Molecular cloning and characterization of three cDNAs encoding allatostatin-like neurosecretory peptides from Pandalopsis japonica

摘要

Three cDNAs encoding allatostatin-like peptides (two myoinhibitory peptides; Paj-MIPI and Paj-MIPII, and one C-type AST; Paj-ASTC) were identified from Pandalopsis japonica through a combination of bioinformatic analysis and PCR-based gene cloning strategy. Paj-MIPI and Paj-MIPII encoded proteins with 189 and 117 amino acid residues, respectively, and a total of 10 mature peptides are putatively produced from the two MIP cDNAs (seven from Paj-MIPI and three from Paj-MIPII). Among the MIPs from various arthropods, their size and organization varied and it was unable to establish the monophyletic evolutionary relationship, which is mainly due to difference in the number and location of the mature peptide W(X _6)W motif of each MIP gene. Based on the sequence similarity of six residues flanked by two conserved tryptophan (W) residues, crustacean MIPs could be further classified into at least four groups. Paj-ASTC cDNA (648bp) encoded a protein with 143 amino acid residues. The prepropeptide of Paj-ASTC showed conserved C-type AST characteristics including a signal sequence, two dibasic cleavage sites, and a mature peptide sequence with two cysteine residues at the 7th and 14th positions, creating a disulfide bridge. Based on the sequence similarity in the mature peptides, the ASTCs in arthropods could be further classified into two subgroups, AVSCF-ASTC and PISCF-ASTC. Phylogenetic and sequence similarity analysis showed that Paj-ASTC belonged to the PISCF-ASTC subgroup. Expression studies revealed that AST-like peptides from P. japonica were mainly expressed in neuronal tissues, and the expression of Paj-ASTC was also detected in the intestine. Eyestalk ablation (ESA) altered the mRNA expression levels of both Paj-MIPs and Paj-ASTC, suggesting that factors from the sinus gland/X organ complex had a transient effect on AST-like peptide transcription. Correlation analysis of three allatostatin-like peptides revealed a strong positive correlation in brain tissues, suggesting that transcriptional regulation of three allatostatin-like peptides from P. japonica is influenced by the similar physiological condition.