Cloning Of Melatonin Receptor Gene And The Distribution And Function In Germ Cells Of Eriocheir Sinensis

Eriocheir sinensis,commonly known as crabs,is Crustaceans,and it is one of the most commercially important aquaculture species,which is widely distributed in freshwater regions in China.Melatonin(MT)was first discovered from the pineal gland extract in 1958.Melatonin is a hormone secreted mainly by the pineal gland of animals and is considered as the key factor to regulate the seasonal reproduction of mammals.MT has been preserved in the course of evolution,which indicating that MT has its own unique function.Melatonin is known to regulating several physiological functions in crustaceans including glucose homeostasis,molting and limb regeneration.What is more,melatonin can affect the development and maturation of ovary by modulating the release of hormones in the X organ-sinus gland complex.And melatonin performs various functions by membrane receptors.But it needs to be further clarified how achieved many of these molecular mechanisms about MT and MTR.Recently,research shows that melatonin receptor gene is signifcantly down-regulated during the development of androgenic gland(AG),which indicates that MTR is involved in the gonad development of E.sinensis.It is not clear that the expression and distribution characteristics and reproduction regulation mechanism of melatonin receptor(MTR)in male crabs germ cells.The characteristics that MTR distributed in the process of acrosomal and germ cells of crab were analyzed by preparation of corresponding antibodies.And analyzing the influence of MT and MTR on sperm acrosomal reaction and the possible mechanism,it can provide the theoretical basis for the further analysis the acrosomal reaction molecular mechanism about MT and MTR.In this experiment,the sequence of immunogen for antibody preparation was determined by analyzing the bioinformatics of the testis transcriptome database of E.sinensis in the laboratory.The gene sequence of the immunogen,which was obtained by polymerase chain reaction(PCR).The gene sequence of MTR with cleavage sites and the expression vector pET-21 b were double-digested in Nde I and Xho l enzymes.And both were ligated with ligase.Then,MTR prokaryotic expression vector was constructed in gene recombination technique and expressed in Escherichia coli BL 21(DE3).So,an engineered strain was prepared which could express MTR protein in large quantities.However,The MTR recombinant protein was expressed in the inclusion bodies.The inclusion bodies were dissolved with urea,and the protein was purified by the affinity of nickel column with histidine of pET-21 b.Using the purified protein to immunizing mice,and detected the antiserum titer of MTR in indirect ELISA.Western blot showed that the MTR polyclonal antibody could effectively bind to the antigen.The polyclonal antibody was used to study the location analysis of MTR in germ cells by immunofluorescence technique.The acrosome reaction rate of sperm was observed by using eosin staining.Fresh sperm of crab were extracted and divided into four groups:(1)control group,the fresh sperm with DMSO;(2)the fresh sperm with MT;(3)the fresh sperm with MT and MTR antiserum;(4)the fresh sperm with MT and negative serum.And the acrosome response rate of sperm was calculated.The results showed that MTR presented throughout spermatogenesis.MTR uniformly presented in plasma membranes in spermatogonium and spermatocyte.And the distribution of MTR in membrane was not uniform in spermatid and metaphase spermatid.However,MTR presented in the acrosomal tubule membrane of sperm.And MTR presented in the acrosomal tubule membrane during acrosomal reaction.The statistical result showed that there were significant difference between the control group and the MT treatment group,significant difference between MT group and MT with MTR antiserum group,significant difference between MT group and the MT with negative serum treatment group.However,there were no difference between the MT treatment group and the MT with negative serum treatment group.It indicated that MTR had an effect on the acrosomal response of the crab sperm and MTR might affect acrosomal response of sperm by binding to MT.