家蚕细胞色素P450基因CYP6AE21的克隆、表达分析及亚细胞定位

摘要

Signal inactivation is a critical step in the olfactory dynamic process, in which various odorantdegrading enzymes are involved. In this study, the cDNA of a cytochrome P450 gene, CYP6AE21, was cloned from the male moth antenna of Bombyx mori by RT-PCR method. CYP6AE21 contains a 1 572 bp open reading frame (ORF), which encodes a putative protein of 523 amino acids. This cDNA-deduced protein has a predicted molecular weight (MW) of 60.5 kD, isoelectric point (pI) of 8.4, and a P450 characteristic structure heme-binding region. A single intron is localized at the same site in each of the P450 gene CYP6AE21 and CYP6AE2 from B. mori, and two corresponding exons with the same size exist in both of the genes. The two genes share 94.5％ nucleotide similarity, and cluster on the same chromosome in a head-to-tail arrangement separated by an approximately 7.6 kb intergenic region. CYP6AE21 was highly expressed in larval head and fat body, and both male and female moth antenna. It was also expressed in other tissues of larvae and some tissues of moth. NADPH cytochrome P450 reductase (CPR), a very important component of monooxygenase system, was also highly expressed in the moth antenna, and expressed in other moth tissues at low levels. Subcellular localization analysis showed that the expression product of CYP6AE21 is localized in cytoplasm. It is so inferred that CYP6AE21 and CYP6AE2 could have been formed by gene duplication of either of them, and CYP6AE21 might participate in the degradation of odorants after these components are internalized into cells.%信号失活是嗅觉动态过程的一个重要步骤,这一过程涉及多样的气味降解酶类.本研究利用RT-PCR方法从家蚕Bombyx mori/雄蛾的触角中克隆了一个细胞色素P450基因CYP6AE21.该基因含有一个1 572 bp的开放阅读框(open reading frame,ORF),编码523个氨基酸,预测分子量为60.5 kD,等电点为8.4,含有细胞色素P450的特征序列血红素结合位点区域.CYP6AE21和家蚕CYP6AE2基因一样在相同位置含有1个内含子序列,且相应的2个外显子大小相同.两者的核苷酸序列相似性达到94.5%,且在基因组上以头尾相连的方式成簇排列,中间由约7.6 kb核苷酸序列隔开.CYP6AE21在幼虫的头部和脂肪体,以及雄蛾和雌蛾的触角中表达量较高;在幼虫的其他组织和蛾的多个组织中也有一定的表达.P450酶系的重要组分之一--NADPH细胞色素P450还原酶(eytochmme P450reductase,CPR)基因也在雌蛾和雄蛾触角中高水平表达,而在其他组织中表达量相对较低.亚细胞定位分析表明CYP6AE21表达产物定位于细胞质中.推测CYP6AE21和CYP6AE2是由其中一个基因加倍复制形成的;此P450的功能之一可能是参与内化进细胞的气味分子的降解清除.