| As an important cultured freshwater prawn,the oriental river prawn(Macrobrachium nipponense)has commercial advantages such as fast growth,good taste,rich nutrition,strong fecundity,and wide adaptability.The growth rate of male M.nipponense is obviously faster than that of female prawns.During the harvest period,the average size of male prawn is 2-2.5 times that of female prawns,so the homozygous culture of M.nipponense has a broad prospect.Therefore,it is of great significance in both theoretical research and production practice to carry out the functional study of genes related to male differentiation and comprehensively analyze the mechanism of male differentiation and development of M.nipponense.It can provide theoretical guidance for the establishment of homozygous breeding technology of M.nipponense.The androgenic gland is a unique endocrine organ of crustaceans,which plays an important role in the regulation of male sex differentiation,the maintenance of male secondary sexual characteristics and reproductive physiology.In this study,using M.nipponense as the research object,the reference genes of M.nipponense were screened and identified systematically.A suitable method and system for reference identification was constructed,through screening of internal reference genes of M.nipponense in different tissues and different experimental conditions.The purpose of this study is to provide a more accurate theoretical basis for the subsequent use of fluorescence quantitative technique to verify the distribution of gene expression and study the mechanism of sex determination.The key genes for male differentiation and development of recombinant M.nipponense were screened,through the comparative analysis of transcriptome and metabolome of the male glands during the breeding and non-reproductive periods.The important differential genes obtained were subjected to gene cloning and spatiotemporal expression pattern analysis,and combined tissue localization,RNA interference and other technical methods are used to study its function in the male differentiation and development mechanism of M.nipponense.1 Comparative analysis of androgenic gland transcriptome and metabolome during reproduction and non-reproductionAfter histological observation of androgenic gland in reproductive stage and nonreproductive stage,it was found that the process of differentiation and development of androgenic gland in reproductive stage was more exuberant than that in nonreproductive stage,and the cells in non-reproductive stage were smaller as a whole.In non-reproductive stage the boundary is clear and the cytoplasm is basophilic,and there are no nucleoli in the nucleus.While in the reproductive stage,the cell membrane is blurred,the cells are closely arranged,many nucleoli can be seen in the nucleus,and the cytoplasm is eosinophilic.The results showed that there was a significant difference in histological morphology between the male-promoting gland in the reproductive stage and the non-reproductive stage,and androgenic gland in the reproductive stage played a more active role in promoting the reproduction and testicular development of male M.nipponense.The results showed that 36,717 non-overlapping transcripts were obtained during the reproductive and non-reproductive androgenic gland transcriptomes;among them,7,958 differentially expressed genes were selected and enriched into 258 KEGG pathways.The most significant pathways are proteasome,eukaryotic ribosomal biogenesis,spliceosome,myocardial contraction,and adrenaline signaling in cardiomyocytes.Metabolome analysis obtained 260 differential metabolites,including147 up-regulated metabolites and 113 down-regulated metabolites.Differential metabolites are mainly enriched in cholesterol metabolism,bio-unsaturated fatty acids,synthesis and metabolism of linoleic acid.2 Screening of reference genes in different tissues and different stress conditions of M.nipponenseWith the development of quantitative technology,real-time fluorescence quantitative PCR(q PCR)has been widely used in the study of gene function.However,unidentified and verified internal reference genes may affect the results of gene quantitative expression.In this study,through searching the common internal reference genes of each model organism and the identified internal reference genes of crustaceans in each transcriptome library of M.nipponense,seven internal reference genes that may be suitable for M.nipponense were screened,including EIF(eukaryotic translation initiation factor 5A eukaryotic translation initiation factor 5A,18S(18S ribosomal RNA18 S ribosomal RNA),EF-1 α(elongation factor-1 α extension factor-1 α).GAPDH(glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase),TUB(α-TUBulin α-TUBulin),β-act(β-act)and RPL18(Ribosomal protein L18 ribosomal protein L18).Expression stability of seven candidate internal reference genes were analyzed by q PCR in different adult tissues(Gill,muscle,eye stalk,hepatopancreas and heart,testis and ovary),five stages of ovary,seven embryonic development stages,five time points of hypoxia stress such as 0,3,6,12 and 24 hours,six time points in white spot syndrome virus(WSSV)infection experiment,and RNA interference test.Four commonly methods of screening internal reference gene(Ge Norm,Norm Finder,Best Keeper,comparison Δ Ct method)were used to analyze the stability of candidate internal reference genes,and Ref Finder method was used to rank the comprehensive stability.The results showed that RPL18 was the best internal reference gene in different adult tissues of M.nipponense,and EIF was the best internal reference gene in different stages of ovarian development,WSSV challenge and embryonic development.In hypoxia stress and RNA interference,β-act is the most stable internal reference gene.Combined with the results of all stability analysis,EIF has the best comprehensive ranking in each group of experiments,and can be widely used in q PCR analysis of M.nipponense.3.Screening of male