斑马鱼prkd1基因在心脏早期发育过程中的作用研究

Role of the prkd1 Gene in Early Cardiac Development in Zebrafish

摘要

为了探究蛋白激酶D (protein kinase D, PKD; 又名PRKD)家族典型成员prkd1基因对早期心脏发育的影响, 选用模式动物斑马鱼为研究对象, 设计了吗啉代反义寡核苷酸敲低实验。表型观察结果显示, prkd1基因敲低会导致斑马鱼心脏发育异常, 出现心包水肿、环化异常、心管线性化等畸形表型; 斑马鱼心率分析数据显示, 与野生型斑马鱼相比, prkd1基因的敲低会导致早期斑马鱼心率降低。利用转录组高通量测序技术对受精后3 d (3 days post fertilization, 3 dpf)的斑马鱼对照组和敲低prkd1组进行测序, 差异基因富集分析表明, prkd1的敲低与心脏相关信号通路如心肌细胞的肾上腺素信号通路等显著相关。进一步通过qRT-PCR对高通量测序结果进行验证, 并对早期心脏发育关键因子进行检测, 结果显示, 部分心血管相关基因显著下调, tbx5a、gata4等心脏调控关键基因同样显著下调。这些表明prkd1基因可能通过调控心脏发育相关信号通路以及心脏发育关键基因来影响早期心脏发育, 在早期心脏发育过程中发挥重要作用。

Abstract

To investigate the role of the prkd1 gene, a typical member of the protein kinase D (PRKD) family, in early heart development, zebrafish was used as an animal model in this study. Through prkd1 knockdown with morpholino antisense oligo (MO), it was found that knockdown of prkd1 resulted in abnormal cardiac de-velopment, such as pericardial edema, cyclization disorder, cardiac tube linearization. Zebrafish heart rate analysis indicated that, compared with the wild type, prkd1 knockdown zebrafish exhibited early arrhythmia. High-throughput transcriptome sequencing was performed for zebrafish embryos at 3 days post fertilization (dpf) in control and prkd1 knockdown groups. Differential gene enrichment analysis revealed that prkd1 knockdown was significantly associated with heart-related signaling pathways, such as the adrenergic signaling pathway in cardiomyocytes. Additionally, the results of high-throughput sequencing were confirmed by qRT-PCR, which showed that some genes associated with the cardiovascular system were markedly down-regulated. Meanwhile, identification of the essential factors in early heart development revealed that key cardiac regu-latory genes like tbx5a and gata4 were significantly down-regulated. These findings suggest that the prkd1 gene may affect early cardiac development by regulating cardiac development-related signal pathways and key genes, and play an important role at the early stage of heart development.

关键词

心脏发育 / prkd1基因 / 斑马鱼 / 吗啉代反义寡核苷酸(MO)

Key words

cardiac development / prkd1 / zebrafish / morpholino antisense oligo (MO)