Expression Characteristics And Function Of Genes Dmrt1 And Foxl2 In The Scallop Patinopecten Yessoensis

Being important cultural scollaps in North China,Patinopecten yessoensis has attracted much attention due to the presence of a certain proportion of hermaphrodite in the cultured population.We are also interested in the molecular mechanism of gonadal development and sex differentiation in the P.yessoensis.In order to explore the expression characteristics and functions of genes for sex formation and gonadal development in the P.yessoensis,the full-length transcriptome sequencing of the female and male gonads of P.yessoensis in the growing and emission stage were performed to screen the differentially expressed genes Dmrt1 and Foxl2,and the c DNA coding region was cloned to analyze their sequence characteristics;The differences in the expression levels of Dmrt1 and Foxl2 in different tissues were detected by Semi-quantitative PCR(RT-PCR);real-time quantitative PCR(qRT-PCR)and in situ hybridization technology ISH(in situ hybridization)were used to reveal the temporal and spatial expression changes of Dmrt1 and Foxl2 in four stages of gonadal development(proliferation,growth,maturation and emission stage)Finally,In order to explore the regulatory effect of Dmrt1 on genes Foxl2 and P450,Dmrt1 was silenced via the RNA interference(RNAi)technology.This study further enriched the contents of researching on the characteristics and functions of genes related to sex differentiation and gonadal development in P.yessoensis,and laid the foundation for exploring the molecular regulation mechanism of sex formation and gonadal development in scallop.The results are as follows:(1)High quality unigenes obtained from the full-length transcriptome were annotated and compared with 8 databases.Among them,23,698 annotated unigenes were compared to the P.yessoensis species in the NR database,accounting for about99.8%.4203 genes were enriched in KEGG Pathway,which were divided into 5 major categories of biochemical pathways,namely Metabolism,Genetic Information Processing,Organic Systems,Cellular Processes and Environmental Information Processing.181 unigenes were enriched to 102 pathways,including ubiquitin mediated proteolysis,oocyte meiosis and other KEGG pathways involved in gonadal development and reproduction.The results of differential expression analysis of unigenes showed that in the mature stage,2379 genes were differentially expressed between male and female,1391 genes were up-regulated and 988 genes were down-regulated in female compared with male.Among them,Dmrt1 has four transcripts,and its transcript XM＿024198039.1 was closely related to male sex;Foxl2 is closely related to female sex.Subsequently,we randomly selected 9 differentially expressed transcripts involved in gender differentiation.Although qRT-PCR quantitative expression results showed ploidy differences with the transcriptome expression levels,the up-and down-regulation trends were similar.Overall,the qRT-PCR results indicated the reliability and accuracy of the RNA-Seq-based transcriptome expression analysis.(2)The full-length transcriptome data were compared with NCBI data,we cloned the coding region sequences of Dmrt1(Transcript number:XM＿024198039.1)and Foxl2,named PyDmrt1 and PyFoxl2.The ORF region of PyDmrt1 was 918 bp,and the ORF region of PyFoxl2 was 1107 bp,encoding 306 and 369 amino acids,respectively.PyDmrt1 had a conserved DM domain;DM domain had a nuclear localization signal sequence KGHKR;PyFoxl2 also had a conserved FH domain;FH domain was a suspected nuclear localization signal sequence(RRRRRMRR).Through phylogenetic tree and homologous sequence analysis,the results demonstrated that the molecular evolutionary status of PyDmrt1 and PyFoxl2 was consistent with their biological taxonomic status.PyDmrt1 had the highest similarity between P.yessoensis with European scallop(Pecten.maximus),which was 79%.PyFoxl2 had the highest similarity between P.yessoensis with Chlamys farreri and European scallop(Pecten maximus)was 98% and 96% respectively,and the similarity with American oyster(Crassostrea virginica)was 73%.(3)The expression of PyDmrt1 and PyFoxl2 in different tissues of growing stage and different stages of gonadal development of P.yessoensis was analyzed by Semi-quantitative RT-PCR and real-time quantitative qRT-PCR.The results showed that a small amount of PyDmrt1 transcripts were detected in the mantle,adductor,kidney,and gills,but not in the hepatopancreas.PyDmrt1 had the highest expression level in the testis at growing stage,and its expression level was significantly higher than that in the ovary.The expression of PyDmrt1 in male gonads was significantly higher than that in female gonads at growing and mature stage(P<0.05).There was no significant difference at the emission stage.PyFoxl2 was detected with a small amount of PyFoxl2 transcripts in the gills,kidneys and hepatopancreas,but not in the mantle and adductor.PyFoxl2 has the highest expression level in the mature ovary,which was significantly higher than that in the testis.PyFoxl2 showed a downward trend in the gonadal development cycle of the testis and it showed a trend of rising first and then decreasing in the ovary,and reaches the maximum at the mature stage.(4)According to the results of in situ hybridization cell localization,PyDmrt1 and PyFox L2 were localized in the cytoplasm of all germ cells except sperm cells in the gonadal of the P.yessoensis.In the ovary,compared with the negative control group,positive signals of PyDmrt1 mRNA were detected in the gonadal differentiated cells at various stages,but there was no significant change in its expression.In the testis,compared with the negative control group,the expression of PyDmrt1 in spermatogonia and spermatocytes was positive.In the spermatocytes,the PyDmrt1 transcript was not detected by in situ hybridization due to the lack of cytoplasm.The positive signals of PyFoxl2 was detected in the gonadal differentiated cells in the ovary at different stages.Among them,the positive signal of oocytes was the weakest.The positive signal of PyFoxl2 in the ovarian proliferation stage was weaker than that in growing and mature stages.In the testis,the expression of PyFoxl2 in spermatogonia and spermatocytes was positive compared with the negative control group.In the mature testis,the positive signal gradually weakened with the differentiation of germ cells,and no signal was detected in the sperm.(5)Partial interference with Dmrt1 gene expression was preliminarily realized by injecting the corresponding dsRNA into the gonadal of male P.yessoensis.The results of fluorescence quantitative analysis showed that the significant effectiveness of PyDmrt1 gene silencing in P.yessoensis was within 48 h.RNA interference of PyDmrt1 may affect the expression of PyFoxl2 and P450.