金鱼etv2基因的克隆及在雌核发育单倍体和自交二倍体胚胎中的差异表达

CLONING AND EXPRESSION OF ETV2 IN GYNOGENETIC HAPLOID AND INBRED DIPLOID EMBRYOS OF GOLDFISH, CARASSIUS AURATUS

  • 摘要: 为研究鱼类雌核发育单倍体循环系统发育异常的原因, 从金鱼(Carassius auratus)中克隆了血管发生主调控基因etv2(Ets variant 2)的全长cDNA序列, 并比较分析了该基因在雌核发育单倍体和自交二倍体中的表达。金鱼etv2 cDNA全长1531 bp, 其开放阅读框为1116 bp, 编码371个氨基酸。序列对比分析表明, 金鱼ETV2蛋白的C端含有ETS(E26 transformation-specific)转录因子家族所共有的ETS DNA结合结构域, 该结构域氨基酸序列与其他脊椎动物ETV2的同源性超过60%。RT-PCR和荧光实时定量PCR分析结果表明, etv2在自交二倍体金鱼成体的肝脏、心脏、肌肉、肾脏、精巢、脑和脾脏等多种器官组织中表达, 但在卵巢和成熟卵子中不表达; 在金鱼胚胎发育过程中, etv2从尾芽期开始表达, 在体节形成后, etv2表达水平随胚胎发育而升高, 在20体节期达到峰值, 随后其表达水平降低。整胚原位杂交显示etv2特异性表达于自交二倍体金鱼胚胎的侧板中胚层、成血管细胞以及血管内皮细胞。在14体节期和20体节期, 雌核发育单倍体胚胎中etv2在躯干及尾部的部分区域表达减弱或缺失, 特别是在胚胎中线表达消失, 并且其整体表达水平显著低于自交二倍体。上述结果说明在金鱼雌核发育单倍体胚胎中成血管细胞数量减少并存在向中线迁移的障碍, 可能是导致雌核发育单倍体血管发生异常的重要原因。

     

    Abstract: All artificially induced haploid fish embryos show extensive lethality and a cluster of developmental defects known as haploid syndrome, in which oedema, expansion of pericardial cavity and distemperedness of blood circulation are the most common characteristics, and its molecular mechanisms remain incompletely understood. In order to study the causes of abnormal development of the circulatory system in haploid fish embryos, the etv2 (Ets variant 2, also known as ER71 or Etsrp), a master regulatory gene for embryonic vasculogenesis, was cloned in goldfish (Carassius auratus) and its expression patterns in gynogenetic haploid and inbred diploid embryos were compared. The full-length cDNA of goldfish etv2 contained 1531 nucleotides with a 1116 bp open reading frame (ORF) that encodes a protein of 371 amino acids. Amino acid sequence alignment showed that the structure of goldfish ETV2 protein consisted an ETS DNA binding domain at the C-terminal, which was conservatively shared with the ETS transcription factor family. The amino acid sequence of this domain has more than 60% homology with other vertebrates ETV2 proteins. In adult goldfish, the etv2 transcripts were expressed in liver, heart, muscle, kidney, testis, brain and spleen, but not in ovary and mature egg. Real-time quantitative PCR (qRT-PCR) showed that etv2 expression started at bud stage, increased as somitogenesis progression, reached a peak at the 20-somite stage and then gradually decreased in later stages of embryogenesis. Whole mount in situ hybridization analysis showed that embryonic etv2 expression was restricted to the lateral plate mesoderm, hemangioblasts, angioblasts and vascular endothelial cells. At the 14-somite stage and the 20-somite stage, the expression of etv2 in gynogenetic haploid embryos was reduced or lost in certain parts of trunk and tail, especially in the midline, and its transcription level was significantly lower than that of inbred diploids. These results indicated that the number of angioblasts in gynogenetic haploid embryos decreased and failed to migrate towards the midline, which may be an important reason for the abnormal vasculogenesis of gynogenetic haploid.

     

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