低频声波频率下瘤背石磺CaM-like、CaMKII基因的表达及功能

长期栖息于潮间带的动物能够感知当地的潮汐规律,形成潮汐记忆的能力。本研究以栖息于潮间带的瘤背石磺(Onchidium reevesii)为对象,研究其通过感知潮汐来临时产生的低频声音来调节其行为与潮汐节律相协调的分子机制。通过RACE-PCR技术克隆到CaM-like基因的cDNA序列,并进行生物信息学分析和qRT-PCR实验。结果显示,瘤背石磺CaM-like基因的cDNA全长2321 bp,包括5′非编码区(UTR)366 bp,3′非编码区(UTR)1337 bp,618 bp的开放阅读框编码206个氨基酸;预测该基因编码的多肽链的原子数量是3165,分子量约为23029.64 kD,理论等电点4.64,分子式是C_(1018)H_(1544)N_(274)O_(320)S_9。N端信号肽由29个氨基酸组成。氨基酸序列比对构建系统进化树,结果显示瘤背石磺CaM-like基因与静水椎实螺(Lymnaea stagnalis)、光滑双脐螺(Biomphalaria glabrata)的CaM-like基因的亲缘关系最接近,这与传统形态学分类相吻合。在实验室内模拟潮汐中低频声音刺激瘤背石磺,应用qRT-PCR检测CaM-like基因在不同组织的分布情况,结果显示CaM-like mRNA在瘤背石磺不同组织均有表达,但神经节部位的表达量显著高于背部皮肤、腹足、肠、肝胰腺、口器和蛋白腺等组织(P0.05),推测其可能参与神经系统的可塑性调节。荧光定量结果显示CaM-like、CaMKII基因分别在25 Hz和50 Hz低频声波下刺激12.4 h表达量较高,初步推测其能感知25～50 Hz声波频率。这可能与栖息于潮间带中的瘤背石磺长期感知12.4 h半日潮潮汐周期节律,形成潮汐记忆有关。本研究将为进一步深入了解瘤背石磺感知潮汐节律的分子机制奠定基础,也为探究海洋动物由海洋向陆地进化过程中对环境的适应机制提供依据。

Expression of CaM-like and CaMKII genes in Onchidium reevesii under low frequency sound wave stimulation

Animals that inhabit intertidal areas long-term can sense the local tidal rhythm, forming a tidal memory. However, to date, no studies have investigated the mechanism of tidal rhythm sensing in Onchidium reevesii. In this experiment, we stimulated O. reevesii in the laboratory using low-frequency tide sounds. The cDNA sequence of the CaM-like gene was cloned by RACE-PCR, after which bioinformatics analysis and qRT-PCR experiments were performed. The results showed that the full-length cDNA of the CaM-like gene was 2321 bp (366 bp 5'' non-coding region (UTR), 1337 bp 3'' non-coding region (UTR), and 618 bp open reading frame (ORF), which encoded a total 206 amino acids. The gene that encodes polypeptide chains was predicted with an atomic number of 3165, a molecular weight of approximately 23029.64 kD, a theoretical isoelectric point of 4.64, a molecular formula of C₁₀₁₈H₁₅₄₄N₂₇₄O₃₂₀S₉, and an N-terminal signal peptide composed of 29 amino acids. The phylogenetic tree was constructed via amino acid sequence alignment. The results suggest that the CaM-like gene of O. reevesii is closely related to the CaM-like gene of Lymnaea stagnalis and Biomphalaria glabrata, which is consistent with traditional morphological classification. The distribution of CaM-like genes in different tissues was detected using qRT-PCR. The results indicated that CaM-like mRNA was distributed in different tissues of O. reevesii, but the expression of ganglion was significantly higher than dorsal skin, pleopod, intestines, liver, mouthparts, and albumen gland (P<0.05). We speculated that it may be involved in the plasticity regulation of the ganglion system. The quantitative fluorescence results indicated that the CaM-like and CaMKII genes were highly expressed, underlying the stimulation of low frequency sound waves at 25 Hz and 50 Hz for 12.4 h, respectively, for which it was presumed that O. reevesii could sense the 25-50 Hz sound frequency. This may be related to the tidal memory formed by the long-term immersion in the 12.4 h half-day tidal cycle rhythm in the intertidal zone. This study will lay the foundation for further understanding of the molecular mechanisms of O. reevesii sensing tidal rhythm, and provide a basis for exploring the environmental adaptation mechanism of marine animals from ocean to land evolution.