| The functions of Acetylcholinesterase include the differentiation, migration and formation of synapses in nerve cells, proliferation and differentiation in hematopoiesis, as well as growth regulation in tumorigenesis. Usually the neurotransmitter acetylcholine (ACh) can be hydrolyzed by Acetylcholinesterase (AChE, EC 3.1.1.7) and terminating neural excitement at postsynaptic membranes. The decrease in acetylcholine neurons and nerve fibers are related to cognitive impairment caused by variety of diseases.Polyrhachis vicina Roger belongs to the genus Polyrhachis, which is important eusocial insect. There are four main stages in the ontogenesis, described as embryo, larva, pupa and adult. Because of the characteristic of caste differentiation, the ant is gradually becoming a special experimental material for the research of development and differentiation.In this paper, the cDNAs encoding acetylcholinesterase 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 acetylcholinesterase (Pv-ace) is 2561bp, and contains a 5'-untraslated region of 529bp and a 3'-untraslated region of 277 bp. The nucleotide sequence contains a stop codon (TGA) at positions 2285-2287. The open reading frame of P. vicina acetylcholinesterase encodes a peptide which includes 584 amino acid residues. The theoretical isoelectric point of the AChE protein in P. vicina is 5.03 and its molecular mass is 66.2 kilodaltons. The complete P. vicina acetylcholinesterase cDNA sequence, named Pv-ace, has been deposited in the GenBank database under accession number JF742990.2. The results of protein sequence alignments indicate that the Pv-AChE protein shares an overall identity of 80% with other known AChE homologues, and is most closely related to that of Apis mellifera L. (96%). Analysis of post-translational modification shows that there are 4 possible N-glycosylation sites,25phosphorylation sites including 10 Serine sites,7 threonine sites and 8 Tyrosine sites; the Pv-AChE protein may be hydrophilic secreted protein. The secondary structure of Pv-AChE protein was determined by bioinformatics software, and the result dispalys that it is a kind of mixed protein. Three-dimensional structure analysis showed that the protein sequence with the best match template is 1qo9A, the best talignmen region locate in the 42～543 amino acid residues, sequence consistency is 59.158%, evalue is 0.00e-1.3. Fluorescent real-time quantitative analysis showed that Pv-AChE protein in P. vicina ants were expressed in different periods. In the larval stage in addition to high expression of the pupal stage, the other stages aer very low. Among the adults, the highest expression is worker ants, followed by the male ants, at least is the female ants. Pv-AChE is highly expressed in the pupal stage. It perhaps are related to the programmed cell death and tissue remodeling. The high expression in worker ants may be related to the amount of complex social functions and grades differentiation. The more investigaion are needed for those specific mechanism and functions.In this work, an acetylcholinesterase homolog named Pv-ace has been cloned from the ant P. vicina, and acetylcholinesterase mRNA expression pattern was provided for the first time. The cloning, mRNA expression and characterization of acetylcholinesterase transcript may exhibit useful molecular information for further investigation on its role in insect developmental processes. |
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