| Eriocheir sinensis is an economic crab with high economic value.Malic dehydrogenase(MDH)is an oxidoreductase and plays a key role in energy metabolism of organisms.The synthesis of malic acid catalyzed by phosphoenolpyruvate carboxylase in cell matrix provides energy for the activity between cytoplasm matrix and organelles.Cathepsin D(CAT-D)belongs to the aspartate protein family in eukaryotic lysosomes,which can hydrolyze proteins and peptides;In terms of male reproduction,cathepsin is directly related to the sclerosis of sperm pods after being expelled in vitro,and is related to the subsequent hydrolysis of sperm pod matrix and the release of sperm.Cathepsin can promote cells through the blood-testis barrier,help sperm release from the spermatogenic epithelium into the lumen,and remove the dead sperm cells and the residues of spermatogenic epithelium in the seminiferous tubules.In this study,the gene fragments of MDH and CAT-D were obtained by polymerase chain reaction,and the expression strains of MDH and CAT-D were constructed.The high-purity proteins were obtained by the specific combination of nickel column and His tag.CAT-D polyclonal antibody was obtained by polyclonal antibody preparation technology,Monoclonal antibody against MDH was obtained by monoclonal antibody preparation technology.The distribution characteristics of MDH and CAT-D in sperm were investigated by Western blot,immunofluorescence and immunoelectron microscopy,and the functions of MDH and CAT-D in sperm acrosome reaction were analyzed.Results : In this study,the expression strains of MDH and CAT-D were successfully constructed,and the in vitro expression proteins of CAT-D and MDH were successfully obtained after induction,which were verified as MDH and CAT-D proteins by mass spectrometry.The titer of monoclonal antibody against MDH obtained by single antibody preparation was 204800;CAT-D polyclonal antibody was obtained by polyclonal antibody preparation technology,and the titer reached 1 : 102400.Western blotting experiments showed that MDH monoclonal antibody could specifically recognize and bind to MDH protein,and CAT-D polyclonal antibody could specifically recognize and bind to CAT-D protein.Immunofluorescence technique showed that CAT-D was distributed in the acrosomal tube or cap of E.sinensis sperm;MDH is distributed in two parts of sperm : acrosomal duct and acrosomal sac.It was found by immunoelectron microscopy that CAT-D was mainly localized under the head cap of E.sinensis sperm and distributed linearly,which was speculated to be related to acrosomal sac valgus and acrosomal membrane rupture;In addition,there is less scattered distribution in the middle layer of acrosomal sac,which is speculated to be related to the abscission of lamellar structure,so that only sperm nucleus,cap and acrosomal tube are left.MDH is located in the sperm acrosomal tube and acrosomal sac of Eriocheir sinensis,suggesting that MDH provides energy for the processes of acrosomal cyst eversion,acrosomal tube extension and lamellar structure shedding.In this study,the position and related functions of MDH and CAT-D proteins in sperm were preliminarily explored and analyzed,which provided basis for exploring the molecular mechanism of the formation,reaction and fertilization of Eriocheir sinensis. |