related genes of M.nipponense based on transcriptomeIn the androgenic gland transcriptome and metabolic groups during the reproductive and non-reproductive stages,16 extremely differential genes were screened.They are proteasome alpha 3,proteasome subunit beta type-5,casein kinase II subunit alpha,Nucleolar protein 56,5 years transcription elongation regulator 1-like isoform 3 ’exoribonuclease 1,RNA exonuclease 1 homolog,DEAD(Asp-Glu-Ala-Asp)box polypeptide 39,U2 sn RNP-associated SURP motif-containing protein-like,transcription elongation regulator 1-like isoform X2,fast-type skeletal muscle actin 7,sodium-potassium ATPase beta,Na+/K+ ATPase alpha subunit isoform 1,protein kinase C,guanine nucleotide-binding protein G(q)subunit alpha-like,phospholipase C,adenylate cyclase type 9 isoform X1).Their tissue distribution in adult prawn was analyzed by q PCR.The results showed that 6 genes had relatively high expression in testes and androgenic glands and there were differences between males and females,which might be candidate genes involved in the mechanism of male differentiation and development of prawns.The previous study showed that REV3 and GEM were important differentially expressed gene between androgenic gland reproductive stage and non-reproductive stage protein group,and SOXE1 was an important sex gene in the homologous study of male-promoting gland library.In the comparison between androgenic gland and other different tissue transcriptome libraries,it was found that SST,STI gene was differentially expressed gene with highly expression.Therefore,these five genes were selected as important male-related genes for further research and analysis.4 Cloning,spatiotemporal expression analysis and functional study of the gonad-related genes REV3,GEM and SOXE1REV3,GEM and SOXE1 were all obtained from androgenic gland and may be important transforming genes involved in the sex differentiation and development of M.nipponense.In this study,the cloning and sequence analysis of these three genes were carried out,and their functions were analyzed by combining the techniques of spatiotemporal expression,in situ hybridization and RNA interference,in order to clarify their functions in the regulation of sex,especially male reproductive differentiation of prawn.The full-length c DNA sequence of REV3 was cloned by RACE technique.The c DNA sequence of Mn REV3 is 6,832 tandem pairs(bp),the open reading frame is 6,102 bp,and it encodes 2,033 amino acids.Mn REV3 shows the highest sequence similarity with P.vannamei.q PCR results of different tissues and developmental stages showed that the highest expression of Mn REV3 in adult tissues appeared in hepatopancreas,followed by androgenic glands and testis,which proved that it is in the immune system of M.nipponense and the male sex differentiation of M.nipponense.In the embryonic development stage,the highest expression level was observed at 15 days hatching,which indicated that it had positive effects on the metamorphosis of M.nipponense.The expression pattern after metamorphosis may be involved in activation and promotion of gonadal differentiation and gonadal development.In situ hybridization was used to study m RNA localization of three genes in the gonadal system.Strong signal Mn REV3 was detected in sperm,spermatocyte and sperm,indicating that REV3 is involved in the whole testis.Strong signal of Mn REV3 was observed in oocytes of stage I,III and V of the ovary,indicating that it may be related to the ovary development is related to yolk deposition.Strong signal of Mn REV3 was observed in the cable structure around androgenic gland cells.The gene function of Mn REV3 was studied by RNA interference technology.q PCR analysis showed that Mn REV3 expression reached its lowest level on the seventh day of the 14-day interference,and was reduced by 75% relative to the control.The expression of IAG,an important androtropic gene,was positively correlated with Mn REV3,and also decreased by 65% on the seventh day.The determination of testis hormone content also proved that with the decrease of Mn REV3 expression,the testis hormone content also decreased,and decreased by 60% on the seventh day which is the lowest point.These results prove that REV3 has a positive effect on the sex differentiation and development of M.nipponense by regulating the expression of IAG and the secretion of testis hormone.The length of the Mn GEM c DNA sequence is 1,018 binary pairs(bp)with the open reading frame is 777 bp in length,and encodes 258 amino acids.A SIP1 superfamily was detected in the protein sequence.Mn GEM has the highest sequence similarity with P.vannamei.In adult tissues,the m RNA expression of GEM is highest in androgenic gland,followed by testis and ovary,indicating that GEM plays an important role in the development of gonads in M.nipponense.In the In situ hybridization,it was found that strong signal of Mn GEM were detected in sperm cells,which indicated that GEM promotion was also observed in the testis.Mn GEM also had strong signals in oocytes of stage II and V,indicating that it may be related to Ovarian development is related to yolk deposition.Mn GEM signals were also detected in the cable structure around the androgenic glandic cells,indicating that GEM may play a role in stabilizing the cable structure,promoting and supporting the formation of androgenic glandic cells.In the Mn GEM interference experiment,the expression of Mn GEM decreased by 79% on the seventh day,while the expression pattern of Mn IAG in the RNAi group was opposite to that of Mn GEM,and rose to twice as much as it in the control group.Similarly,the determination of testis hormone content reached the same level on the seventh day.These results prove that Mn GEM inhibits the male sex differentiation and development of M.nipponense by inhibiting the expression of IAG and the secretion of testis hormone.The length of Mn SOXE1 was 1,748 bp,and the open reading frame was 1,533 bp,encoding 510 amino acids contained