| The oriental river prawn,scientific name Macrobrachium nipponense,is one of the important freshwater cultured shrimp species in China.It has the characteristics of fast growth,wide adaptability,strong reproductive power,and high economic value.There is significant difference between males and females of M nipponense in the growth.The average size of males in the same harvest period is about 2-2.5 times that of females.The whole male culture will help increase the yield and economic benefits of M nipponense.The further research on the sex determination and differentiation mechanism can provide technical guidance to realize the monoculture of M.nipponense.This study intends to use M.nipponense as the research object.The key period of gonad differentiation and development was determined by histological section.We used high-throughput sequencing technology to construct a full-length and second-generation transcriptome Library of the critical period of gonad development in M nipponense.We selected for sex-related genes by homology analysis and differential expression analysis.We conduct functional analysis of these selected genes,through the temporal and spatial expression rules,the histological location in gonads and the results of RNA interference.1 The libraries construction in the key stages of gonad differentiation and screening for the sex-related genes in M.nipponenseWe studied the initiation time and developmental process of gonadal differentiation in M nipponense by histological sectioning.The results of histological sectioning showed that there are no germ gland anlag in Post-larvae(PL-5).Germ gland anlag was present in PL-10.Spermatogonia and oogonium were present in PL-15.In PL-25,ovaries had reached OⅡ stage,mature sperm had appeared in the testis.The results indicated that PL5-PL25 was a critical period for gonadal differentiation and development.A full-length transcriptome and a second-generation transcriptome library for the gonad development process of M.nipponense larvae were constructed using the PacBio and Illumina NovaSeq 6000 platforms.A total of 49,840 non-redundant transcripts were obtained.The average nucleic acid sequence is 3,057bp in length.A total of 14,726 alternative splices were identified.A total of 14,339 SSRs were obtained.A total of 31,180 ORF(Open reading frame)were obtained.2,903 transcription factors are predicted.A total of 7,802 LncRNAs were predicted.There were 37,355 non-redundant transcript sequences in the M.nipponense gonad differentiation critical period,which were functionally annotated in the Nr database.However,12,485 non-redundant transcripts did not receive functional annotations.There were 19,673,13,395,18,618 non-redundant transcripts in the GO,COG,and KEGG databases.Based on the above results,more comprehensive and effective differential transcripts were obtained,and the expression characteristics of transcripts in M.at different developmental stages were preliminarily summarized.Through the phase by phase comparison between PL-5 and PL-25,we screened 1028 differentially expressed genes in the critical period of gonad differentiation.During the development of PL-5 to PL-10,there were 340 up-regulated transcripts and 142 down-regulated transcripts.The function of differential transcripts was related to organ growth and energy metabolism.During the development of PL-10 to PL-15,20 transcripts were up-regulated and 78 transcripts were down-regulated.The function of differential transcripts was related to energy metabolism,organogenesis,hormone synthesis and reproductive behavior.During the development of PL-15 to PL-25F,187 transcripts were up-regulated and 53 transcripts were down-regulated.The function of differential transcripts was related to environmental adaptation and immune activity.During the development of PL-15 to PL-25M,126 transcripts were up-regulated and 82 transcripts were down-regulated.The function of differential transcripts was related to environmental adaptation,immune activity and fat synthesis and metabolism.2 Expression analysis of key genes(JHEH,ALY,DHP and SMA6)in M.nipponense for gonad differentiationAmong the differentially expressed genes in the transcriptome library during the critical period of gonadal differentiation,we screened 1028 sex-related genes.Four candidate genes involved in gonadal differentiation and development were selected through functional annotation of the differential genes and cluster analysis of expression patterns.The full-length fragments of DHP,ALY,and SMA6 genes were screened from the transcriptome library at the critical stage of gonad differentiation.The expression patterns of JHEH,DHP,ALY and SMA6 genes were studied,in order to analyze whether they are sex-relatedgenes in M.nipponense.The results of qRT-PCR analysis in different adult tissues showed that MnJHEH was highly expressed in hepatopancreas and gonad in both male and female(P<0.05).In the same tissue,the expression level in male was higher than that in female(P<0.05).During different developmental stages of the ovary,the expression of MnJHEH gene was lower in OⅠ,OⅢ stages,and higher in OⅡ,OⅣ,OⅤ stages(P<0.05).The results indicated that MnJHEH was not directly related to ovarian development.Quantitative analysis results at different developmental stages showed that the MnJHEH gene was expressed the highest level at cleavage stage during embryonic development,and gradually decreased with cell differentiation(P<0.05).The expression of MnJHEH gene was low during the development of juvenile.With the metamorphosis of juveniles to larvae,the expression of MnJHEH gene in PL 10 to PL15,which was the key to gonad differentiation of larvae,began to increase sharply.Later,as the development of larvae,there were gender differences in PL25(P<0.05).The above results indicate