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

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.