| The occurrence of germ cells is one of the important contents of reproductive biology research,and the formation of gametes in the gonads is an extremely important period in individual development.Compared with other organisms,the research on the molecular mechanism of reproduction in decapods is relatively weak,and the presentation of genetic resources of freshwater shrimps is particularly lacking.Therefore,it is of great significance and potential application value to excavate and obtain a large amount of genetic resources related to the reproduction of freshwater shrimps.In this paper,based on high-throughput sequencing,transcriptome analysis of the ovarian tissue of siberian prawn,Exopalaemon Modestus was carried out,and 71 genes related to reproduction and development were identified.Then,the gonad development of E.Modestus was used as the main line to obtain related gene.Three genes related to the occurrence of germ cells and gamete formation in E.Modestus,and their sequence characteristics and expression patterns were analyzed.The experimental results are as follows:1.Analysis of ovary transcriptome,identification of reproductive related genes and preliminary identification of molecular markers in E.Modestus.In order to study the genetic information carried or stored during the ovary development of E.Modestus,108,789,834 reads were obtained through library construction and high-throughput sequencing using ovaries as experimental materials,and 91,921,438 effective reads were obtained after screening,34,099 contigs were assembled using Trinity,with an average length of 1198 bp.Each contig was compared with the sequence published on GenBank,and 11965 annotation genes were obtained.Using blast2go software to annotate go,71 genes related to reproduction and development,such as cathepsin B and 1,dead box family,cyclin,cyclin dependent kinase,nanos,piwi,Kazal like serine protein,ribosomal protein L24,were found.Through online KEGG analysis of the annotated contrags,1306 genes were found to be involved in the metabolic pathway of KEGG database.1956 microsatellite sequences were obtained by scanning contrags with mreps software,and 85590 SNPs were obtained by SNP analysis of each contrags with varscan software,including 47150 conversions(A/G,C/T)and 38440 transversions.These results provide useful basic data for functional genomic research and germplasm resources research of the E.Modestu and even the whole shrimp and crab.2.Molecular cloning,characteristic analysis,spatiotemporal expression and evolution analysis of vasa gene in E.ModestuRT-PCR and rapid amplification of cDNA ends(race)were used to clone the full cDNA sequence of vasa gene of E.Modestu.The protein structure and function of vasa gene were analyzed,and its tissue expression characteristics and genetic evolution were studied.The results show that the full-length cDNA of the vasa gene of E.Modestus(Em-vasa)is 2509 bp,consisting of a 69 bp 5 ’untranslated region(UTR),a 631 bp 3’untranslated region,and a 1809 bp open reading frame frame(ORF),encoding 602 amino acids.Vasa protein contains 9 conserved domains and GG repeats of dead box family protein,and has zinc finger structure and glycine(g)and arginine(R)enriched regions at N-terminal.Subcellular localization predicted that vasa protein was located in cytoplasm.The homology of vasa protein of E.Modestus is 72%,52%and 50%,respectively,with that of Macrobrachium nipponense,Penaeus chinensis and Eriocheir sinensis.In the process of evolution,purification is the main choice.The phylogenetic analysis showed that the molecular evolutionary status of E.Modestus based on vasa gene was completely consistent with the biological classification status.The results of semi quantitative RT-PCR showed that Em-vasa was only expressed in the gonads of E.Modestus.Therefore,Em-vasa is expected to be a molecular marker for the study of the origin and differentiation of its germline.The results of real-time fluorescent quantitative PCR showed that the expression of vasa gene transcripts in immature ovary was lower than that in mature ovary,and significantly higher in immature testis than that in mature testis;the expression of vasa gene transcripts increased first and then decreased in embryonic development,and reached the highest level in blastocyst stage.3.Molecular Cloning,Characteristic Analysis and Expression Patterns of the Subunit Genes of Maturation Promoting Factor(MPF)in E.ModestusCyclin can combine with cyclin-dependent protein kinase(CDK)molecules to form a protein kinase complex called MPF,which dominates the process of animal mitosis and meiosis.In this experiment,we obtained the full-length cDNA sequence of cyclinA gene and the full-length cDNA sequence of cdc2 gene by RT-PCR and RACE methods,and studied the expression pattern of cdc2 in different tissues,gonads and embryos at different development stagesThe full-length cDNA sequence of cyclinA gene was obtained,which was 2150 bp long,1356 bp open reading frame,451 amino acids,128 bp 5 ’ untranslated region and 626 bp 3’ untranslated region.Through bioinformatics analysis of this sequence,it was found that the predicted encoded amino acid sequence is highly conserved and contains a cyclinA-specific destruction box and a cyclin recognition box.The results of multiple sequence alignment and homology analysis showed that the homology of cyclinA protein in E.Modestus is 73%,72%and 63%,respectively,with that of Penaeus vannamei,Penaeus monodon,and Eriocheir sinensis.Analysis of the three-dimensional structure of the predicted protein shows that the central structure region of Em-cyclinA protein is composed of two interconnected spherical structures,which mainly include a-helix and random coil.Real-time fluorescence quantitative PCR method was used to determine the expression of cyclinA gene in 6 different tissues,immature and mature gonads,and embryos at different developmental stages.The results showed that the expression of cyclinA gene was highest in the ovary,and the expression in the testis was relatively high,but in the blood,gills,Heart,muscle and other tissues,the expression level is very low.The expression in immature ovary is significantly higher than that in mature ovary,and there is almost no difference in the expression of cyclinA mRNA in immature testis and mature testis;In the process of embryonic development,the highest expression was found in the Gastrula,and then dropping to a lower level in the nauplius,protozoea and zoaea stage.The total length of the obtained cdc2 gene cDNA of E.Modestus is 1589 bp,the open reading frame was 701 bp,encoding 299 amino acids,the 5’non-coding region was 298 bp,and the 3’ non-coding region was 689 bp.The predicted amino acid sequence have typical sequence characteristics of cdc2,including the serine/threonine protein kinase active site vlhrdlkpqnlli,the basic characteristic sequence of cyclin family EGVPSTAFREISLKE,protein kinase ATP binding region and three phosphorylated or dephosphorylated amino acid residues.Multi-sequence alignment,three-dimensional structure prediction and phylogenetic tree analysis were carried out.Multi-sequence alignment and homology analysis showed that the homology of CDC2 protein in E.Modestus is 99.67%、97.66%、93.65%,respectively,with that of Palaemom carinicauda,Macrobrachium nipponense,and Penaeus vannamei.Real-time fluorescence quantitative PCR method was used to determine the expression of cdc2 gene in 6 different tissues,immature and mature gonads,and embryos at different developmental stages.The results show that,the expression in the testis was highest,the expression in the ovary was relatively high,and the expression in the blood was relatively low,it is hardly expressed in gills,heart and muscles.The expression in mature testis is about 1/2 of immature testis.The expression of cdc2 mRNA in immature ovary is slightly higher than that in mature ovary.During embryonic development,the expression of cdc2 gradually increased first,then gradually decreased,and was up-regulated during the gastrointestinal period to the highest value.The amino acid sequences of Em-cyclinA and Em-CDC2 proteins are highly conserved,which ensures their structural function in cell division.The higher expression level of them in immature gonads indicates that they play an important role in gonadal development.In embryonic development,the gastrula is an important stage in the formation of germ layer.At this stage,the highest expression level of Em-cyclinA and Em-cdc2 suggests that they play a key role in the embryonic development of E.Modestus. |
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