| Macrobrachium nipponense,a decapod crustacean with high economic value,is one of the main species of freshwater aquaculture in china.In recent years,with the sharp drop of natural resources and the scale expansion of aquaculture of M.nipponense,the farmed M.nipponense showed a decline in production performance and the degradation of germplasm resources,especially the emergence of the phenomenon of sexual precocity.The precocious M.nipponense usually reach breeding period in advance,when the gonad has matured and the growth has stopped,the prawn has not yet reached the size of the commodity specifications,and thus decreasing the commodity rate and devaluing the commercial production of M.nipponense.The precocious female prawns often produce low-quality offspring,which aggravate the degradation of germplasm resources and restrict the healthy development of aquaculture of M.nipponense.So,it is important for elucidating the molecular mechanism of sexual precocity and solving the problem of degradation of germplasm resources of M.nipponense to study on the sexual precocity of female M.nipponense,screen sexual precocity related genes and analyze the function of these genes.In this study,the differentially expressed genes(DEGs)between ovaries of sexually precocious and normal sexually mature M.nipponense were identified by differential expression analysis through transcriptome sequencing.Through annotation,cluster analysis and significant enrichment analysis on these DEGs,genes that could be related to the sexual precocity of M.nipponense were screened out.The full-length cDNA sequences of 5 genes that could be related to the sexual precocity of M.nipponense were obtained using gene cloning and rapid-amplification of cDNA ends(RACE)PCR techniques,and their sequencs were analysized through bioinformatical methods.The expression patterns of these 5 genes were detected and the roles of these 5 genes in ovarian development of M.nipponense were explored by RNA interference technique.The main results were as follows: 1.Transcriptome sequencing of ovaries of sexually precocious and normal sexually mature M.nipponenseThrough transcriptome sequencing,63,633 unigenes as well as a large number of reproduction-related unigenes and metabolic pathways were obtained from ovaries of sexually precocious and normal sexually mature M.nipponense.Moreover,26,008 SSRs were identified from 18,133 unigenes.80,529 and 80,516 SNPs were yielded from ovarian libraries of sexually precocious and normal sexually mature prawn,respectively,and 29,851 potential SNPs between these two groups were also predicted.For the first time,549 DEGs were identified from the ovary of sexually precocious M.nipponense compared with the ovary of normal sexually mature prawn,including 212 up-regulated genes and 337 down-regulated genes.In addition,187 DEGs specifically expressed in the ovaries of normal sexually mature prawn and 90 DEGs specifically expressed in the ovaries of sexually precocious prawn were identified from the 549 DEGs.Through GO classification analysis,KEGG pathway analysis,GO and KEGG significant enrichment analysis of these 549 DEGs,12 DEGs,namely,Vitellogenin(Vg),Cathepsin L(CTSL),Cystatin(CST),Insulin-like receptor(INSR),Insulin-like growth factor 1 receptor(IGF1R),Cyclooxygenase(COX),Copper and zinc superoxide peroxidase(SOD),Fatty acid synthase(FASN),Diphosphate nucleoside kinase(NM23),Ribosomal protein L24(RPL24),Glutathione peroxides(GPX)and ribonucleoside reductase subunit M1(RRM1)were identified as genes related to sexual precocity of M.nipponense.2.Cloning and sequence analysis of genes related to sexual precocity of M.nipponenseThe full-length cDNA sequences of MnNM23,Mn RPL24,MnCOX,MnCST and MnRRM1 genes were cloned.The full-length cDNA of these 5 genes were 829 bp,564 bp,3238 bp,6199 bp and 2941 bp,respectively,and their ORF were 531 bp,486 bp,1845 bp,2709 bp and 2436 bp,respectively,encoding 177,162,615,903 and 812 amino acids,respectively.Based on the bioinformatics analysis,it was found that MnNM23 protein had a conserved NDK domain and NDK active site motif(NXXH [G/A] SD),and had the highest similarity with the amino acid sequence of NM23 protein of Macrobrachium rosenbergii(MrNM23);The MnRPL24 protein has a conserved Trash domain,20 phosphorylation sites and two