大黄鱼(Pseudosciaena crocea)细胞色素P450 CYP4F7基因的克隆及表达分析

Molecular cloning,characterization of a homologue of cytochrome P450s 4F7 in Pseudosciaena crocea

CYP4F7是细胞色素P450超基因家族中CYP4F亚家族中的同工酶之一,而细胞色素P450在通过电子传递参与生物体代谢和转化内源和外源化合物方面起着重要的作用。以本实验室构建的大黄鱼消减杂交cDNA文库中623bp的CYP4F7片段设计引物,以大黄鱼的总RNA为模板,利用RACE-PCR的方法,克隆鉴定出了大黄鱼细胞色素P450 CYP4F7基因。结果表明:基因全长1934bp,5′端非编码区59bp,3′端非编码区264bp,开放阅读框1611bp,编码536个氨基酸。序列分析表明大黄鱼的CYP4F7氨基酸序列与舌齿鲈的相似性为91%。利用RT-PCR方法研究CYP4F7在大黄鱼各组织中的表达特征,结果表明CYP4F7在肝脏、脾脏中表达量最高,在心脏、肾脏、头肾、鳃丝中表达量次之。另外,CYP4F7在注射了细菌脂多糖的大黄鱼各组织中的表达量均低于正常的大黄鱼,认为细菌脂多糖可能是CYP4F7表达的抑制剂,说明CYP4F7可能与鱼类免疫反应有一定相关性。

CYP4F7 is a member of the 4F subfamily of the Cytochrome P450, which plays a vital role in the course of either catalyzing and regulating the metabolism of endogenous substances or activating and degrading exogenetic substances by transferring electrons. A CYP4F7 cDNA sequence （GenBank accession number： EU169141 ）was isolated and identified from the total RNA of Pseudosciaena crocea kidney using the combined methods of BD SMARTTM cDNA synthesis and RACE--PCR. The sequence of CYP4F7 cDNA is 1 934 bp which contains a 5＇ UTR of 59 bp and a 3＇ UTR of 264 bp, and an open reading frame with 1611 bp that encodes a protein of 536 amino acids. The amino acid sequence of P. crocea CYP4F7 shared 67%-71% of residue identity with the CYP4F subfamily of other high mammals such as human and mice, and shared the highest identity of 91% with sea bass, Dicentrarchus labrax. Semi--quantitative RT--PCR analysis showed that mRNA expression of CYP4F7 can be detected in the spleen, heart, liver, anterior kidney, kidney and gills, but not in the intestine and muscle in the normal group and the lipopolysaccharide （LPS）--stimulated group of P. crocea, in which the higher expression was found in the spleen and liver. The expression of the CYP4F7 in all these tissues examined showed some decrease in the LPS--treated P. crocea compared with normal P. crocea, suggesting that LPS might be an inhibitor of cytochrome P450 CYP4F7 gene and CYP4F7 might be related to large yellow croaker P. crocea immune response.