瘤背石磺低频听力相关基因OrNXN克隆及表达分析

Cloning and expression analysis of OrNXN gene associated with low frequency hearing in Onchidium reevesii

摘要:

从瘤背石磺不同频率声音基因转录组中将有显著差异表达的基因与IMPC人类听觉感知和耳聋相关基因对比发掘出与听力相关的基因NXN。利用cDNA末端快速扩增(RACE)技术克隆获得OrNXN基因cDNA全长序列,并对其序列进行生物信息学分析;利用实时定量PCR(qPCR)技术分析OrNXN在瘤背石磺不同组织、不同声音频率及不同潮汐节点的表达水平。结果显示,OrNXN基因cDNA全长为1 593 bp,开放阅读框长度为432 bp,共编码143个氨基酸,相对分子质量为15.57 ku;结构域预测分析表明,OrNXN含有Thioredoxin家族典型的硫氧还蛋白结构;多序列比对以及进化树构建结果表明,OrNXN与其他物种的相似度较高,保守性较强;荧光定量结果表明,在瘤背石磺7个组织中OrNXN在肠、神经节、性腺和腹足中均有表达,在肠中表达量最高,其次为神经节。不同声音频率刺激下,OrNXN在200和50 Hz处有差异表达,在不同潮汐节点,OrNXN表达量均不同,表达量基本与潮汐水位变化一致。研究表明,OrNXN可能在瘤背石磺参与感知潮汐节率时对潮汐发出的低频声音产生了响应。

Abstract:

The genes with significantly different expressions were selected from the different frequency sound gene transcriptomes of Onchidium reevesii. We compared these genes with the genes related to human auditory perception and deafness in IMPC, and the hearing-related gene NXN was discovered. The cDNA sequence of OrNXN gene was cloned by rapid amplification of cDNA ends (RACE) technology, and the sequence was analyzed by bioinformatics; real-time quantitative PCR (qPCR) technology was used to analyze the relative expression in different tissues, different frequency sounds, and different tide nodes. The results show that the length of OrNXN is 1 593 bp with 432 bp of open reading frame. OrNXN protein is encoded by 143 amino acids. The molecular mass of OrNXN is 15.57 ku. Domain prediction analysis shows that OrNXN contains a typical sulfur redox protein structure of the Thioredoxin family. The results of multiple sequence alignment and the phylogenetic tree construction showed that OrNXN has high similarity with other species. The qPCR results show that OrNXN is expressed in the intestine, ganglia, gonads and gastropods of O. reevesii. The expression is highest in the intestine, followed by ganglia. OrNXN is expressed differently at 200 and 50 Hz under different sound frequency stimulation. The relative expression level of OrNXN is different at different tidal nodes, and it basically changes with the tide water level. Studies have shown that OrNXN may respond to the low-frequency sound emitted by the tide when O. reevesii senses the tidal node rate.