蜜蜂表皮蛋白apidermin(apd)基因家族3个新成员的特性鉴定及昆虫APD家族序列特征的分析

Characterization of three new members of the apidermin(apd) gene family from honeybees and sequence analysis of the insect APD family

Apidermin(APD)蛋白家族是一个新的昆虫结构性表皮蛋白家族。本研究结合生物信息学和RT-PCR扩增,对意大利蜜蜂Apis mellifera ligustica(简称"意蜂")的apd-1-like,apd-3-like和中华蜜蜂Apis cerena cerena(简称"中蜂")的apd-2等3个新的apd基因的结构特征和表达进行了分析,并分析了昆虫APD蛋白家族的序列特征。结果显示,在西方蜜蜂Apis mellifera(简称"西蜂")中,apd基因家族的6个成员串联排列在基因组序列第4号连锁群上,它们在A.m.ligustica雄蜂头部中的转录水平差异明显,且其启动子序列所含顺式元件也不同。中蜂apd-2和意蜂apd-1-like都含有3个外显子和2个内含子,而意蜂apd-3-like则由4个外显子和3个内含子组成。蛋白序列分析结果显示,目前已知的10条APD蛋白序列N末端均具有相似的信号肽序列,其成熟蛋白分子量为6.0～37.0kD,pI为6.2～10.8。其中西蜂的APD1-3、APD-like和东方蜜蜂Apiscerena的APD-2等5条较短的多肽中疏水氨基酸残基达52%～67%,且Ala含量最为丰富(占25%～34%);而丽蝇蛹集金小蜂Nasonia vitripennis的APD1-3和西蜂APD-1-like,APD-3-like等另外5条APD多肽富含Gly(21%～30%),其序列中疏水氨基酸残基含量为35%～41%。多肽序列多重比对和系统进化分析结果显示,APD家族可划分为2个亚家族。亚家族Ⅰ含有西蜂APD1-3和东方蜜蜂APD-2等4条较短的多肽序列,其N末端为一个长33aa的保守基序;亚家族Ⅱ由另外6条相对较长的多肽序列组成,其N末端保守基序长50aa,C末端保守基序长16aa。本文所描述的APD蛋白家族序列特征有助于以后从其他昆虫中鉴定新的apd基因。

The apidermin（APD） protein family is a novel structural cuticular protein family of insects.To gain a better understanding of this protein family,by using bioinformatics and RT-PCR amplification,we identified three novel apd genes（apd-1-like,apd-3-like from Apis mellifera ligustica,and apd-2 from Apis cerana cerana） and investigated their structural features,and then we revealed the characteristic motifs of the APD family.The results showed that a cluster of six apd genes were tandemly arranged on chromosome 4 of Apis mellifera.These apd genes were differentially expressed in drone head of A.m.ligustica,and their expression pattern was consistent with that of the cis-acting elements in their promoter sequences.The genomic DNA of apd-2 from A.c.cerana and apd-1-like from A.m.ligustica contain three exons and two introns,while that of apd-3-like from A.m.ligustica contain four exons and three introns.As the deduced proteins of the apd genes were analyzed,it was found that the presently available 10 APDs possess similar N-terminal signal peptide sequences.The mature APD proteins ranged in size from 6.0 to 37.0 kD,and their pI ranged from 6.2 to 10.8.Intriguingly,five small APD proteins,including APD-2 from A.cerana,and APD 1-3 and APD-like from A.mellifera,were rich in hydrophobic amino acid residues（52%-67%） with Ala being the most abundant（25%-34%）.However,the other five larger APDs,including APD 1-3 from Nasonia vitripennis,and APD-1-like and APD-3-like from A.mellifera,were Gly-rich（21%-30%） proteins with hydrophobic amino acid residues constituting 35%-41% of their amino acid sequences.Multiple alignment and phylogenetic analysis revealed that the APD protein family could be classified into two subfamilies.Subfamily Ⅰ,which contains four low complexity sequences（APD 1-3 from A.mellifera and APD-2 from A.cerana）,was characterized by a 33-aa N-terminal conserved motif.The other six larger APD proteins were classified as subfamilyⅡ,which was featured by a 50-aa N-terminal motif and a 16-aa C-terminal motif.The diagnostic features of APD protein family described here will be very helpful for identifying novel apd genes from arthropods.