that the MnJHEH can promote the differentiation and development of the gonadal gonads.The full-length cDNA of the MnDHP gene is 2,557bp and encodes 566 amino acids.Phylogenetic tree results showed that the MnDHP gene was clustered with Procambarus clarki DHP.The results of qRT-PCR analysis in different adult tissues showed that the expression of MnDHP had obvious gender differences.The expression levels in brain and eyestalk in male were significantly higher than those in female(P<0.05).The expression in male shrimp hepatopancreas was also higher than that in female shrimp.In the gonads,there was not much difference between male and female(P<0.05).In different ovarian development stages,the expression of MnDHP was the lowest in the OI stage,and reached the highest in the OIV stage with the ovarian development(P<0.05).The results showed that MnDHP can promote ovarian maturation.MnDHP was expressed in different developmental stages of M.nipponense.The expression of MnDHP was the lowest at the cleavage stage(P<0.05).As the cell differentiation expression gradually increases,the expression level of flea larvae reaches the highest(P<0.05).MnDHP gene had the lowest expression at L5 during the development of juvenile,and then it began to increase(P<0.05).MnDHP had the highest expression at PL1 during the development of larvae,and then it gradually decreased with the development of larvae(P<0.05).The above results indicate that the MnDHP gene may be involved in the differentiation and development of the gonadal gonads,but its results at different developmental stages of the ovary indicate that the MnDHP gene can promote the maturation of the ovary.The full-length cDNA of the MnALY is 1,385bp,encoding a total of 397 amino acids.Phylogenetic tree results showed that the MnALY gene was clustered with ALY/REF of Galendromus occidentalis.The results of qRT-PCR showed MnALY had the highest expression in the gonads in both males and females(P<0.05).During different ovarian development stages,MnALY expression was lower in OⅢ and OⅣstages,and higher in the other three stages(P<0.05).The MnALY expressed in different developmental stages of M.nipponense.The MnALY gene had the lowest expression at the blastocyst stage,L1,and PL 10,and then the expression level began to increase(P<0.05).There was a gender difference in the expression of PL25.The above results indicate that the MnALY gene can promote the differentiation and development of gonads in females.The full length cDNA of MnSMA6 is 1299 BP,encoding 377 amino acids.The phylogenetic tree showed that MnSMA6 gene and the SMA6 gene in Ricciaris exocalata were clustered into one branch.The results of qRT-PCR showed MnSMA6 was highly expressed in muscle and gills in both males and females,and low in gonads(P<0.05).There was significant gender difference in hepatopancreas,and the expression level in male hepatopancreas was much higher than that in female(P<0.05).During different developmental stages of the ovary,the expression of MnSMA6 was highest during the egg proliferative stage(OⅠ)(P<0.05),and the expression was lower in the other four stages(P<0.05).MnSMA6 expressed in different developmental stages of M.nipponense.The expression level of MnSMA6 gene was relatively low during embryonic development,and significantly increased during the zoea stage(P<0.05).In PL10-PL25,MnSMA6 decreased with the differentiation of the gonads.The above results indicate that the MnSMA6 gene may be involved in gonadal differentiation and development of larvae,but its results at different stages of ovarian development indicate that the MnSMA6 gene can promote the proliferation of eggs in the ovary.3 Identification,characterization and function analysis of DMRT11E gene in M.nipponenseAmong the differentially expressed genes in the transcriptome library at the critical stage of gonadal differentiation,dmrtlle gene,which is highly homologous with vertebrate sex determining gene,was selected.We determined the function in M.nipponense by cloning,analyzing the spatiotemporal expression,and positioning histology.The full-length cDNA sequence of MnDMRT11E gene was cloned by using RACE technology.The full-length cDNA of MnDMRT11E gene was 2,585bp,which encoded and synthesized a 542aa protein.The results of sequence analysis,amino acid comparison and phylogenetic tree analysis showed that MnDMRT11E gene was highly similar to DMRT11E of Macrobrachium rosenbergii(93%),and its key DM domain was 100%.The results of qRT-PCR analysis in different adult tissues showed the expression of MnDMRT11E was the highest in the gonads(P<0.05).During the different stages of ovarian development,the MnDMRT11E expression gradually increased from OⅠ to OⅢ and decreased to the lowest level at the end of OⅣ(P<0.05).In situ hybridization analysis showed that MnDMRT11E was mainly located in the oocytes at various stages of ovarian development and spermatogonia,eosinophilic matrix in vas deferens and glandular cells in androgenic gland.It was speculated that the gonad is the main functional site of MnDMRT11E.Quantitative analysis results at different developmental stages showed that MnDMRT11E gene increased sharply during cleavage stage and PL5(P<0.05).The results showed that it could promote the division of embryonic cells and the development of primordial gonads.Silence of DMRT11E gene by RNAi,the expression of MnDMRT11E gene decreased by 86%compared with the control group(P<0.01),the expression of MnVG was down-regulated by 60%in the ovary(P<0.01),and down-regulated by 94%in the hepatopancreas(P<0.01).RNAi results showed that MnDMRT11E can positively regulate the expression of VG gene.the expression of MnIAG in male shrimp was increased by more than 2.5 times(P<0.01).VG is a precursor of yolk protein and is essential for the development and