Molecular Cloning And Expression Pattern Analysis Of JHBP And JHE Gene Family In Scylla Paramamosain

Scylla paramamosain(mud crab),belonging to the Crustacea,Scylla,is one of the main precious marine crab species aquacultured in the southeastern coastal areas of China.Due to the nutritional,economical,and pharmaceutical values of sexually mature female crabs,they are increasingly favored by consumers and researchers.At present,precocious puberty during the process of breeding crabs is aways a key problem to be solvedin the mud crab breeding process,which greats limits the development of the mud crab aquaculture industry.Therefore,the molecular regulation mechanism of the endocrine system of S.paramamosain is one of the important research contents of Crustacea.In recent years,a lot of researches have been done on how to obtain high quality crabs in the process of crab breeding,and made some progress.Methyl farnesoate(MF)is an important endocrine hormone regulating the growth,development and plays a crucial role in sexual maturity.After the synthesis of MF in the Mandibular organ(MO),it takes the help of the Methyl farnesoate binding protein(MFBP)during its entry into the target organ,protecting and transporting MF against degradation by specific esterases.Specific carboxylesterases in the hemolymph are able to hydrolyze MF and play a crucial role in controlling the titer balance of MF.Therefore,the molecular biological studies of these related genes in the MF metabolism of S.paramamosain have good guiding significance for understanding the endocrine control mechanisms of crustaceans.From the perspective of molecular biology,this study is based on the genomic data of S.paramamosain,and intends to analyze the juvenile hormone binding protein(JHBP)and juvenile hormone esterase(JHE)associated with MF metabolism during the development and reproduction of S.paramamosain.The JHBP and JHE gene family were cloned,and different tissues and developmental stages expression patterns of S.paramamosain were analyzed.The main research results obtained were as follows: 1.Molecular cloning and expression analysis of the Sp-JHBP in S.paramamosainJHBP protein,a class of protein family that plays an important role in protection/transportation of juvenile hormone(JH)signaling pathway,is a key component of JH metabolism system.In this study,seven JHBP sequences were cloned and obtained from S.paramamosain.The multiple sequence comparisons showed high similarity with JHBP domain in insects.They were named as Sp-JHBPs after analysis.The lengths of the seven ORFs were 663 bp to 1,326 bp,encoding 221-441 amino acids,the predicted protein molecular weight is 24,157-48,188 Da,isoelectric point 4.10-5.77.Only Sp-JHBP1 contains no signal peptide.The consistency of the seven Sp-JHBPs sequences was 38.96%.The secondary structure of the seven Sp-JHBPs includes 26.0-39.7% alpha helix,28.5-47.6% strand,and 25.0-31.8% loop,except for Sp-JHBP5.All contain 2-6 protein binding regions.Under simulated conditions,the 3D structure of seven Sp-JHBP proteins could bind to MF.Quantitative analysis showed that seven genes almost expressed in zoea stages(Z1-Z5),megalopa stage(M),and juvenile crabs stages(C1-C2)during the developmental stages of S.paramamosain,but the expression levels of Sp-JHBP1 and Sp-JHBP5 are very low.The expression of Sp-JHBP2 was the highest in Z1 stage,but no expression was detected during the molting of Z2-Z4 zoea,and then a higher expression level was observed during Z5-C1 metamorphosis stages.Sp-JHBP3 and Sp-JHBP4 have similar expression patterns.During the metamorphosis stage of Z5-C2,there was only high expression in the C1 stage.The two stages of Z5 and M are the metamorphosis transition stages of juvenile crabs.We speculated that Sp-JHBP3 and Sp-JHBP4 may be related to MF function.In the tissue-distribution expression patterns of Sp-JHBPs,it was found that Sp-JHBP2 is mainly expressed in ovarian tissues and Sp-JHBP3-7 is highly expressed in the hepatopancreas.Adults induction experiment of MF indicates that MF can inhibit the expression of Sp-JHBP2 in ovary,and has no significant effect on the expression of Sp-JHBP3,4,5,6,7 in hepatic pancreas.The experimental results of MF induced megalops showed that MF significantly up-regulated the expression of Sp-JHBP s,and the upregulation effect was the most significant with 0.1 ?M.The above results indicate that Sp-JHBPs of S.paramamosain is regulated by MF.2.Molecular cloning and expression analysis of the Sp-JHE in S.paramamosainJHE is a member of carboxylesterase family presenting in arthropods,and plays an important role in maintaining the titer balance of endocrine hormone-JH in organisms.In this study,four JHE sequences were cloned and obtained from our database.Combined with previous studies in our laboratory,eleven JHEs were obtained.The similarity of the eleven gene sequences was 52.56%.It is closely related to JHE in insects and named Sp-JHEs.Their cDNA sequences ranged in length from 1,594 to 4,371 bp,and ORFs ranged from 720 bp to 1,797 bp,encoding 239-598 amino acids.The molecular weight of the predicted protein was 26,488-66,729 Da and the isoelectric point was 5.02-6.29.Multiple sequence alignments showed consensus typical conserved motif of the JHE protein.The Sp-JHEs transcript results showed different distribution characteristics.In summary,Sp-JHBP and Sp-JHE gene sequences related to MF metabolism were cloned in this study,and then related bioinformatics analysis was performed.Their expression patterns in were studied S.paramamosain.The results showed that Sp-JHBP and Sp-JHE were involved in the larval growth,development and reproduction of S.paramamosain.These basic studies provided data support for further revealing the endocrine control mechanisms of crustaceans.