Cloning, expression and cellular localization of Daphnia pulex senescence-associated protein, DpSAP

Abstract

Daphnia (water fleas) are small crustaceans that undergo an unusual switch from asexual to sexual reproduction that is dependent on environmental conditions. In this study, a senescence-associated protein (SAP) from the common freshwater species Daphnia pulex was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). Real-time PCR was employed to quantify the expression of D. pulex SAP (DpSAP) in individual organisms. The role of DpSAP in the reproductive transformation was further investigated in both parthenogenetic and sexual females by using digoxin-labeled SAP RNA probes and RNA whole-mount in situ hybridization. DpSAP was more highly expressed in sexual females, indicating a role in growth and reproduction. Cellular localization studies using RNA whole-mount in situ hybridization showed specific expression in the second tentacle joints. These expression patterns suggest an important role for DpSAP in the reproductive transformation of D. pulex.

Keywords: CAT; Daphnia; Daphnia pulex SAP; DpSAP; Gene cloning; MDA; MW; NJ; ORF; PCR; POD; RACE; RNA whole-mount in situ hybridization; ROS; RT-PCR; Reproductive transformation; SAGs; SAP; SDGs; SOD; Senescence-associated protein (SAP); catalase; isoelectric point; malondialdehyde; molecular weight; neighbor-joining; open reading frame; pI; peroxidase; polymerase chain reaction; rapid amplification of cDNA ends; reactive oxygen species; reverse transcription polymerase chain reaction; senescence-associated genes; senescence-associated protein; senescence-downregulated genes; superoxide dismutase.