CLONING AND EXPRESSION ANALYSIS OF KAISO IN GOLDFISH, CARASSIUS AURATUS

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.