Cloning, sequence analysis of the NR1 gene of Onchidium reevesii and low frequence sound stimulation on gene expression

NMDA receptors (N-methyl-D-aspartic acid receptor, NMDAR) are members of ionotropic glutamate receptor family, widely distributed in central nervous system, and play a pivotal role in auditory pathway. NR1 is the fundamental subunit necessary for the NMDAR complex. The life activities of Onchidium reevesii are closely related to tidal cycles. In this study, we used low-frequency sound to stimulate the O. reevesii and investigated the expression of NR1 receptor gene in its ganglion. The NR1 gene's cDNA sequence was cloned using RACE-PCR on nervous tissue from O. reevesii, followed by bioinformatics analysis and qRT-PCR experiments. We then stimulated O. reevesii in the laboratory by simulating the low-frequency sounds of the tides, and measured NR1 gene expression under different frequencies of sound stimulation. The full-length cDNA sequence of NR1 gene was 2 434 bp, comprising a 357 bp 5′non-coding region, a 184 bp 3′non-coding region, and a 1 893 bp open reading frame encoding 630 amino acids. The cloned receptors contain a signal peptide, predicted binding sites for glycine and glutamate, a recognized transmembrane region. Multi-sequence alignment revealed high conservation of NR1 across species. A phylogenetic tree indicated that the NR1 gene of O. reevesii is related to that of Aplysia california, aligning with traditional morphological classification. The results of qRT-PCR showed that OrNR1 was expressed in different tissues, but the relative expression level in ganglion was significantly higher than that in other tissues, followed by the pleopod and liver, and the expression level in the mouthparts and dorsal skin were low (P<0.05). The expression level of OrNR1 was higher than the control group at 200 and 160 Hz (P<0.05); the expression of OrNR1 gene was inhibited at 120 Hz, the expression levels of different frequencies were significantly differently(P<0.05). We hypothesize that the OrNR1 gene plays an important role in the low-frequency sound perception of O. reevesii.