Cloning Of Cytochrome CYP4 Gene And Expression In Yellow Sea And Bohai Sea Coasts In Sternaspis Scutata(annelida:polychaeta) Responded To PAHs

In the present study, the partial c DNA of CYP4 was amplified from the total RNA of the polychaete Sternaspis scutata by reverse transcription with the primers based upon the conserved motifs of P450 family. Specific primers were designed according to the partial gene of Sternaspis scutata CYP4, and two fragments about 423 bp and 958 bp were obtained by using 3’RACE and 5’RACE. Sequence analysis revealed a 1829 bp c DNA containing the 27 bp 5’-untranslated region, 272 bp 3’-untranslated region and 1529 bp open reading frame encoding 509 amino acid. The deduced amino acid sequence of CYP4 contained the conserved motifs of P450 family(Fxx Gxxx Cx G) and had K-helix motifs of P450 family(Exx R). In addition, it has the characteristic sequences of CYP4 family(EVDTFMFEGHDTT). The identity rate of deduced amino acid sequence of CYP4 was subjected to Blast searching in NCBI, which showed that the CYP4 shared 58% identity with CYP4 V of Nereis aibuhitensis, 29% identity with CYP4BB1, 31% identity with CYP342A1 of Alitta virens and 35% identity with CYP4AT1 of Capitella capitata. Therefore, it is inferred that the c DNA sequence might belong to the subfamily of CYP4 V.The Real–Time fluorescent quantitative PCR of Sternaspis scutata CYP4 was designed with the primers based upon CYP4 c DNA full–length sequence and actin m RNA sequence to detect expression characteristics and regularities of CYP4 exposed to PAHs in marine sediment, what’t more, meteorological chromatograph- mass spectrometry analysis of PAHs in sediments. According to the data, the expression of CYP4 correlated with the concentration of PAHs in different sediments. The expression level were obviously different(p<0.05) between Dalian Bay and Hongyanhe, and it was also in Changxingdao and Qinhuangdao, what’s more, there are significant difference(p<0.01) among the rest of the sea. The concentration of PAHs in sediments gradually reduced in Dalian Bay, Changxingdao, Hongyanhe, Jinzhou Bay, Zhuanghe and the Qinhuangdao. Correlation between the expression of CYP4 in Sternaspis scutata and the concentration of PAHs in sediment was analysed. The relative expression of CYP4 in Sternaspis scutata was positively correlated with the concentration of PAHs in sediments in Yellow sea and Bohai sea(p=0.037). That is, the expression levels of CYP4 increased the concentration of PAHs. Therefore, the expression levels of CYP4 in sternaspis scutata responses to the PAHs, and the PAHs was degraded by biotransformation to reduce its ecological toxicity.