Cloning and characterization of nicotinic acetylcholine receptor γ-like gene in adult transparent Pristella maxillaris.

Abstract

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating the development and function of nervous system. The muscle AChR is composed of four homologous glycoprotein subunits with a stoichiometry α2βγδ in fetal or α2βεδ in adult. But the mechanism controlling the transition of fetal AChR γ-subunit to adult AChR ε is still unknown. Here a gene annoted AChR γ-like in Pristella maxillaris was first cloned by rapid amplification of cDNA ends (RACE) based on a transcriptome of dorsal fins. The full length of AChR γ-like was 1984 bp and it encoded 518 amino acids from 100 bp to 1653 bp. The multiple alignment analysis showed that AChR γ-like had 98% protein identity to AChR γ-like in Astyanax mexicanus. Then an 11647 bp DNA from 5'-UTR to 3'-UTR was cloned based on gene structure of AChR γ-like in A.mexicanus. Additionally a 2768 bp DNA upstream 5'-UTR was cloned by chromosome walking method. Furthermore, the results from semi-quantitative PCR showed that AChR γ-like was highly expressed in embryo and adult tissues, such as the muscle, eye, heart and intestine. While it showed low expression in the brain and gill. Significantly, the results of in situ hybridization showed strong diffused expression of AChR γ-like in the muscle of 1 dpf (day post-fertilization) embryo. And weak signal was observed in the muscle of 2-4 dpf embryos. All these data indicated that AChR γ-like could be one subunit of AChRs in the muscle and it could be used to study the development of the neuromuscular junction in adult transparent Pristella maxillaris. Thus our work will lay the foundation for using Pristella maxillaris to analyze the in vivo function of the nAChRs in adult vertebrate.