关键词: 中华按蚊, 表皮蛋白, CPF家族, 保守基序, 进化

Abstract: 【Aim】 This study aims to identify the CPF family (CPFs) of cuticular protein genes in Anopheles sinensis genome, to analyze their structure and characteristics, to deduce their possible biological functions, and to investigate and compare the CPFs of representative mosquito species so as to provide information frame for the family of genes. 【Methods】 We identified the CPFs in the genomes of An. sinensis, An. gambiae, An. minimus, Aedes aegypti, Culex quinquefasciatus and Drosophila melanogaster using BLASTP, TBLASTN and HMM with An. gambiae CPFs as query, predicted the structure and splicing variation of An. sinensis CPF gene and the signal peptide, transmembrane region, structural domain and 3D structure of An. sinensis CPF proteins using bioinformatics techniques, and constructed phylogenetic relationships using maximum likelihood (ML) method and deduced the origin and evolution of CPFs in these species. 【Results】 There are 4, 4, 4, 3, 3 and 3 CPFs in An. sinensis, An. gambiae, An. minimus, Ae. aegypti, Cx. quinquefasciatus and Dr. melanogaster genomes, respectively. The CPFs in An. sinensis were named as AsCPF1, AsCPF2, AsCPF3 and AsCPF4, respectively. Their full-length cDNA sequences are 736, 2 021, 531, and 1 001 bp, respectively, encoding 219, 345, 148 and 185 amino acids, respectively. AsCPF1, AsCPF2 and AsCPF3 only have one intron, but AsCPF4 contains three introns, which all have phase “0”. There are 3, 2, 1 and 2 selective spicing variants for AsCPF1, AsCPF2, AsCPF3 and AsCPF4, respectively. AsCPF3 has the highest expression quantity, followed by AsCPF4, AsCPF2 and AsCPF1. The theoretical molecular weights of AsCPF1, AsCPF2, AsCPF3 and AsCPF4 are 22.86, 36.47, 15.08 and 18.66 kD, and their isoelectric points are 9.08, 8.97, 9.44 and 9.16, respectively. These AsCPFs contain a 44-amino-acid conserved region and C-terminal region, and all are secretory proteins with signal peptide sequences except for AsCPF2 that is non-secretory protein and lacks a signal peptide sequence. All the four AsCPFs have alpha helix, random coil and extended strand, and only AsCPF4 has a transmembrane region that is located between amino acid 5 to 27. Phylogenetic analysis showed that CPF3 might be the earliest derived CPF gene, CPF1 and CPF2 might originate from a common ancestor and consequently experienced a gene duplication event, and CPF4 might be unique for Anopheles mosquitoes and the latest derived CPF gene. The Ka/Ks ratio of CPFs are all less than 1 in An. sinensis in reference to An. gambiae, suggesting the purification selection of these genes in evolution. 【Conclusion】 The whole-genome identification and characteristics analysis of CPFs in An. sinensis and the comparison of CPFs in representative mosquito species revealed the diversity, structure and amino acid characteristics and the origin and evolution of the CPF family of genes in mosquitoes, which provides a comprehensive information frame for further research and utilization of the CPF gene family.

Key words: Anopheles sinensis, cuticular protein, CPF family, conservative motif, evolution