Gene cloning and transcriptional regulation of the alkaline and acid phosphatase genes in Scylla paramamosain.

In crustaceans, innate immunity serves as the frontline of defense against microbes. Alkaline phosphatases (ALPs) and acid phosphatases (ACPs) are essential enzymes that play a significant role in crustaceans' immune defenses. However, the function and transcriptional regulation of the alp and acp genes in the Scylla paramamosain, an important aquaculture species in China, have not been elucidated. In this study, the full-length cDNAs of Spalp and Spacp were identified, which consist of 2,718 bp and 3,768 bp, encoding 579 and 452 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that these two genes were conserved among different species and shared high homology with crustaceans. The mRNA expression of Spalp and Spacp were examined in eight tested tissues, with the highest levels in the hepatopancreas. The 5'-flanking regions of Spalp and Spacp were cloned and sequenced. The core promoter region of the Spalp and Spacp was -39 bp∼+8 bp and -39 bp∼+10 bp, respectively. Potential binding sequences for SOX-2, c-fos, SP1, NF-κB, GATA-1, YY1, and AP-1 transcription factors were found in the 5'-flanking regions of Spalp and Spacp. The NF-κB binding site located between -1,223 bp and -972 bp in Spalp while SP1 and AP-1 binding sites located between -1,249 bp and -514 bp in Spacp. Mutation analysis confirmed that NF-κB negatively regulated the expression of Spalp gene, and SP1 and AP-1 positively regulated Spacp gene expression. These results provide us with essential information to elucidate the function of the Spalp and Spacp in S. paramamosain. This study is the first one to analyze the activity of Spalp and Spacp promoters.
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