鲫Kaiso基因cDNA的克隆和表达分析

CLONING AND EXPRESSION ANALYSIS OF KAISO IN GOLDFISH, CARASSIUS AURATUS

  • 摘要: 在爪蟾和斑马鱼中, Kaiso是一种在整个基因组范围内与甲基化CpG序列特异性结合的转录抑制因子, 在调控被甲基化基因表达的时间模式中起重要的作用。为深入研究DNA甲基化对我国重要养殖鱼类生殖和发育的影响, 我们克隆了鲫Kaiso基因的cDNA序列, 并对其时空表达模式进行了分析。该cDNA全长3145 bp, 5-非翻译区132 bp, 3-非翻译区1117 bp, 开放阅读框1896 bp, 编码631个氨基酸。鲫Kaiso蛋白与其他物种Kaiso蛋白的同源性分析表明, 与其他物种一样, 其 N端和C端分别有高保守性的BTB/POZ结构域和锌指结构域。整胚原位杂交结果显示, Kaiso mRNA在早期胚胎发育的各个时期均广泛表达, 信号均一, 但从尾芽期开始出现组织特异性表达差异。对不同发育阶段胚胎的实时定量PCR检测结果表明: 卵子中有高丰度的母源Kaiso mRNA存在; 在卵裂期至囊胚中期胚胎中Kaiso mRNA的丰度逐渐降低; 从囊胚中期至原肠早期都维持在最低水平状态; 原肠后期其表达水平又逐渐升高, 至尾芽期达到与未受精卵中相当的高水平后在器官发生期的整体水平又稍有下降。Kaiso mRNA丰度在胚胎发育早期的这种变化过程提示在卵裂期检测到的mRNA可能都是母源mRNA, 合子核Kaiso基因可能是在囊胚晚期后才开始转录。对成体不同组织的实时定量PCR检测结果表明Kaiso的表达存在明显的组织特异性差异, 在鲫肌肉、视网膜、心脏和脑中表达水平较高, 而在肾、胰、肝等器官中表达水平很低。Kaiso表达的时间和组织特异性提示其作为甲基化基因的转录抑制因子参与了胚胎和成体基因表达时空模式的调控。这些结果为进一步研究Kaiso和DNA甲基化修饰在鲫发育调控和遗传育种中的作用提供了基础资料。

     

    Abstract: Previous studies have demonstrated that Kaiso, a methyl-CpG specific binding protein and a global transcriptional repressor, plays an important role in timing the expression of methylated genes during early embryogenesis in amphibian and zebrafish. To investigate the reproductive and developmental functions of DNA methylation in goldfish (Carassius auratus), an important cultural fish, we cloned its full-length Kaiso cDNA by reverse transcription and rapid amplification of cDNA ends (RACE). The spatiotemporal expression pattern of goldfish Kaiso was examined by whole mount in situ hybridization and real-time quantitative reverse-transcriptional polymerase chain reaction (qRT-PCR). The entire Kaiso cDNA was 3145 bp long, including a 132 bp long 5-UTR, a 1117 bp long 3-UTR and a 1896 bp long open read frame, which encoded a protein with 631 amino acids. Amino acid sequence alignment of Kaisos of Carassius auratus, Danio rerio, Xenopus laevis, Gallus gallus, Mus musculus and Homo sapiens revealed that the structure of goldfish Kaiso protein also consisted a highly-conserved BTB/POZ domain at the N-terminal and zinc finger domains at the C-terminal. Whole-mount in situ hybridization examination showed that Kaiso was ubiquitously expressed during early embryogenesis but tissue-specifically expressed from bud stage onward. qRT-PCR examination revealed that high abundance maternal Kaiso mRNA existed in the eggs. During embryogenesis, the level of Kaiso mRNA gradually decreased during cleavage and remained low from late blastula stage to early gastrula stage, and then gradually increased from late gastrula stage and reached to the highest level at bud stage. These results suggested that the Kaiso transcripts detected in cleavage stage might be the maternal mRNA and the transcription of zygotic Kaiso might start at late blastula stage. qRT-PCR analysis of different adult tissues revealed that the transcriptional levels of Kaiso in the muscle, retina, heart and brain were much higher than those in the kidney, pancreas and liver. The spatiotemporal expression pattern of Kaiso suggested that Kaiso as a methyl-CpG specific binding protein and a global transcriptional repressor was involved in regulating the spatiotemporal pattern of gene expression both in the embryo and in the adult. These results provided useful information for studying of the roles of Kaiso and DNA methylation in goldfish reproduction and embryogenesis.

     

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