maturation of the ovary.IAG gene is a typical male gene in M nipponense.RNAi results show that MnDMRT11E can positively regulate the expression of VG gene and negatively regulate the expression of IAG gene.The above results indicate that MnDMRT11YE promotes the accumulation of yolk in the ovary and inhibits the expression of the IAG gene.MnDMRT11E is a feminization gene that can promote the development of the feminization of M.nipponense.4 Cloning and expression of transformer-2 a and doublesex gene in M.nipponense and the relationship with each otherDrosophila and M.nipponense belong to the arthropod phyla,and the relationship between them is relatively close.Among the differentially expressed genes in the transcriptome library at the critical stage of gonad differentiation,the transformator-2a(tra-2a)and doublesex(DSX)genes were selected,which were highly homologous with the major sex determining genes of Drosophila.We determined their function in M.nipponense by cloning,analyzing the spatiotemporal expression,and positioning histologyThe full-length cDNA of two different Mn TRA-2 submits and MnDSX gene were cloned by using RACE technology.The total length of Mn TRA-2A cDNA is 1,293 bp,encoding 270 amino acids.The results of sequence analysis and amino acid alignment analysis showed that the length and sequence composition of RRM and linker region of MnTRA-2A gene were relatively conservative among different species.The phylogenetic tree results showed that MnTRA-2A was clustered with the TRA-2 gene of Fenneropenaeus chinensis and Eriocheir Sinensis.The full-length cDNA sequence of MnDSX gene is 1,487 bp and encodes 252 amino acids.The results of sequence analysis and amino acid comparison showed that the MnDSX gene contained a conserved DM domain of DMRT gene family.Phylogenetic tree results showed that MnDSX and that in Daphnia were clustered into one branch,DMRT genes and DSX genes were clustered into one branch respectively.However,different DSX gene subtypes of various species were clustered into one branch.The results of qRT-PCR analysis in different adult tissues showed that the expression of MnTRA-2A reached the highest levelin the gonads(P<0.05).In situ hybridization analysis showed that MnTRA-2A was mainly located in the oocytes at various stages of ovarian development and the spermatogonia.During different developmental stages in ovary,MnTRA-2A expressed the highest level in the OV,but decreased with the maturation of the ovary and reached the lowest level in the OIV(P<0.05).All of the above results suggested that MnTRA-2A may promote gonadal development and gamete production.Quantitative analysis results at different developmental stages showed that MnTRA-2A gene expressed the highest in PL5(P<0.05).The results indicated that it may play an important role in the gonad differentiation of M.nipponense.The results of qRT-PCR analysis in different adult tissues showed the expression level of MnDSX in testis was significantly higher than that in other tissues(P<0.05).It speculated that testis was the main functional site of MnDSX.At different developmental stages in ovary,the expression of MnDSX gene is lower in OⅤ,OⅠ,OⅡstages(P<0.05).While the expression level begins to increase in OⅢ stage,and it reached the highest level in OⅣ(P<0.05).The results suggested that MnDSX may promote ovarian development.Quantitative analysis results at different developmental stages showed that MnDSX is highly expressed in the gastrula stage and PL25M(P<0.05).The results indicated that MnDSX is involved in the formation of embryonic organs and the differentiation and development of male sex.In this study,the RNAi technology was used to inhibit the expression of MnSXL,MnTRA-2 and MnDSX genes.The relationship between the three genes and MnIAG gene was studied at the transcription level.The interference results showed that dsRNA of all three genes could reduce the expression level of target genes by more than 90%.The results of mutual regulation showed that after interfering with MnSXL,MnTRA-2 decreased by 61.0%(P<0.01);MnDSX decreased by 99.7%(P<0.01).After interfering with MnTRA-2,the expression of MnSXL decreased slightly which had nosignificant difference compared with the control group(P>0.05),and MnDSX decreased by 95.1%(P<0.01).After interfering with MnDSX,MnSXL decreased by 29.0%(P<0.05),and MnTRA-2 decreased by 54.0%(P<0.01).The mutual regulation relationship among MnSXL,MnTRA-2 and MnDSX genes in green shrimp was determined.After interfering with MnSXL,MnTRA-2,and MnDSX,the expression of MnlAG gene increased by 247%(P<0.01),175%(P<0.01),and 21%(P<0.01)compared with the control group.IAG gene is a typical male gene in M.nipponense.MnSXL,MnTRA-2 and MnDSX can negatively regulate MnIA G.The results indicated that the MnSXL,MnTRA-2 and MnDSX genes may be involved in the development of female.The above results indicate that the MnSXL,MnTRA-2 and MnDSX genes are all feminization genes,which can promote the development of the female development of M.nipponense.This experiment identified the critical period of gonadal differentiation in M.nipponense.The full-length transcriptome and second-generation transcriptome Library of the gonadal development of prawn were constructed.The genes involved in sex determination and differentiation were screened systematically.Cloning,spatial and temporal expression and functional analysis of the selected genes were carried out.The functions of MnDMRT11E,MnSXL,MnTRA-2 and MnDSX genes were preliminarily determined.The results of this study provided an important reference for the study of sex determination and regulation in M.nipponense,and also provided a reference for the study of sex determination mechanisms in other shrimp and crab species. |
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