The Expression Of CYP4 In Perinereis Aibuhitensis Under Benzo(a)pyrene And Phenanthrene Exposure

In order to reveal the PAHs detoxification mechanism in CYP4 Perinereis aibuhitensis,and provide information for the application of P. aibuhitensis in ecological pollution risk assessment, in this study the P. aibuhitensis CYP4 ORF fragments was amplicated according to the whole cDNA sequence of P. aibuhitensis CYP4 by cloning technology, prokaryotic expression system was constructed to obtain the recombinant protein. Furthure, the gene and protein expressions regulation of P. aibuhitensis CYP4 under B(a)P and phenanthrene exposure were examined. The results are showed as follows:(1) Prokaryotic expression system was constructed with pET-28 a and E.coli BL21 competent cells. The best inducing temperature was 37 ℃and the best IPTG inducing concentration was 0.8mmol/L. The recombinant protein was confirmed by SDS-PAGE. The results revealed that the expressed recombinant protein was inclusion bodies. In order to obtain antibody of CYP4, the recombinant protein was purified and renaturated, and was used to immune rabbit after the concentration was determined.(2)The gene expression characteristics of CYP4 in P. aibuhitensis under B(a)P exposure were detected through real-time fluorescence quantitative PCR. Results showed that the gene expression levels at 0.5 μg/L and 50 μg/L groups were increased, while the other groups were not obviously different with that of the control group.The 0.5 μg/L group expression levels of CYP4 was significantly different with control group(P<0.01). The expression levels of CYP4 increased obviously in day 7, and showed obvious dose-effect relationship. Gene expression in 10 μg/L concentration group was obviously different with that of the control group(P <0.05). The expression levels of CYP4 decreased in day 14, and expression levels of CYP4 in 5 μg/L and 50 μg/L groups were obviously different with that of the control group(P<0.05), while the other groups were not obviously different with the control group.(3) The gene expression characteristics of CYP4 of P. aibuhitensis under phenanthrene exposure were detected through real-time fluorescence quantitative PCR.The expression levels of CYP4 decreased gradually with the increase of concention in day 4, and all groups expression levels of CYP4 was significantly lower than the control group(P<0.01). In 7 days, the gene expression level in 50μg/L group was significant lower than the control group(P<0.01).However, CYP4 gene expression in the other concentration groups was not obviously upregulated. In 14 days, CYP4 gene expressions in all groups were lower than the control group with no significant difference.(4) The protein expression characteristics of CYP4 of P. aibuhitensis under B(a)P exposure were detected by indirect ELISA. In 4 days, the protein expression in all groups had no obviously difference.In 7 days, The protein expression levels of CYP4 in 5 μg/L group was extremely different with that of the control group(P<0.01), while the other groups were not obviously different with that of the control group.In 14 days, the protein expression level of CYP4 in 50μg/L group was the highest, the protein expression levels of CYP4 in 5μg/L group was higher than that of control group with significant difference(P<0.05), and protein expressions in the other groups were not obviously different with that of the control group.(5) The protein expression characteristics of CYP4 of P. aibuhitensis under phenanthrene exposure were detected by indirect ELISA. In 4 days, protein expression levels in all groups were lower than the control group, and exhibit negative relationship with concentration of phenanthrene. The protein expression levels of CYP4 in 5μg/L and 10μg/L groups were obviously different with that of the control group(P < 0.05). The protein expression level of CYP4 in 50μg/L group was the lowest(P < 0.01).In 7 days, The protein expression levels of CYP4 in 5μg/L and 10μg/L groups were decreased with significant difference(P < 0.05).In 14 days, the protein expression level of CYP4 in 5μg/L group was also decreased(P < 0.01), while in the other groups the expression had no difference with that of the control group.All in all, the transcription and translation of CYP4 in P. aibuhitensis were different, so protein expression and gene expression is not a linear relationship. Protein expression is modulated by multiple levels and the transcription is only one part of this. This research will lay the foundation for the deep research of protein expression in P. aibuhitensis.