太平洋真宽水蚤CYP4C和CYP44基因克隆及分子特征分析

采用RACE技术首次克隆太平洋真宽水蚤CYP4C和CYP44全长cDNA序列,并命名为EpCYP4C和EpCYP44。EpCYP4C基因全长序列2032 bp,5’非翻译区60 bp,3’非翻译区487 bp,开放阅读框1485 bp,编码494个氨基酸;EpCYP44全长cDNA序列1752 bp,开放阅读框1485 bp,编码494个氨基酸,5’非翻译区85 bp,3’非翻译区182 bp。经预测发现EpCYP4C和EpCYP44蛋白具有CYP450家族蛋白特有的血红素结合区FxxGxxxCxG;而且EpCYP4C蛋白具有CYP4家族基因特征序列EVDTFMFEGHDTT。通过多序列比对和系统进化树分析发现EpCYP4C和EpCYP44蛋白与近亲真宽水蚤同源性更高,亲缘关系更近。蛋白二级结构预测显示EpCYP4C和EpCYP44蛋白主要以α螺旋和无规则卷曲为主。亚细胞定位预测发现EpCYP4C和EpCYP44蛋白分布在细胞质和线粒体的可能性较大,这可能与CYP450酶系的解毒代谢机制相关。研究结果说明太平洋真宽水蚤EpCYP4C和EpCYP44基因均为CYP450酶系,且太平洋真宽水蚤EpCYP4C基因属于CYP4家族。对太平洋真宽水蚤EpCYP4C和EpCYP44基因的序列分析为进一步研究EpCYP4C和EpCYP44基因在污染物暴露下的表达奠定理论基础;同时有助于了解海洋桡足类CYP450超家族的分子特征。

Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

Objective: To clone cytochrome P450 from Aedes aegypti(Ae. aegypti) and determine the characteristics using bioinformatics tools. Methods: Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction, cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation, signal peptide, transmembrane helix, phosphorylation, O-glycosylation, secondary and tertiary structures of the deduced protein. Results: Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P(Gen Bank accession number KF779932). The pseudogene has 1537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the Wxxx R motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide, transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated. Conclusions: A cytochrome P450 pseudogene, CYP4H44 P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.