| Paralichthys olivaceus,belongs to Paralichthys,Pleuronectiforms,is an important marine economic fish in China.Female individuals grow faster than male individuals,and therefore female breeding period is short,and its economic value is higher.Sex differentiation and gonadal development have always been the important research direction of fish development and reproductive biology.CBX2 is one of the key members of polycomb group complex(PCG).It plays a key role in mammalian sexual development as an important transcription factor.If the gene mutation or loss of function will lead to sexual reversal,however,the function of cbx2 in sex differentiation and gonadal development in vertebrates except mammals is still unclear.So the cbx2 gene was identified in Paralichthys olivaceus,and its expression profile and its relationship with miR-200 family and sex-related genes were analyzed by various molecular biological techniques.It lays a foundation for revealing the molecular mechanism of miRNA involved in gonadal development of Paralichthys olivaceus by regulating the expression of cbx2 gene.In this study,the cDNA sequence of cbx2 gene was cloned by molecular cloning technique.The obtained cbx2 cDNA sequence of Paralichthys olivaceus contains an open reading frame of 1 584 bp,encoding 527 amino acids.The physicochemical properties and spatial structure of CBX2 protein were analyzed online by bioinformatics methods,the results showed that the molecular weight and isoelectric point of the reduced protein is 56578.98 and 10.03,respectively.The CBX2 protein was an alkaline and hydrophilic protein,which had no signal peptide and was not transmembrane protein,and the amino acid encoding the protein had the highest content of serine(Sr).The spatial structure analysis found that the CBX2 protein is mainly composed of ?-helix and random coil,and its secondary structure contains one chromatin domain and four low complexity regions,which chromatin domain is often binding to methylated amino acids.The gene structure,amino acid homology and phylogeny of cbx2 in vertebrates were compared.Theses indicated that the cbx2 gene is more conserved in the evolution of vertebrate.The gene structure of cbx2 was more similar to that of Oreochromis niloticus and Oryzias latipes,and its amino acid homology was the highest with Seriola dumerili,which is 89%.The expression of cbx2 gene in different tissues of Paralichthys olivaceus was analyzed by RT-PCR technique,the result showed that the expression of cbx2 mRNA was the highest in the testis,and its expression was the second one in the ovary,but in other tissues there were a lower expression.The expression of cbx2 gene in different tissues and early development stages of Paralichthys olivaceus was detected by real-time PCR.The results showed that the expression of cbx2 gene was the highest in the testis of Paralichthys olivaceus,followed by ovary.But the expression the other tissues was lower,which was similar to the result of RT-PCR,There was a certain maternal expression of cbx2 gene in the unfertilized eggs and during early embryo development of Paralichthys olivaceus,its expression was the highest in the nerve embryo,however,the cbx2 gene remained a low expression level after hatching;The localization of CBX2 protein in testes and ovary were detected by immunohistochemistry.The results showed that cbx2 was expressed in the germ cells of II,III and IV stages.Among them,there was strongly immunopositive in the spermatogonial cells of testis at III stage.However,the expression of cbx2 protein could hardly be detected in spermatozoa of V stages.At the same time,the expression of cbx2 protein in II stage to V stage ovaries was detected.It was found that CBX2 protein was mainly expressed in oocytes of II and III stage ovaries,but in the same period,the expression of CBX2 protein in the testis was higher than that in the ovary.This result is similar to the quantitative result.These results suggested that cbx2 gene may be involved in the proliferation and meiotic division of spermatocyte,indicating that cbx2 gene plays an important role in the male reproduction of Paralichthys olivaceus.In order to investigate the relationship between cbx2 and sex-related genes,the expression of cbx2 and four known sex-related genes(sf1,sox9,foxl2 and wnt4)in the gonads of Paralichthys olivaceus was detected by real-time fluorescence quantitative PCR.The results showed that cbx2,sf1 and sox9 in the testis was significantly higher than that in the ovariy,while the expression of foxl2 and wnt4 in the ovariy was higher than that in the testis.In order to further explore the regulation of the expression of sex-related genes by cbx2 gene,the siRNA interference technique was used to interfere the cbx2 gene in embryonic cells of Paralichthys olivaceus,and the optimal interference sequence and time of siRNA were screened,the relative expression of cbx2,sf1,sox9,foxl2 and wnt4 was detected.The results of RNAi showed that cbx2 and Testicular development related genes(sf1and sox9)are inhibited,and the genes(foxl2 and wnt4)expressed in ovary were promotedFinally,in order to confirm the relationship between cbx2 gene and miRNA,the targeting relationship between miR-200 family and cbx2 was studied by double luciferase reporter gene technique.Double luciferase reporter gene experiment demonstrated that the cbx2 is a target gene of miR-200 family,which is directly regulated by pol-miR-200 a and pol-miR-200b;however,pol-miR-429 and pol-miR-141 have no obvious regulated role to the expression of cbx2 gene in Paralichthys olivaceus.These results suggest that pol-mir-200 a and pol-mir-200 b may be involved in the gonadal development of Paralichthys olivaceus by directly regulating the expression of cbx2 gene.In conclusion,the results from this study lay an important foundation for further study on the role of miRNA-cbx2-sex-related genes in gonadal development of Paralichthys olivaceus. |
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