Genomic structure of the alpha-amylase gene in the pearl oyster Pinctada fucata and its expression in response to salinity and food concentration

摘要

Amylase is one of the most important digestive enzymes for phytophagous animals. In this study, the cDNA, genomic DNA, and promoter region of the alpha-amylase gene of the pearl oyster Pinctada fucata were cloned by using reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends, and genome-walking methods. The full-length cDNA sequence was 1704 bp long and consisted of a 5'-untranslated region of 17 bp, a 3'-untranslated region of 118 bp, and a 1569-bp open reading frame encoding a 522-aa polypeptide with a 20-aa signal peptide. Sequence alignment revealed that P. fucata alpha-amylase (Pfamy) shared the highest identity (91.6%) with Pinctada maxima. The phylogenetic tree showed that it was closely related to P. maxima, based on the amino acid sequences. The genomic DNA was 10850 bp and contained nine exons, eight introns, and a promoter region of 3932 bp. Several transcriptional factors such as GATA-1, AP-1, and SP1 were predicted in the promoter region. Quantitative RT-PCR assay indicated that the relative expression level of Pfamy was significantly higher in the digestive gland than in other tissues (gonad, gills, muscle, and mantle) (P < 0.001). The expression level at salinity 27%, was significantly higher than that at other salinities (P < 0.05). Expression reached a minimum when the algal food concentration was 16 x 10(4) cells/mL, which was significantly lower than the level observed at 8 x 10(4) cells/mL and 20 x 10(4) cells/mL (P < 0.05). Our findings provide a genetic basis for further research on Pfamy activity and will facilitate studies on the growth mechanisms and genetic improvement of the pearl oyster P. fucata. (C) 2016 Elsevier B.V. All rights reserved.