Expression and Functional Analysis of <i>AMT1</i> Gene Responding to High Ammonia Stress in Razor Clam (<i>Sinonovacula constricta</i>)

Abstract

Ammonium transporter 1 (AMT1), a member of ammonia (NH3/NH4+) transport proteins, has been found to have ammonia transport activity in plants and microorganisms. However, the functional characteristics and molecular mechanisms of AMT1 in mollusks remain unclear. The razor clam (Sinonovacula constricta) is a suitable model species to explore the molecular mechanism of ammonia excretion because of the high concentration of ambient ammonia it is exposed to in the clam–fish–shrimp polyculture system. Here, the expression of AMT1 in S. constricta (Sc-AMT1) in response to high ammonia (12.85 mmol/L NH4Cl) stress was identified by real-time quantitative PCR (qRT-PCR), Western blotting, RNA interference, and immunofluorescence analysis. Additionally, the association between the SNP_g.15211125A > T linked with Sc-AMT1 and ammonia tolerance was validated by kompetitive allele-specific PCR (KASP). A significant upregulated expression of Sc-AMT1 was observed during ammonia exposure, and Sc-AMT1 was found to be localized in the flat cells of gill. Moreover, the interference with Sc-AMT1 significantly upregulated the hemolymph ammonia levels, accompanied by the increased mRNA expression of Rhesus glycoprotein (Rh). Taken together, our findings imply that AMT1 may be a primary contributor to ammonia excretion in S. constricta, which is the basis of their ability to inhabit benthic water with high ammonia levels.

Keywords

• AMT1
• <i>Sinonovacula constricta</i>
• ammonia excretion
• RNA interference
• gene expression
• SNP validation