| Paralichthys olivaceus(P.olivaceus),belongs to Paralichthys,Pleuronectiforms,is an important marine economic fish in China.During growth and development of P.olivaceus,there are obvious differences between male and female,and the female individuals are large and growing rapidly.Therefore,the study on gonadal differentiation and development of P.olivaceus is beneficial to parthenogenetic culture,which has an important prospect of economic application.In recent years,cloning genes related to gonadal development of P.olivaceus have gradually become the focus of aquatic genetics and biology,but the molecular mechanism of gonadal differentiation in P.olivaceus is still not clear.The study of differential protein expression between female gonad and male gonad of P.olivaceus is even scarce.Therefore,the study on differential proteomics of female gonad and male gonad in P.olivaceus will have important theoretical significance in elucidating its molecular mechanism.iTRAQ(isobarictags for relativeand absolute quantitation)is a new biological technique based on the proteomics.It can be used to relative and absolute quantitative analysis for eight different samples simultaneously.This technique could be used to qualitative and quantitative analysis synchronously,and has the characteristics of high sensitivity,high throughput,strong separation ability and reliable results.In recent years,it has been applied in many disciplines and domains of science.Quantitative and qualitative analysis of proteome in male and female gonads of P.olivaceus was carried out by using iTRAQ technique.The TripleTOF 5600 mass spectrometer system was used for mass spectrometer analysis.The obtained number of secondary spectra and analytic secondary spectra was 361050,51910 respectively.The number of containing proteins with two unique peptides at least was 1444,accounting for 51.36% of the total protein.The identified most peptide fragment length was 11,and average length of all peptide fragments was 13.28,which fits the reasonable range of peptide length.Under the condition that the difference multiple is greater than or equal to 1.5 or 0.67 times,a total of 191 significant differentially expressed proteins was detected,of which 118 were up-regulated and 73 were down-regulated.The differential proteins were enriched by GO and the following Gene Ontology terms were found in the biological process: female sex differentiation,embryonic morphogenesis,positive regulation of cell morphogenesis involved in differentiation,regulation of ERK1 and ERK2 cascade,regulation of steroid hormone receptor signaling pathway,spermatogenesis,male gamete generationgerm,cell development and so on.The differential proteins are mainly found in the cytoplasmic membrane,sex chromatin and cytoskeleton.The Pathway enrichment analysis of differential proteins showed that some proteins may be associated with gonadal development in these pathways: Steroid biosynthesis,MAPK signaling pathway,Wnt signaling pathway,Progesterone-mediated oocyte maturation.The following proteins have preliminarily been screened out: heat shock protein family A member 8(HSPA8),?-catenin,cytochrome P450 11beta(CYP11B),17-beta hydroxysteroid dehydrogenase type 1(17?-HSD1),calretinin(CALB2),fragile X mental retardation 1(FMR1)and WD40 repeat protein 1(WDR1).CALB2 protein was down-regulated in Phosphatidylinositol signaling system and GnRH signaling pathway;FMR1 protein was up-regulated in RNA transcriptional signaling pathway;and WDR1 protein was up-regulated in p53 signaling pathway and RNA degradation pathway.The three proteins have been reported in studies of mammalian reproduction,but their functions in the development and differentiation of gonads in fish was rarely studied.Therefore,the three proteins(CALB2,FMR1,WDR1)were selected for verification and functional study.According to the NCBI database information,the primers of corresponding gene were designed.The gene sequences of the three proteins were cloned and verified,and then the biological information analysis of alignment,phylogenetic and syntenic relationship were carried out by using the softwares of DNAMAN,MAGE and so on.The obtained cDNA of calb2 was 1456 bp,including a 816 bp open reading frame(ORF),encoding a polypeptide of 271 amino acids,and its molecular weight is about 31.52 kDa.The amino acid sequence alignment analysis displayed that the calb2 from P.olivaceus shows the highest 97% homology with that of Oreochromis niloticus.The predicted secondary structures showed that it has six EFh hand structures.The obtained cDNA of fmr1 was 2826 bp,including a 1833 bp ORF,encoding a polypeptide of 610 amino acids,and its molecular weight is about 66.27 kDa.The amino acid sequence alignment analysis displayed that the fmr1 from P.olivaceus shows the highest 94% homology with that of Lates calcarifer,and the predicted secondary structures show that it has two KH domains,and when the mutation of KH domain occurs,FMRP loses the ability to bind RNA.The obtained cDNA of wdr1 was 2988 bp,including a 1821 bp open ORF,encoding a polypeptide of 606 amino acids,and its molecular weight is about 65.95 kDa.The amino acid sequence alignment analysis displayed that the wdr1 from P.olivaceus shows the highest 87% homology with that of Seriola dumerili,and the secondary predicted structuresshowed that it has 11 WD domains.Real-time quantitative PCR revealed that the calb2,fmr1,wdr1 mRNA were expressed in all detected tissues of P.olivaceus.The calb2 gene had the highest expression in the brain,followed by the gonads,moreover,there was a significant difference in expression between the two tissues(P<0.01),and the expression level of calb2 in the testis was about three times that in the ovary.The fmr1 gene was highly expressed in the ovary compared to other tissues,and the expression is about 9 times higher than that in the testis.The result of wdr1 is similar to that of fmr1,and the highest level of wdr1 was found in the ovary and relatively high level was in the testis.Western blot analysis for CALB2,FMR1 and WDR1 proteins revealed that specific strong signals are found in the position near the 31 kDa,71 kDa and 66 kDa,and the expression of CALB2 in testis was higher than that in ovary,however,the expression of FMR1 and WDR1 in testis were lower than that in ovary.Immunohistochemistry showed that the CALB2 is expressed in ovarian germ epithelium in ovary and the mesenchymal cells in testis.Temperature and hormones are important environmental factors that affect the gonadal differentiation of fish.To explore the role of calb2,fmr1 and wdr1 during gonad differentiation of P.olivaceus,We investigated the effects of temperature and hormone on the there genes in the early gonad differentiation of P.olivaceus.The larvae during gonad differentiation(30~105 days post hatching,dph)were treated under high temperature(28?),estrogen(10 ?g/L),androgen(10 ?g/L)and normal temperature(20?).The real-time quantitative PCR results showed that high temperature could inhibit the expression of calb2 gene,and androgen could induce the calb2 gene expression in advance,but at the same time,it could reduce the expression of calb2 gene.The expression of fmr1 was inhibited by external stimulation.Estrogen and androgen significantly affected the expression of wdr1 gene and promoted the expression of wdr1 during gonadal differentiation of P.olivaceus. |
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