中华按蚊血红素过氧化物酶家族基因的全基因组鉴定、特征和进化

Genome-wide identification,characterization and evolutionary analysis of genes of the heme peroxidase family in Anopheles sinensis(Diptera:Culicidae)

【目的】在全基因组上鉴定中华按蚊Anopheles sinensis血红素过氧化物酶(heme peroxidase,HPX)家族基因,并预测其基本特征,探究双翅目中5种代表性昆虫HPX基因的系统发育关系和进化。【方法】以NCBI数据库中黑腹果蝇Drosophila melanogaster和家蚕Bombyx mori等昆虫HPX基因编码的氨基酸序列为询问序列,通过本地Blast搜索鉴定中华按蚊基因组上HPX家族基因,并鉴定了冈比亚按蚊Anopheles gambiae、致倦库蚊Aedes aegypti和埃及伊蚊Culex quinquefasciatus基因组上的HPX基因;基于冈比亚按蚊HPX基因的命名系统,对中华按蚊HPX基因进行命名;运用生物信息学方法预测了中华按蚊HPX基因的特征,包括基因的结构及在scaffold的定位,氨基酸的替换率和保守结构域,蛋白质3D结构等,通过与冈比亚按蚊共线性分析定位了中华按蚊HPX基因在染色体上的位置;基于HPX核苷酸序列,采用PAUP4.0和MEGA6.0软件利用最大相似法构建了5个双翅目代表性种HPX基因的系统发生树。【结果】中华按蚊基因组共有20个HPX基因,冈比亚按蚊、埃及伊蚊和致倦库蚊基因组分别有18,14和12个HPX基因。这4种蚊虫的HPX蛋白都被分类进入Peroxinectin,Peroxidasin,DBLPX和DUOX 4个亚家族,其氨基酸的分子量介于61.6~186.6 k D之间(除As HPX8为29.6 k D)。中华按蚊20个HPX基因共具有98个外显子,75个内含子,其外显子与内含子分布模式在基因间差异较大。中华按蚊HPX基因被定位到10个scaffold上,对应到冈比亚按蚊的2R,3R,2L,3L和X染色体。这些中华按蚊HPX基因编码的氨基酸序列(除DUOX)都具有1个血红素结合位点和5个Ca^(2+)结合位点,在N端和C端各具有2个半胍氨酸位点并界定了两个二硫键。中华按蚊与冈比亚按蚊同源基因对的ω值都小于1,说明HPX基因在进化过程中没有受到明显的环境选择压力。系统发育关系研究表明,5种双翅目昆虫的HPX基因可以分为16个组,其中11个组在进化上呈明显的单系,具有至少83%的bootstrap支持。【结论】本研究提供了中华按蚊的HPX基因的基础信息。不同蚊虫种具有相似分子量的HPX蛋白,这与HPX家族蛋白结构的高度保守性有关。蚊虫的DUOX随着主要功能位点的缺失逐渐失去其功能,这与其特殊环境适应相关。DBLPX亚家族的peroxidase结构域在HPX家族所有亚家族中是最保守的。

【Aim】 To identify genes of the heme peroxidase （HPX） family of Anopheles sinensis at the wholegenome level, to predict the basic features of these HPX members, and to explore the phylogenetics and evolution of the HPX members of five representativedipterans. 【Methods】 The amino acid sequences encoded by HPX genes in Drosophila melanogaster, Bombyx mori and other insects were downloaded from NCBI databases and used as queries to search for the HPX genes in An. sinensis genome using the local Blast program. The HPX genes in Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus on the whole genome were also identified by using the same method. The HPX genes identified in An. sinensis were named following the nomenclature system established for An. gambiae HPX genes. The characteristics of these HPX genes in An. sinensis, including the structure and scaffold location of these genes, the substitution rate and conserved domain of their amino acids, and the 3D structure of their proteins, were predicted using bioinformatics analysis. These An. sinensis HPX genes were also located on chromosome through the synteny analysis with An. gambiae HPX genes. The phylogenetic relationships of the HPX genes of five representative species of Diptera were constructed based on nucleotide sequences using maximum likelihood method with PAUP 4.0 and MEGA 6.0. 【Results】 The An. sinensis genome contains 20 HPX genes, while those of An. gambiae, Ae. aegypti and Cx. quinquefasciatus contain 18, 14 and 12 HPX genes, respectively. The HPX proteins of the four mosquito species were all classified into Peroxinectin, Peroxidasin, DBLPX and DUOX subfamilies, with the molecular weights of their amino acids ranging from 61.6-186.6 kD, except AsHPX8 （29.6 kD）. The 20 HPX genes of An. sinensis contain 98 exons and 75 introns, and the distribution patterns of their exons and introns are quite diverse among different genes. These HPX genes of An. sinensis are mapped on 10 scaffolds, and syntenied to the chromosomes 2R, 3R, 2L, 3L and X in reference of An. gambiae genome. All the amino acid sequences of HPX genes in An. sinensis （except DUOX） each has one heme and five Ca2＋ binding sites, and contains two cysteine sites in each of N-terminus and C-terminus, which define two disulfide bonds. The ω values of An. sinensis and An. gambiae orthologousgene pairs were all less than 1, suggesting that HPX genes has no obvious environmental selective pressure during their evolution. The HPX genes of the five representative dipteran species were divided into 16 groups on the phylogenetic tree, 11 of which showed to be monophyletic with 〉83% bootstrap support and homologous genes clustered together. 【Conclusion】 This study provides the basic information frame for the HPX genes of An. sinensis. The HPX proteins in different mosquito species have similar molecular weights, and this is associated with a high degree of structure conservation of the HPX family proteins. The DUOX of mosquitoes gradually lost its function with their deletion of the main functional loci, and this is related to its specific environmental adaptation. The peroxidase domain in the DBLPX subfamily is the most conserved in all subfamilies of the HPX family.