Cloning And Expression Determination Of Cytochrome P4503A137Gene Complementary DNA In Silver Carp

Cytochrome P450（CYPs） enzymes are members of the hemoprotein superfamily, and they areinvolved in the mono-oxygenation reactions responsible for metabolism of various kinds of endogenousand exogenous compounds, such as chemicals, drugs, hormones in mammals. CYP3A is an importantmember of CYP450and plays a key role in the detoxification of toxicants. The present study aims to cloneCYP3A137from the hepatopancreas of silver carp and to determine its expression at mRNA level when thefish is exposed to rifampicin, chlorpyrifos or ionic liquids.A full-length sequence of CYP3A137cDNA in silver carp was cloned and sequenced, and then aphylogenetic tree of CYP3A was structured. Meanwhile, sequence analysis revealed that CYP3A137washighly conserved in fish. The results of bioinformatics prediction showed that the full length of CYP3A137cDNA was1810base pair （bp） long and contains an open reading frame of1539bp encoding a protein of513amino acids. The molecular weight of CYP3A137protein was58.7995ku and the isoelectric pointwas7.96. This protein was stable and hydrophilic, and it had45.22%of a-spin and4.29%of β-fold, andtwo binding-domain of crossing-membrane according to the bioinformatics analysis.The results of quantitative real-time polymerase chain reaction （Q RT-PCR） revealed that CYP3A137of silver carp expressed in all tissues examined and the sequence of expression rate was as the following ofhepatopancreas> intestine> kidney> spleen> brain> heart> muscle.The results of Q RT-PCR showed that CYP3A137mRNA levels in the fish liver fromrifampicin-treated groups were significantly higher than that of control after24or72h of exposures,suggesting that [C8mim]Br can increase the transcription of CYP3A137. Liver microsomal enzymaticactivities were shown that, CYP3A activities in the treatment groups were higher than that of control,except for50mg Kg-1of rifampicin exposure at72h of time interval, suggesting that rifampicin can inducethe enzyme activity of CYP3A in silver carp.The results of acute toxicity tests show that chlorpyrifos was highly toxic to silver carp and the96hLC50was0.172mg L^-1. According to the LC50obtained, chlorpyrifos-exposure concentrations weredesigned to be0.0172mg L^-1（1/1096h LC50）or0.103mg L^-1（3/596h LC50）for silver carp.After24hof exposure, the results of Q RT-PCR indicated that chlorpyrifos significantly down-regulated the expression of CYP3A137in silver carp hepatopancreas.The acute toxicity of the ionic liquid1-octyl-3-methylimidazolium bromide （[C8mim]Br） on silvercarp and expression of CYP3A137were also determined in this study. The results of acute toxicity testsshowed that [C8mim]Br was low toxic to silver carp and the72h LC50was218.52mg L^-1. According to theLC50obtained,[C8mim]Br-exposure concentrations were designed to be22mg L^-1（1/10LC50） or109mgL^-1（1/2LC50） for silver carp. For24h or72h of exposure, hepatopancreas were collected and immediatelyfrozen in liquid nitrogen and stored at-80C for total RNA isolation and Q RT-PCR. The results of PCRindicated that [C8mim]Br significantly up-regulated the expression of CYP3A137in fish hepatopancreas,and the results of ERND indicated that [C8mim]Br significantly up-regulated the CYP3A activities in thetreatment groups in fish hepatopancreas, suggesting that [C8mim]Br may be the substrate of CYP3A137insilver carp.