed to 25 mg/L CdCl₂·5H₂O.It was found that ceruloplasmin expression was significantly up-regulated at 96 h after exposure.Ammonium thiomolybdate（TTM）,an inhibitor of polycopper oxidase,was added to determine that ceruloplasmin was related to cadmium adsorption and accumulation of Dp815.In order to further explore the mechanism of ceruloplasmin of Dp815 in cadmium tolerance,the domain and full-length gene of ceruloplasmin of Dunaliella Dp815 were amplified by PCR to construct recombinant ceruloplasmin domain CP-D and full-length CP-Full expression vector pET-28a-CP-D and pET-28a-CP-Full,respectively.The constructed recombinant plasmid was transformed into E.coli BL21（λDE3）,the recombinant was screened by kanamycin resistance,and the recombinant bacteria BL21（pET-28a-CP-D）and BL21（pET-28a-CP-Full）were identified by colony PCR and sequencing.The expression was induced by IPTG and the protein expression was detected by SDS-PAGE electrophoresis.The recombinant strain BL21（pET-28a-CP-D）was exposed to 1,2 and 4 mg/L CdCl₂·5H₂O,and the recombinant strain BL21（pET-28a-CP-Full）was exposed to 0.5,1,5 and 10 mg/L CdCl₂·5H₂O,and the transformed strain BL21（pET-28a）obtained from E.coli BL21 transformed with empty plasmid pET-28a was used as control to determine its adsorption and cumulative effect on cadmium.The results showed that the recombinant strain BL21（pET-28a-CP-D）successfully expressed the target protein with a molecular weight of about 35 k Da at 18℃and induced by 0.05 m M IPTG for 6 h,and the adsorption and accumulation of cadmium decreased significantly,indicating that the ceruloplasmin domain CP-D of Dp815 could reduce the adsorption and accumulation of cadmium by Dp815.The recombinant strain BL21（pET-28a-CP-Full）successfully expressed the target protein with a molecular weight of about 130 k Da at 25℃and induced by 0.4 m M IPTG for 20 h,and its adsorption and accumulation of cadmium increased significantly,indicating that the full-length CP-Full of ceruloplasmin from Dunaliella Dp815 could increase the adsorption and accumulation of cadmium by Dp815.This study explored the mechanism of ceruloplasmin in cadmium tolerance of Dunaliella tumefaciens and provided an experimental basis for the development of related biological agents to remove cadmium pollution in the environment.