Molecular Cloning And Expressive Characterization Of Gonadotropin-releasing Hormone Receptor Homolog In The Chinese Mitten Crab,Eriocheir Sinensis

As an essential mediator in the Gonadotropin-releasing hormone(GnRH)signaling pathway,GnRH receptor(GnRHR)coupled to GnRH,plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction.At present,we have selected the genes related to GnRH signaling pathway in the Chinese mitten crab cerebral and ovarian transcriptome.These sequences of the genes screened were highly similar to sequences in most insect and other crustaceans.Our laboratory has successf?lly isolated the GnRH analogue from the Chinese mitten crab.So far,we have not seen the research on GnRHR gene in the Chinese mitten crab.In order to study whether there is a pathway similar to the hypothalamus-pituitary gonadal axis in invertebrates to reg?late gonad,the experiment selected the important aquac?lture specie the Chinese mitten crab(Eriocheir sinensis)as an object to study gonadotropin-releasing hormone receptor molec?lar identification and expression in the invertebrate crustaceans.To detect the existence of GnRHR,we cloned and characterized GnRHR in the Chinese mitten crab,Eriocheir sinensis(named EsGnRHR).The f?ll-length EsGnRHR cDNA is 2038 bp in length,including an open reading frame(ORF)of 1566 bp,a 57-bp 5?-untranslated region(5?-UTR)and a 415-bp 3?-UTR.Analysis of transmembrane prediction showed that the predicted EsGnRHR protein contained seven ? hydropHobic transmembrane regions(TMs).This feature is consistent with the gonadotropin release hormone receptors that had been identified in vertebrates.The analysis of cluster evolution tree shows that the relationship between the the Chinese mitten crab GnRHR and the macrobrachium nipponense GnRHR are relatively close.Analysis of the expression of EsGnRHR gene in various tissues of the Chinese mitten crab ad?lts by reverse transcription PCR.Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary,and weakly distributed in the testis and brain.Further studies in situ hybridization was used to verify the distribution of EsGnRHR gene at the mRNA level.It showed that the hybridization signals of EsGnRHR mRNA were mainly distributed in the protocerebrum(pro),deutocerebrum(deu)regions of the brain.In the ovarian tissue,the situ hybridization signals were mainly in the cytoplasm of pVt and the cytoplasm of the Vt;In the testis,EsGnRHR mRNA is mainly distributed in spermatocyte and spermatocyte.These res?lts show that the GnRHR gene is present in the Chinese miter crab.Real-time fluorescent quantitative PCR res?lts showed that EsGnRHR mRNA expression in whole testis and ovary development period,suggesting that EsGnRHR was involved in controlling gonadal development.Finally,the res?lts of fluorescence qnantitative analysis about injection of GnRH polypeptide in the Chinese mitten crab suggested that the effect on the expression of GnRHR,according the res?lts of GnRHR expression in male crab,in 15d(day)appeared significant difference,however,in the female crab in 24h(hour)and 7d point have significant difference,suggested that GnRH can stim?late GnRHR mRNA expression in the Chinese mintten crab.In conclusion,this study presents the first report of characterization of EsGnRHR cDNA of the Chinese mitten crab.Molec?lar characterization of the gene was supplemented by phylogenetic analysis and researching its evolutionary relationship with other species.The EsGnRHR mRNA was distributed in the nervous and gonad.In partic?lar,the EsGnRHR was expressed throughout testic?lar development,and early and medium ovarian development,implying the existence of diverse functions of EsGnRHR.Although further investigations are necessary,our information of EsGnRHR wo?ld provide support for the hypothesis of brain-thoracic ganglion-gonadal axis in the Chinese mitten crab.