Molecular 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, an important cultural fish, we cloned the full-length Kaiso mRNA of goldfish(Carassius auratus) 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 Ploymerase Chain Recaction (qRT-PCR). The entire Kaiso cDNA is 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 encodes a protein with 631 amino acids. Amino acid sequence alignment of Kaisos of Carcasses auratus, Danio rerio, Xenopus laevis. Gallus gallusy Mus musculus and Homo sapiens revealed that the structure of goldfish Kaiso protein also consists 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 is ubiquitously expressed during early embryogenesis but tissue-specifically expressed from bud stage onward. qRT-PCR examination revealed that high abundance maternal Kaiso mRNA exists in the eggs. During embryogenesis, the level of Kaiso mRNA was gradually reduced during cleavage and was maintained at the lowest from late blastula stage to early gastrula stage, but was 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 level of Kaiso in the muscle, retina, heart and brain is much higher than that in the kidney, pancreas and liver. The spatiotemporal expression pattern of Kaiso suggests that Kaiso as a methyl-CpG specific binding protein and a global transcriptional repressor is 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.