Molecular cloning and expression of purple acid phosphatase in Haliotis diversicolor under stress

The small abalone Haliotis diversicolor is one of the most commercially important cultured abalone in southern coastal areas in China. However, the frequent occurrence of infectious disease, especially during hot summers, is a major problem that has threatened the abalone aquaculture industry for a long time. As an invertebrate, abalone lacks an adaptive immune system and relies exclusively on innate immunity to defend against bacterial challenge. However, research on gastropod immune responses is limited. Purple acid phosphatase(PAP)belongs to a large family of dinuclear metalloenzymes and is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron(Ⅲ) charge transfer transition. PAPs are a group of tartrate resistant,molybdate sensitive, iron containing acid phosphatases with a molecular weight of about 35 40 kD and a high activity towards activated phosphoric acid monoesters and anhydrides. They catalyze the hydrolysis of a wide range of phosphate esters. PAPs play important roles in response to different stresses in plants and mammals.However, to date it has not been investigated in molluscs. In this study, the first molluscan PAP gene, HdPAP from H. diversicolor, was cloned by combining the expressed sequence tag(EST) and rapid amplification of cDNA end(RACE) methods. Its full length cDNA sequence is 1 215 bp, with a 969 bp open reading frame encoding a protein of 322 amino acids(GenBank: KC337074). The 5′ and 3′ untranslated regions(UTR) of HdPAP contain 28 bp and218 bp, respectively. Pairwise analysis results revealed that the HdPAP amino acid sequence has the highest identity, 59%, to the invertebrate Saccoglossus kowalevskii PAP. The calculated molecular mass of deduced HdPAP is36.8 kD with a theoretical isoelectric point(PI) of 5.27. Multiple sequence alignment of the HdPAP amino acid sequence with other known vertebrate PAPs and invertebrate PAP family proteins revealed that it was conserved,while their lengths varied among species. Predicted three-dimensional modeling(3D) of HdPAP from Swiss-model identified 7 α-helices and 12 β-sheets. These important secondary structures contribute to the function of this enzyme. HdPAP expression profiles in various tissues and at different time phases after thermal, hypoxia, or bacteria challenge were determined by real-time quantitative PCR. HdPAP was detected in all tissues examined, with the highest levels being in the hemocytes and hepatopancreas, suggesting that purple acid phosphatase is an important enzyme with a vital function in metabolic stress. HdPAP expression was suppressed to varying degrees in the gill,hemocytes, and hepatopancreas by thermal stress. Its expression was significantly down-regulated after 4 h and 24h in the gill, 4 h, 24 h, 96 h, and 192 h in the hemocytes, and 24 h, 96 h, and 192 h in the hepatopancreas at 30℃.HdPAP expression levels in the hemocytes of the hypoxic group was significantly lower than that of the control group after 4 h of exposure, then rose to the same level as the control group after 24 h. After 96 h the expression level was significantly higher than that of the control group and subsequently dropped to the same level as the control group after 192 h. Expression inhibition was also observed in the hemocytes after Vibrio parahaemolyticus(isolated from diseased H. diversicolor) injection, with the level being significantly lower than that of the control group after 3 h and 6 h. These results suggest that HdPAP plays an important role in environmental stress and bacterial challenge responses in abalone.