typical nuclear localization signals.The MnRPL24 protein has not the tag sequence that mitochondrial type RPL24 should possess,so MnRPL24 protein is a cytoplasmic type ribosome protein;MnCOX has an EGF-like domain,a transmembrane helix domain and eight conserved amino acid residues which play an important role in cyclooxygenase activity,the mRNA of the 3'untranslated region(3'UTR)sequence has many AU-rich elements(AREs),so the structure and function of MnCOX should be closest to COX2 of vertebrates;The amino acid sequence of MnCST contains 6 Cystatin-like domain,so MnCST is a multicystatin protein;MnRRM1 has conserved enzyme active sites,the two ?-helical domains(necessary for the polymerization of subunits)that class I ribonucleotide reductases should possess,MnRRM1 belongs to the large subunit of class I RR.3.The mRNA expression analysis of genes related to sexual precocity of M.nipponenseThe expression levels of MnNM23,MnRPL24,MnCOX,MnCST and MnRRM1 genes were lower in fertilized eggs among six growth stages including fertilized eggs,nauplius,zoea,mysis,postlarva and adult prawn;In stage ?-? ovaries of M.nipponense,the expression levels of MnNM23,MnRPL24,MnCOX and MnRRM1 were the highest in stage ?ovary,and the expression level of MnCST gene was the highest in stage ?ovary;The MnNM23 gene was highly expressed in hepatopancreas and muscle among the nine tissues including ovary,stomach,gill,heart,abdominal ganglion,muscle,intestines,hepatopancreas and eyestalk of M.nipponense.The expression levels of MnRPL24 and MnCST gene was the highest in the intestines of M.nipponense,and the expression levels of MnCOX and MnRRM1 was the highest in the gill of M.nipponense;There were significant differences in the expression levels of these five genes in the ovaries between sexually precocious and normal sexually mature M.nipponense(P<0.05).4.Functional verification of genes related to sexual precocity of M.nipponenseAfter MnNM23 gene silencing,the expression levels of Vg,Vgr,Cyclin B and Cdc2 genesin ovary of M.nipponense,and the GSI of prawn were always peaked in advance during an ovarian development cycle compared with the control group.The expression levels of Vg,Vgr,Cyclin B and Cdc2 genes and the GSI of prawn were significantly decreased at many time points during an ovarian development cycle after MnRPL24,MnCOX and MnRRM1 gene silencing compared with the control group(P<0.05).After MnCST gene silencing,the expression levels of CTSL gene was significantly decreased at many time points during an ovarian development cycle compared with the control group(P<0.05),while the expression levels of Vg,Vgr,Cyclin B and Cdc2 genes and GSI of prawn showed no significant difference between sexually precocious and normal sexually mature M.nipponense(P>0.05).Ovarian histological observation showed that ovarian development of M.nipponense was accelerated by MnNM23 gene silencing,while was delayed by MnRPL24,MnCOX and MnRRM1 gene silencing,and was not affected by MnCST gene silencing.Therefore,MnNM23 has a negative correlation with vitellogenesis and oocyte maturation of M.nipponense,and the low expression of MnNM23 gene promotes the rapid development of ovary.MnRPL24,MnCOX and MnRRM1 had a positive correlation with the vitellogenesis and the oocyte maturation.The low expressions of MnRPL24,MnCOX and MnRRM1 genes delay the ovarian development.MnCST had no effect on vitellogenesis and oocyte maturation,and thus had no effect on ovarian development.The lack of MnNM23,or the excess of MnRPL24,MnCOX and MnRRM1 during ovarian development may be the important causes of sexual precocity of female M.nipponense,but the exact molecular mechanism needs further study.In summary,this study identified the DEGs of ovaries between sexually precocious and normal sexually mature M.nipponense,and screened out the genes that could be related to sexual precocity of M.nipponense.Cloning,sequence analysis and mRNA expression analysis of five genes that could be related to sexual precocity of M.nipponense were carried out.Finally,the role of these five genes in ovarian development of M.nipponense was investigated.The results of this study will provide a basis for elucidating the molecular mechanisms of sexual precocity of M.nipponense. |
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