| The PBC family includes homeodomain proteins encoded by elegans ceh-20, Drosophila extradenticle(exd), and vertebrate Pbx genes. Extradenticle, as a Hox cofactor, was first implicated in the fly. The evidence indicated that extradenticle homologues play a crucial role in appendage and notum patterning and identity of arthropod.Polyrhachis vicina Roger belongs to the genus Polyrhachis, which is important eusocial insect and resource insect. Because of their own unique features, they are different from other insects in possessing strict and mysterious hierarchical division of labors.In this paper, the cDNAs encoding extradenticle of the ant, P.vicina, have been amplified, cloned and sequenced by RT-PCR and RACE, which is a normal technique in modem molecular biology. The results of these investigations are offered as follows:1. The full-length cDNA of P. vicina extradenticle (Pvexd) is 1854 bp, and contains a 3'-untraslated region of 136 bp and a 5'-untraslated region of 542 bp. The nucleotide sequence contains a stop codon (TGA) at positions1716-1718. The open reading frame of P. vicina extradenticle encodes a peptide which includes 391 amino acid residues. The theoretical isoelectric point of the EXD protein in P. vicina is 6.53 and its molecular mass is 42.7 kilodaltons. The complete P. vicina extradenticle cDNA sequence, named Pvexd, has been deposited in the GenBank database under accession number GU223153.2. The results of protein sequence alignments indicate that the PvEXD protein shares an overall identity of 58.5%-98.2% with other known EXD homologues, and is most closely related to that of Apis mellifera L. (Hymenoptera:Apidae). Acording to the dendrogram of several EXD sequences. The PvEXD clusters together with that of A. mellifera with high bootstrap support. Analysis with the SignalP program showed that the PvEXD protein may be nonsecreted protein. The secondary structure of PvEXD protein was determined by bioinformatics software, and the result is that it's a kind of mixed protein. A series of predicted function motifs are found in the PvEXD protein:three N-glycosylation sites, two cAMP-and cGMP-dependent protein kinase phosphorylation sites, four casein kinaseâ…¡phosphorylation sites, seven N-myristoylation site, two protein kinase C phosphorylation sites, one Tyrosine kinase phosphorylation site.3. The relative expression level of the extradenticle mRNA in P. vicina was detected by real-time quantitative RT-PCR which is a kind of techniques that possess characteristic of sensitiveness, quickness, speciality and can quantitate the nucleic acid based on different fluorescence. During P. vicina developmental processes, extradenticle was highly expressed in the embryos, as well as the fourth instar larvae, and then decreased to the third instar in the whole, while the lowest expression level of Pvexd was observed in the pupae. An increase occurred in adults.The extradenticle expression pattern is different between in the young and in the adult, which suggests that Pvexd may regulate the complicated behaviors in the adult. In the three castes, the Pvexd gene expressed at higher levels in workers than in other two castes, and there were no statistical differences between levels in the male and in female ants. But the heads of males have about eight times more extradenticle mRNA than the heads of females. These results may show a function of the Pvexd in the regulation of spermatogenesis, oogenesis or differential processes in male or in female.In this work, an extradenticle homolog named Pvexd has been cloned from the ant P. vicina, and Pvexd mRNA expression pattern was provided for the first time. The cloning, mRNA expression and characterization of Pvexd transcript may exhibit useful molecular information for further investigation on its role in insect developmental processes. |
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