刺参TRAF7基因克隆及其响应高温胁迫下的表达特征分析

Cloning, characterization and expression regulation in response to high-temperature stress of TRAF7 gene in the sea cucumber Apostichopus japonicus

关键词:  刺参  TRAF7  高温胁迫  基因克隆  表达特征

Key words:  Apostichopus japonicus  AjTRAF7  High temperature stress  cDNA cloning  Characteristics of expression

摘要:

肿瘤坏死因子受体相关因子7 (tumor necrosis factor receptor associated factor 7, TRAF7)作为一种细胞内蛋白，参与信号转导，并介导宿主应激防御调控过程。为研究TRAF7在刺参(Apostichopus japonicus)高温胁迫中的作用，本研究利用cDNA末端快速扩增技术(RACE)和实时荧光定量PCR技术(RT-qPCR)，克隆了刺参TRAF7 (AjTRAF7)全长cDNA序列，分析了其结构特征和在基因组中的分布，并检测了该基因在刺参不同组织和高温胁迫过程中体壁组织中的表达变化情况。结果显示，AjTRAF7全长2 576 bp，开放阅读框(ORF)长1 770 bp，5´UTR长345 bp，3´UTR长461 bp，编码589个氨基酸，预测蛋白分子量为65.6 kDa，理论等电点(pI)为7.52。蛋白序列包含1个RING finger结构域、1个Coiled coil结构域和6个WD40结构域，N端包含1个螺旋区域。该基因预测蛋白包含41个丝氨酸磷酸化位点、20个苏氨酸磷酸化位点和3个酪氨酸磷酸化位点，蛋白N端具有3个天冬酰胺糖基化位点。刺参基因组中比对到2个AjTRAF7基因拷贝，第一个拷贝包含18个外显子和15个内含子，第二个拷贝包含17个外显子和14个内含子。系统进化学分析表明，刺参TRAF7氨基酸序列与蝠海星(Patiria miniata)和紫色球海胆(Strongylocentrotus purpuratus)相似性较高，分别为40.58%和38.37%，刺参与蝠海星和紫色球海胆聚在一支。RT-qPCR结果显示，AjTRAF7在健康刺参各组织中均有表达，其在雌性性腺中表达量最高，体腔细胞中表达量次之，在呼吸树、体壁、肠道、雄性性腺和纵肌中表达量依次降低，各组织间表达量差异显著(P<0.05)。在响应高温胁迫的过程中，与对照组相比，体壁组织中AjTRAF7基因表达量在第2天和第4天显著上升(P<0.05)，第6天基因表达开始下降。综上，AjTRAF7基因可能参与刺参应对高温胁迫的表达调控过程，相关结果将为解析刺参应对高温胁迫的分子机制提供科学依据。

Abstract:

As an intracellular protein, tumor necrosis factor receptor-associated factor 7 (TRAF7) is involved in signal transduction and regulates host stress defense. In a previous transcriptome sequencing analysis of the high-temperature-stressed sea cucumber body wall, TRAF7 was significantly differentially expressed between healthy sea cucumbers and ulcered skin in high-temperature-stressed sea cucumbers. However, the structure of the TRAF7 gene and its specific expression pattern under high-temperature stress have not been reported. In this study, based on the results of sea cucumber genome sequencing, the full-length sequence of sea cucumber TRAF7 (named AjTRAF7) cDNA was obtained using RACE technology. Its structural features and distribution in the genome were analyzed, and gene expression was examined in different tissues and under high-temperature stress. AjTRAF7 has a total length of 2,576 bp, an ORF length of 1,770 bp, a 5´UTR length of 345 bp, a 3´UTR length of 461 bp, and encodes 589 amino acids; the molecular weight of the predicted protein is 65.6 kDa, the theoretical isoelectric point (pI) is 7.52, the instability coefficient is 32.34, and the total average hydrophilicity is –0.150. The AjTRAF7 protein contains a RING finger structural domain, a coiled-coil structural domain, six WD40 repeats, and the N-terminus contains a helical region. The predicted protein contained 30.05% α-helices, 6.62% β-turns, 36.84% irregular coils, and 26.49% extended strands and was predicted as a cytoplasmic protein. It had 41, 20, and 3 serine, threonine, and tyrosine phosphorylation sites, respectively, and the N-terminus of the protein had 3 asparagine glycosylation sites. Aligned with the sea cucumber genome sequences, evm.model.chr8.1363 and evm.model.chr8.1742 served as two copies of the gene. Evm.model.chr8.1363 contained 18 exons and 15 introns, and evm.model.chr8.1742 contained 17 exons and 14 introns. The evolutionary tree was constructed with the amino acid sequence of TRAF7. The results showed that the sea cucumber, bat starfish (Patiria miniata), and purple globular sea urchin (Strongylocentrotus purpuratus) clustered into one branch, and the sea cucumber was more similar to the bat starfish and the purple globular sea urchin, at 40.58% and 38.37%, respectively. RT-qPCR showed that AjTRAF7 was expressed in all tested tissues of healthy sea cucumbers. Its expression was highest in female gonads, followed by coelomocytes; it decreased in respiratory trees, body walls, intestines, male gonads, and longitudinal muscles in that order, and the difference in expression among tissues was significant (P<0.05). Under high-temperature stress, AjTRAF7 expression in body wall tissues increased during the first four d and decreased in the subsequent four d. The results indicated that AjTRAF7 might be involved in regulating expression in response to high-temperature stress in sea cucumbers, and the results provide a scientific basis for analyzing the molecular mechanism of high-temperature stress in sea cucumbers.