MOLECULAR CLONING OF TP53INP1 IN GRASS CARP AND ITS EXPRESSION IN RESPONSE TO MICROCYSTINS

In order to investigate its structure and expression changes induced by microcystin-LR (MC-LR), the cDNA sequence of TP53INP1 was amplified by using rapid-amplification of cDNA ends (RACE) (GenBank accession number: MG797689). TP53INP1 was 1154 bp in length, containing a 216 bp 3’ untranslated region (UTR) and a 759 bp open reading frame that encodes a protein of 252 amino acids. TP53INP1 of grass carp belongs to the type β subgroup and possesses four PEST motifs. The maximum identity between the derived TP53INP1 of grass carp and Anabarilius graham TP53INP1 was 98%, and the maximum identity with Homo sapiens TP53INP1α and TP53INP1β were 58% and 55%, respectively. Phylogenetic analyses showed that TP53INP1 fell into well-supported clades with Anabarilius graham, Danio rerio and other teleost. The results of qRT-PCR revealed that TP53INP1 gene was ubiquitously expressed in all examined tissues of healthy grass carp, and it was highly expressed in liver, blood and other tissues, and its relative expression was significantly higher than that in the head kidney (P<0.05). The livers of grass carp injected with the dose of 25 µg MC-LR/kg BW, 75 µg MC-LR/kg BW and 100 µg MC-LR/kg BW were analyzed by Western blot. TP53INP1 protein was significantly down-regulated in the 100 µg MC-LR/kg BW group at 96h (P<0.05) compared with control group with a fold change of 0.07. These results provide a basis for further research on the function of TP53INP1 and its mechanism in response to MC-LR.