Cloning and expression pattern of alkaline phosphatase during the development of Paralichthys olivaceus

Abstract

Alkaline phosphatases are ubiquitous enzymes involved in many important biological processes. Mammalian tissue-nonspecific alkaline phosphatase has long been thought to feature in embryonic development and bone formation. In this study, an alkaline phosphatase (ALP) gene from Paralichthys olivaceus was identified by rapid amplification of cDNA ends and genome-walking PCR. The ALP gene extends 10,141 bp and contains 11 exons and 10 introns. The open reading frame of the ALP transcript consists of 1,431 bp, which encodes 476 amino acids products named as POALP. An analysis of its secondary and tertiary structure revealed that the POALP was conserved in different species, but one disulfide linkage made it possible to adapt to low-temperature environment. The ALP activity was found to be first detectable in the embryo before hatching. The POALP was distributed ubiquitously in the body of P. olivaceus and was particularly high in the digestive tract. These findings suggest the potential role of POALP in nutrient absorption and transportation. During the pre-metamorphosis (F stage), ALP gene expression is 2.5-folds of that in the pro-metamorphosis (E stage); but in the post-metamorphosis (I stage), it was 1.8-folds of that of pro-metamorphosis. Exogenetic thyroxine (T4) and thiourea (TU) influenced the ALP gene expression significantly during the metamorphosis. Bioinformatics analysis showed that Japanese flounder ALP promoter region contained promoter sequence and putative recognition site for several transcriptional factors, including SREBP-1, SYR, and CdxA. In vitro promoter assays employing EGFP reporter system demonstrated that the promoter of ALP was active.