a HMG domain,showing the highest sequence similarity with P.vannamei.The expression results of Mn SOXE1 gene in different tissues of adult prawns showed that the expression level of Mn SOXE1 gene in testis was twice that of ovary.During embryonic development,the expression of Mn SOXE1 was significantly higher in the gastrul stage,suggesting that Mn SOXE1 may be involved in the production of mesoderm in early embryonic cell differentiation,which is related to early gonadogenesis.The expression of PL10 was the highest during the metamorphosis of juvenile prawn,which proved that Mn SOXE1 was actively involved in early gonadal differentiation of M.nipponense.On the 25 th day after metamorphosis,the production of mesoderm during early embryonic cell differentiation of Mn SOXE1 in male prawn was related to early gonadal formation.However,in situ hybridization,strong signal of Mn SOXE1 gene was observed in testis supporting cells and spermatocytes,suggesting that Mn SOXE1 gene may actively participate in the differentiation of testis supporting cells,to participate in maintaining testis development and promoting sperm formation.Due to the low expression of Mn SOXE1 in adult gonads,no interference experiments were conducted on it.The other results showed that Mn SOXE1 was more involved in the early gonadal formation and the promotion of early male differentiation in M.nipponense.5 Study on functions of androgenic gland-specific protein SST and STIIn the laboratory’s previous research on androgenic glandic glands,it was found that slow tension S2 tropomyosin(SST)and slow tropomyosin subtypes(STI)are two important specific proteins of androgenic gland.Their both have specific high expression.In order to study the functions of the two genes in the M.nipponense further,RNA interference technology was used.However,within 12 hours,half of the experimental M.nipponenses had acute deaths.Therefore,this experiment combines the observation of HE stained sections and comparative analysis of transcriptome to explore the potential mechanism of acute death after injection of tropomyosin-related gene ds RNA in prawns.The section results of HE staining showed that the gill filaments became thinner,and the number was reduced,accompanied by obvious deformation and necrosis of the secondary gill pieces.There is no obvious pathological change in other conventional tissues.The results indicate that changes in gills and heart may be the direct cause of interference and death.Transcriptome analysis was performed on the experimental dying group after RNA interference,the experimental survival group,and the control group which targeted tissues is gills and heart.A total of 175,694 unigenes were obtained,of which 65,537 genes can be annotated in the NR database.The screening results showed that in the gills,the dying group and overlapping differentially expressed genes are the most,and the number of up-regulated and down-regulated differential genes are 3,844 and 7,184respectively;in the heart,there are 6,007 in the dying group and the surviving group.Up-regulated genes and 7,951 down-regulated genes are the comparison group with the most differential genes.KEGG analysis is used to study the enrichment pathways of differentially expressed genes.This experiment combines the p value and the number of co-DEG to select the main enrichment metabolic pathways.The results show that in the gills,the main metabolic pathways are lysosomes,phagosomes and peroxisomes;in the heart,glycolysis/gluconeogenesis and oxidative phosphorylation are considered to be the key and abundant metabolic pathways.The results showed that different genes have more functional roles,and energy metabolism is related to oxidative stress.In addition,screening and analysis of high abundance expression of 10 differentially expressed genes,suggesting that they may be closely related to the tolerance and stress resistance of M.nipponense.Subsequent studies on them in hypoxia showed that fructose 1,6-bisphosphate aldehyde acetal esterase(FBA),glyceraldehyde 3-phosphate dehydrogenase(GDPDH),alcohol dehydrogenase-3(ADC3),ATP synthase subunit 9(ATPS9)and acid ceramidase-like(ACL)five genes are closely related to hypoxia,which can provide basic data for subsequent research on the stress tolerance and hypoxia tolerance of M.nipponense.In this experiment,Tissue sections were used to study the structural differences of androgenic gland tissue morphology during the reproductive and non-reproductive periods,and construct a transcriptome library of androgenic gland during the reproductive and non-reproductive stages.In the androgenic gland library,systematically screened the differentially expressed genes,especially nine genes that were highly expressed in androgenic glands and testis were identified,which may be new male-related genes in prawns.We screened and determined the internal reference genes applicable to fluorescent quantitative PCR in the routine experiments of M.nipponense.The androgenic gland-related genes Mn REV3,Mn GEM and Mn SOXE1 were cloned,sequenced,and temporally and spatially expressed and functionally tested,and their functions in the process of male sex differentiation and development of M.nipponense were determined.Through the transcriptome study and analysis of the lethality after STI and SST gene interference,the target tissues that interfered and the cause of lethality were determined,and the differentially expressed genes related to the stress resistance and tolerance of the M.nipponense were identified and screened.Five genes related to hypoxic stress.The results of this study are helpful to improve the accuracy and scientificity of fluorescence quantification in M.nipponense,and provide basic scientific data for the study of sex determination,sex differentiation,especially male differentiation and development of M.nipponense.It can also be used to provide reference for related research in other crustaceans. |
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