异育银鲫P450家族CYP3A136基因的克隆与表达

Cloning,sequencing and tissue expression of the cytochrome P450 3A136 in crucian carp(Carassius auratus gibelio)

根据5种硬骨鱼已知CYP3A序列保守区设计兼并引物,以异育银鲫cDNA为模板扩增得到CYP3A基因片段,根据得到片段序列设计特异引物并利用cDNA末端快速扩增技术(RACE)获得全长cDNA。得到异育银鲫CYP3A基因全长为1 769 bp,开放阅读框为1 545个核苷酸,编码514个氨基酸。其预测蛋白质分子量为58.624 ku,理论等电点为6.30。将异育银鲫CYP3A序列理论编码氨基酸序列提交细胞色素P450命名委员会(Cytochrome P450 NomenclatureCommittee)并由其命名为CYP3A136。氨基酸序列分析显示,其与稀有鲫、草鱼、鲦同源性较高,并且具有高度保守的血红素结合区域FXXGXXXCXG。异育银鲫CYP3A基因的半定量RT-PCR显示,在肝和肠组织中转录水平最高,在肾和鳃中次之,其余组织较低。

Cytochrome P450s（CYPs）are important xenobiotic metabolizing proteins.Their gene feature and protein function have been well understood in mammals,while their potent drug metabolic activity in freshwater fish aroused aquatic biologists＇ attention.Many DNA sequences of CYP3As have been discovered in teleosts such as killifish,rainbow trout and minnow.However,little has been referred to important freshwater economic fish in China.This study focused on CYP3A＇s cDNA sequence of crucian carp and predicted its protein structure and function preliminarily.In this study,degenerate primers for CYP3A of crucian carp were designed on the basis of conserved regions of known CYP3A sequences from five teleosts.Using crucian carp cDNA as a template,the partial fragments of crucian carp CYP3A cDNA were amplified.An antisense primer for 5′-RACE and a sense primer for 3′-RACE were designed to obtain the full-length CYP3A cDNA sequence by 5′ and 3′-RACE.The full-length of CYP3A cDNA for crucian carp is 1 769 bp with open reading frame（ORF）of 1 545 bp encoding 514 amino acids.The calculated MW was of 58.624 ku and a theoretical pI of 6.30.The Cytochrome P450 Nomenclature Committee has named this cDNA CYP3A136.The deduced amino acid sequence of crucian carp＇s CYP3A136 showed high similarity with those of rare gudgeon,grass carp and fathead minnow.It contained the conserved heme-binding motif of cytochrome P450 monooxygenases（FXXGXXXCXG）.Furthermore,we examined the gene expression levels of CYP3A136 in tissues by reverse transcription-polymerase chain reaction（RT-PCR）assay.The results demonstrated that the highest levels of CYP3A136 mRNA were seen in liver and intestine,followed by kidney and gill,and lower level were seen in other tissues.