Molecular Structure And Function Of Ecdysone Receptor EcR In Eriocheir Sinensis

Chinese mitten crab(Eriocheir sinensis),commonly known as river crab,is an important aquaculture crustacean in China.Molting is a unique biological phenomenon of crabs,which directly determines the growth and development.Ecdysone receptor(EcR)is a key regulator in the molting process and exists widely in crustaceans and insects which is a nuclear receptor and regulate molt.There are widely studies of EcR gene in molting process of insects,however,the molecular structure and function of EcR in molting of crabs have not been well researched.Here,the technologies of molecular biology,cellular biology,evolutionary biology,bioinformatics were used to study the EcR gene of Eriocheir sinensis in the following four aspects:(1)Molecular structure and phylogenetic evolution of EcR gene.(2)The function of EcR alternative isoforms in the molting and development of crab.(3)Regulation of EcR on metabolic pathway related to molting process of crab.(4)The function of EcR-RXR transcription complex in regulation of molting of crab.The main results and conclusions are presented as follows: 1.Molecular structure and phylogenetic evolution of EcR geneEcdysone receptor(EcR)of Eriocheir sinensis,similar to other nuclear receptors,has four functional domains: A/B domain,DNA binding domain(C domain),Hinge domain(D domain),and ligand binding domain(E domain).In this study,on the basis of previous laboratory transcriptome data,the full-length sequence of EcR gene was obtained by using the method of chromosome walking.The total length of EcR gene is 45,481 bp,which contains nine exons.Four alternative splicing isoforms of EcR gene were identified in hepatopancreas of crab at different molting stages by molecular cloning.They were named Es-EcR-1,Es-EcR-2,Es-EcR-3 and Es-EcR-4 with open reading box lengths of 1,638 bp,1,626 bp,1,545 bp and 1,638 bp,respectively.Based on the location of alternative splicing,three types of alternative splicing in these four transcripts: 5’splicing site type,5’ splicing site and exon spanning type,exon spanning,and intron retention type were identified.Four isoforms of EcR were predicted by sequence alignment and protein three-dimensional structure prediction.The four isoforms were conserved in the A/B and C domains,and the variation regions were located in the D and E domains.The 12 bp deleted in Es-EcR-2,12 bp deleted in Es-EcR-3 and the whole exon was located in the D domain,and the intron retained in Es-EcR-4 was located in the E domain,which showed the different EcR isoforms have different structure and may regulate different biological processes.The analysis of EcR gene isoforms revealed that the alternative splicing sites of insect were mainly in A/B domain,while the alternative splicing sites of crustacean species were mainly in D domain and E domain.The phylogenetic tree constructed by maximum likelihood method(ML)indicated that EcR genes of crustaceans are clustered into one group and insects are clustered into another group.Those results proved that EcR gene may have different evolutionary pressures in crustaceans and insects.2.The function of EcR alternative isoforms in the molting and development of crabTo explore the biological functions of different EcR isoforms,the expression of EcR gene in different tissues,hepatopancreas at different molting stages and ovaries at different developmental stages of crab were studied.qRT-PCR showed that different EcR isoforms had different expression patterns at different process.Es-EcR-2,Es-EcR-3,and Es-EcR-4 were expressed in different molting stages of crab.The expression of Es-EcR-2 and Es-EcR-4 did not change significantly in the intermolt,postmolt and premolt stages.However,the expression of Es-EcR-3 was nine times higher in the premolt stage than in the intermolt stage.Western blot showed that the protein level of Es-EcR-3 increased significantly in the premolt stage,which was consistent with the level of mRNA.The results showed that Es-EcR-3 is a key isoform in the regulation of the molting process of crab.With the siRNA interference technique,Es-EcR-3 isoform was successfully interfered.The results showed Es-EcR-3 interference significantly prolonged the molting time of crabs.This further indicated that EsEcR-3 isoform played an important role in the molting process.The expression patterns of other EcR isoforms were also different in different tissues.Es-EcR-2 and Es-EcR-4 were significantly higher in the ovary of crabs than in other tissues(except hepatopancreas).The expression of Es-EcR-1 was low in hepatopancreas,eyestalk and stomach tissue,but relatively high in muscle,heart,thoracic ganglion and testis.The expression of Es-EcR-2 isoform increased gradually with the development of ovary,which was significantly higher in primary oocyte and primary oocyte growth stage than in oogonium stage,suggesting that Es-EcR-2 may play a role in ovary development.3.Regulation of EcR on metabolic pathway related to molting process of crabIn order to explore how Es-EcR-3 gene regulates molt process,RNA interference technique was used to successfully interfere with the expression of Es-EcR-3 gene in the early stage of molting.The hepatopancreas of crab on EcR knockdown group and control group were sequenced with high throughput sequencing technology and their expression profiles were analyzed.There were 2,017 differentially expressed genes in the two groups.Compared with control group,1,124 genes were down-regulated,and 947 genes were upregulated in the knockdown group.Further GO enrichment analysis of differentially expressed genes showed that the genes expressed lowly in knockdown group mainly focused on redox reaction and reproductive regulation genes,and genes expressed highly mainly focused on steroid hormone-mediated signaling pathway.The interference of Es-EcR-3 may hinder the redox reaction process and cause a reduction in energy supply,then prolonged molting.This result was consistent with the above content 2.Meanwhile,the knockdown of EcR and the decrease of reproductive related gene expression indicated that EcR is related to reproduction.KEGG enrichment analysis showed the decreased expression genes in the experimental group were mainly concentrated in the process of insect hormone synthesis,histidine metabolism and the metabolism of exogenous substances by P450 cytochrome enzymes,while the highly expressed genes were mainly concentrated in immune-related metabolic pathways.Interference with EcR caused the decrease of the expression of genes related to insect hormones synthesis,which may lead to the blockage of hormone synthesis related to ecdysone signaling pathway and further lead the decrease of the expression of cytochrome P450 related genes involved in hormones metabolism.4.The function of EcR-RXR transcription complex in regulation of molting of crabIt was reproted that EcR and RXR must be combined to form EcR/RXR transcriptional regulatory complex to activate the expression of downstream genes and regulate the molting process in insects.In order to investigate the regulatory relationship between EcR and RXR during the molting process of crab,the transcriptional regulatory activity of EcR/RXR was studied.Six alternative splicing isoforms were identified in RXR of crab.They were Es-RXR-1,Es-RXR-2,Es-RXR-3,Es-RXR-4,Es-RXR-5 and Es-RXR-6 with the open reading box lengths of 1,350 bp,1,299 bp,1,236 bp,1,200 bp,870 bp and 915 bp,respectively.Studies on EcR and RXR during the molting cycle showed that the relative expression of EcR in hepatopancreas,epidermis and muscle tissues increased significantly at the premolt stage,but the relative expression of RXR in the three tissues did not increase significantly at the premolt stage.Analysis of absolute expression showed that RXR expression was 269 times(hepatopancreas),209 times(epidermis)and 299 times(muscle)of EcR at the postmolt stage,and 8 times(hepatopancreas),42 times(epidermis)and 28 times(muscle)at the premolt stage,respectively.It further proved that the expression of RXR was rich and stable during the molting cycle of crab,while the expression of EcR increases sharply at the premolt stage,which indicated that EcR is the main limiting factor in the process of molting and decide whether the molting starts or not.Dual fluorescence reporting system experiment found that only EcR or only RXR could not activate the expression of their downstream gene E75,but EcR and RXR together could activate the transcription of downstream gene E75.It suggested that EcR and RXR must form EcR/RXR transcriptional regulatory complex to regulate the expression of downstream genes and initiate molting processes.