长期栖息于潮间带的瘤背石磺能感知潮汐来临时产生的低频声音，本研究模拟潮汐的低频声音刺激瘤背石磺，探讨NR1型受体基因在瘤背石磺感应低频声音的行为中发挥的作用。首先通过cDNA末端扩增法(RACE)得到瘤背石磺NR1基因(OrNR1) cDNA全长，进行生物信息学分析，并通过实时荧光定量PCR (qRT-PCR)检测OrNR1基因在各组织以及不同低频声波刺激下神经节中的表达。结果显示，OrNR1基因全长2 434 bp，其中5'非编码区 (UTR) 长357 bp, 3'UTR长184 bp，1 893 bp开放阅读框共编码630氨基酸。瘤背石磺OrNR1蛋白分子质量约为70.29 ku，等电点为5.93，分子式为C₃₁₅₃H₄₉₁₁N₈₃₃O₉₂₄S₃₂。OrNR1基因含有信号肽，一个预测的甘氨酸和谷氨酸结合位点，一个跨膜域，多序列比对结果表明各物种间NR1具有较高的保守性。 系统进化树结果显示，瘤背石磺OrNR1基因与加州海兔亲缘关系最高，符合传统形态学分类。qRT-PCR结果显示，OrNR1在瘤背石磺不同组织均有表达，在神经节中相对表达量最高，其次是肝胰腺、腹足，而在口器和背部皮肤中表达量较低OrNR1在频率为200、160 Hz的低频声音刺激下表达量在不同时间组均显著高于对照组，40 Hz的低频声音刺激下2 h后表达量显著高于对照组；在120 Hz时OrNR1基因表达量受到抑制，不同组的表达量有显著差异。推测该基因在瘤背石磺低频声音感知中起到了重要作用。

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.

Q 786；S 968.3

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