Cloning And Functional Analysis Of Ecdysis Triggering Hormone Receptor(ETHR) And Gametocyte-specific Factor 1(Gtsf1) In Mcrobrachiumnipponense

The oriental river prawn Macrobrachium nipponense(Arthropoda;Crustacea;Decapoda;Palaemonidae)is widely distributed in freshwater areas of China,Japan,Korean and north of Vietnam.Due to its flavor and high nutritive value,it is widely farmed in China and has been an economically important freshwater prawn in China.Recently the important genes about M.nipponense have been widely studied,and it also has become one of research focus of our lab.So far,nearly 20 important genes about reproduction,development,sex of M.nipponense have been studied in our lab.Previous study showed that Ecdysis triggering hormone receptor(ETHR)play an important role in initiating the molting behavior in insects and Gametocyte-specific factor 1(Gtsfl)plays a crucial role in regulation of germ cell development in insect and mammal,while these are unknow in crustacean.Based on the normalized cDNA library of ovary and testis of M.nipponense in our lab,a partial fragment of ETHR cDNA and an ES’T of Gtsfl were identified.To further our understanding of ETHR and Gtsfl in crustaceans,we cloned the full-length ETHR and Gtsfl cDNA from the freshwater prawn M.nipponense,and investigated their spatial expression.We also used RNA interference(RNAi)technology to investigate the function of ETHR in molting.ETHR is a member of G protein-coupled receptor and plays crucial physiological roles in arthropod molting.In this study,the full-length cDNA of ecdysis triggering hormone receptor gene of M.niponense(ETHR)was first cloned and the expression of the gene was investigated.The transcript encoded 390 amino acids and has the seven transmembrane domains and specific functional sites which were highly conserved with those in other arthropod species.It was highly expressed in brain among ten different examined tissues,indicating that brain is the major function site of ETHR.During embryo development,ETHR displayed a relatively low expression level in the early stages(cleavage stage,blastula stage,gastrula stage)and then increased gradually from nauplius stage.A significantly increase was detected in zoea stage(P<0.05).There was a rapid elevation of ETHR in the two examined tissues(brain and epidermis)in the premolt stage(P<0.05).In dsRNA-mediated RNA interference experiment,the expression level of ETHR in experimental team was significantly decreased and the observed molting frequency was nearly half of the control team,which prove that ETHR can promote molting in M.nipponense.Eyestalk ablation(ESA)could also significantly up-regulation of the expression and molting,which show that eyestalk has inhibitory effect on ETHR mRNA expression and molting.Gtsfl is involved in gonadal development and plays an important role in maintaining normal proliferation of germ cells in Mus musculus and Drosophila melanogaste.In order to study precocity in Macrobrachium nipponense,the homologous cDNA sequence of Gtsfl was screened from the ovary and testis differentially gene expression profile,and the full-length cDNA sequence was cloned by RACE technique(KR349325).The length of its cDNA sequence was 349 bp,containing a 240 bp open reading frame(ORF),encoded a 79-amino acid protein.MnGtsfl contained a zf-U11-48K domain that shared highly with its homologues in other vertebrates.The sequence did not contain signal peptide,indicating it is a non-secreted protein.According to the phylogenetic tree analysis,MnGtsfl has the closest evolutionary relationship with its homologous in Lygus hesperus.MnGtsfl wasexclusively expressed in the gonads,especially in ovary,which is 6 times higher than that in testis.The MnGtsfl expression increased with the ovarian development.MnGtsfl expression in the early embryonic development was higher than that in the late embryonic development.The MnGtsfl expression showed the highest in blastula stage,and its expression stayed at a low level from gastrula stage to the 10th day after metamorphosis.These results indicate that MnGtsfl might be involved in early embryonic development.Thereby these results proved basic data for further function research of Gtsfl in M.nipponense.