| Sex size dimorphism refers to the phenomenon that individuals of different sexes have different sizes in the same species.This phenomenon is very common in fish.It is found that it exists in at least 600 kinds of fish.Among them,Cynoglossus semilaevis,which is widely distributed in the Yellow Sea and Bohai Sea,is one of the typical representatives.Cynoglossus semilaevis is an important economically cultured fish in China and has high nutritional and economic value.The adult female of Cynoglossus semilaevis is much higher than the male in both body length and body weight,At the same time,the female growth process will be reversed due to external environmental factors,which greatly reduces the proportion of female Cynoglossus semilaevis,which seriously restricts the sustainable development of Cynoglossus semilaevis breeding industry.Early stage transcriptome sequencing of the growth growth axis tissue of Cynoglossus semilaevis showed that the genes of insulin μ Lin like growth factor binding protein(IGFBP)showed a significant differential expression trend in male and female tissues.igfbp7 was a member of the IGFBP superfamily,it is a representative gene in the IGFBP family,which is involved in the regulation of insulin-like growth factor(IGF)and insulin signal transduction.The transcriptome data found that igfbp7 is highly expressed in the female gonads of Cynoglossus semilaevis,which aroused our interest.Therefore,this study carried out the cloning,expression pattern analysis The purpose of promoter transcription regulation analysis and RNA interference knockdown analysis is to explore the effect of igfbp7 on gender dimorphism of Cynoglossus semilaevis.The main results are as follows:The cloning and evolutionary analysis of igfbp7 gene of Cynoglossus semilaevis showed that the total length of igfbp7 m RNA was 1012 bp,the length of coding region was 666 bp,encoding 221 amino acids,including 4 exons and 3 introns,and its spatial structure was highly conservative.Phylogenetic analysis showed that Cynoglossus semilaevis and flounder,Atlantic salmon and zebrafish were clustered into one branch,which had close genetic relationship and contained a common domain IGFBP,which was relatively conservative in evolution.By analyzing the expression patterns among different tissues at the age of 3,it was found that the igfbp7 gene of Cynoglossus semilaevis was overexpressed in the female gonad,and the female gonad showed differential expression with any other tissues(P <0.05),followed by intestine,liver and muscle.The expression of igfbp7 gene in females was higher than that in males,and there was almost no expression in male gonads and other tissues.In the differential analysis of gonads in different periods,the expression of female gonads has been much higher than that of male gonads.In the expression pattern of gonads in different periods,it was found that the expression of female gonads was the highest at 6 months old,showing differential expression with male and female gonads in other periods(P < 0.05).Among them,the expression of female gonads and male gonads in 6m,1.5y and 3y periods showed differential expression(P < 0.05).This expression pattern may suggest that igfbp7 may play an irreplaceable role in ovarian development.The results of in situ hybridization showed that there were hybridization signals in female gonads and the signals were obvious,mainly concentrated around the ovary cells,while there were only weak signals in male gonads,which was more consistent with the results of real-time fluorescence quantification.There was no hybridization signal in sense probe.Through the analysis of the methylation results of 1.5-year-old,it can be seen that in female tongue sole,igfbp7 was 1282 bp upstream of the gene and the coding region,And 1459 bp downstream of the gene showed a low methylation level,the lowest in females,the highest in males,followed by pseudomales.Generally,the higher the methylation level,the lower the expression.This result is also consistent with the quantitative results,suggesting that methylation can regulate the expression of igfbp7 in Cynoglossus semilaevis.The igfbp7 promoter of Cynoglossus semilaevis was constructed.The promoter was located 1215 bp upstream of the coding region.The transfection results showed that the promoter had strong promoter activity compared with the negative control group p GL3 basic,and then through promo(http://alggen.lsi.upc.es/cgibin/promo＿v3/promo/promoinit.cgi?dir DB=TF＿8.3)After transfection,the transcriptional activity of Cynoglossus semilaevis was significantly enhanced by CO transfection with the transcription factor of POU1F1.The addition of steroid hormones estradiol(E)and testosterone(T)also promoted the transcriptional activity,and there was a significant difference after the addition of T(P < 0.05).The results of si RNA transfection in female ovarian cells of Cynoglossus semilaevis showed that when igfbp7 was knocked down,the downstream genes serine / threonine kinase 1(akt),heat shock protein 60(hspd1)and insulin-like growth factor receptor(igf1r)were activated in varying degrees.Among them,the activation of akt and igf1 r was super significant(P < 0.001),and the activation of hspd1 was very significant(P < 0.01),The activation of these genes suggests that igfbp7 may play an inhibitory role in the ovarian development of Cynoglossus semilaevis.Pc DNA3.1-igfbp7 plasmid containing igfbp7 coding region was constructed,with PNF-κB was co-transfected with PNF-κB,luciferase activity showed significant difference(P<0.05),indicating that Cynoglossus semilaevis igfbp7 activated this pathway.In order to explore the role of igfbp7 in the partial female sex expression of Cynoglossus semilaevis,we analyzed the expression patterns of igfbp7 in different tissues and gonads of Cynoglossus semilaevis at different stages.The results showed that igfbp7 was highly expressed in gonads and almost not expressed in other tissues,indicating its possible role in growth and gonadal development,In addition,when the transcription factor f1 akr and its downstream transcription factor were silenced,it was confirmed that the transcription factor f1 akr had strong activity,In order to identify the possible regulatory pathways of igfbp7 in the gender dimorphism of Cynoglossus semilaevis,these preliminary studies on igfbp7 gene of Cynoglossus semilaevis will help us to better understand the fish igfbp gene family. |
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