CYP2K CDNA Cloning, Tissue Expression And Effects Of Microcystins On The Expression Of CYP2K Gene In Silver Carp

Microcystins (MCs) are common algal toxins mainly produced by Microcystis aeruginosa.Because of the frequent occurence of blue-green algae blooms, the danger of MC on human healthattracts more and more attention in recent years. As an important species of aquaculture, silvercarp (Hypophthalmichthys molitrix) is frequently under the exposure of MC in thebloom-occurrence water. It has been reported that silver carp can bear the toxicity of MC.Cytochrome P450superfamily is an important detoxication enzyme involved in the metabolism ofchemicals in the phaseⅠreaction of animal liver and it is widely distributed in all the species offish. To determine the expression of the CYP2K gene in silver carp and to investigate therelationship between CYP2K expression and detoxicification of MC in the liver of the silver carp,we conducted the following experiments. First of all, we designed the degenerate primersaccording to the conserved sequence of the CYP2K gene from other species of fish and cloned thecoding sequence of cDNA of CYP2K gene in the liver of silver carp by using PCR. Then, themRNA levels of CYP2K cDNA in the liver, kidney, spleen, heart, brain, muscle, and intestine fromsilver carp were detected by using the quantitative real-time quantitative PCR (qPCR). Moreover,an acute toxicity test was conducted and the i.p.LD50of MC on silver carp was obtained. After that,the fish were exposed to MC by intraperitoneal injection at the concentration of1/2or1of LD50,the fish were sacrificed after6h of exposure and the liver were excised for histologicalexamination.Finally the mRNA levels of CYP2K cDNA in the liver of silver carp were detected byusing qPCR after the fish were intraperitoneally injected with the MC at the concentration of1/5and1/10of the i.p.LD50. The main results obtained in the this study are as follows:(1) The coding sequence of cDNA of CYP2K gene in the liver of silver carp was cloned in thepresent study..(2) The results of the real-time PCR showed that the CYP2K gene expressed in all the tissuesof silver carp, in which liver is the most expressed one.(3) The i.p. LD50of MC was calculated to be432.7μg·kg-1.(4) Histological examination revealed that relatively damage occurred in the liver of fish exposed to MC at1/2of i.p.LD50while serious injure was observed in the group of high dose ofMC (LD50).(5) The results of qPCR showed that the transcription of CYP2K gene was up-regulated in theliver of fish exposed at the lower dose of MC (1/10of LD50), but repressed at the higher dose ofMC (1/5of LD50).(6) Our result suggestes that MC-exposure can alter the expression of CYP2K gene in theliver of silver carp.