白鲢肝脏CYP3A序列分析及毒死蜱对其抑制作用研究

SEQUENCE ANALYSIS OF THE ENZYME CYP3A IN SILVER CARP AND ITS EXPRESSION INHIBITION BY CHLORPYRIFOS

CYP3A是I相解毒酶系CYP450家族中的重要成员,在肝脏解毒过程中发挥重要作用。首次克隆了白鲢肝脏CYP3A基因并研究了毒死蜱对该基因表达的影响。生物信息学分析预测结果表明,CYP3A基因编码513个氨基酸,其蛋白质分子量为58.8 ku,理论等电点为7.96。该蛋白是一个稳定蛋白且具有一定的亲水性。二级结构预测可知,CYP3A包含45.2%的α-螺旋、12.3%的延伸链、4.3%的β-折叠和38.2%的无规则卷曲,具有2个显著的跨膜结合区。急性毒性实验结果表明,毒死蜱对白鲢具有很高的毒性,其96h LC50为0.172 mg/L。另外,荧光定量PCR检测发现,毒死蜱对白鲢CYP3A基因表达有明显的抑制作用。

Cytochrome P450 （CYPs） enzymes are responsible for the metabolism of various kinds of endogenous and exogenous compounds in mammals. In fish, CYP3A plays an important role in the detoxification of xenobiotics. In the present study, a full-length sequence of CYP3A cDNA in silver carp was cloned and sequenced, and then a phylogenetic tree of CYP3A was constructed. Additionally, the acute toxicity of chlorpyrifos on silver carp and the effect of chlor- pyrifos on CYP3A expression were also evaluated in this study. The results of bioinformatics prediction revealed that the full CYP3A encoded a protein of 513 amino acids and the molecular weight of CYP3A protein was 58.8 ku and the isoelectric point was 7.96. This protein was stable and hydrophilic, and it had 45.2% of α-spin, 12.3% of extended strand, 4.3% of β-fold, 38.2% of random coil and two binding-domain of crossing-membrane. Meanwhile, the results of acute toxicity tests indicated that chlorpyrifos was highly toxic to silver carp and the 96h LC50 of chlorpyrifos on silver carp was 0.172 mg/L. Finally, the results of Q RT-PCR indicated that chlorpyrifos significantly down regulated the expression of CYP3A at mRNA level in the liver of silver carp.