Identification,Expression And Function In Gonadal Development Of Igf3 Gene In Scophthalmus Maximus

Scophthalmus maximus is an important marine economic animal industrialized in China,which has high nutritional value.The growth of turbot shows that the female individual is larger than the male,and the growth rate and survival rate of female are higher than that of male.In addition,turbot cannot lay eggs naturally in the artificial breeding project,so it is necessary to inject hormones to promote its ovulation.Therefore,it is very necessary to study the gonadal development of turbot.Sex differentiation and gonadal development are of great significance in the study of fish biology and genetics.The differentiation and development of fish gonads are related to many factors.As a member of insulin growth factor family,igf3 has been proved to be found only in bony fish,and it is up to that in gonads.It is involved in germ cell proliferation,follicular activation,follicular cell survival Oocyte maturation and ovulation are involved in the proliferation and differentiation of spermatogonia in the testis,but the expression and function in different fish are not exactly the same,and its effect on the gonadal development of turbot remains to be studied.Therefore,in this study,igf3 gene was cloned and identified in turbot,its structure was analyzed by bioinformatics technology,its function in sex differentiation and gonadal development was studied,and prokaryotic expression plasmid of Igf3 was prepared,which laid a foundation for understanding the molecular mechanism of igf3 gene regulating gonadal development and preparing efficient production inhibitors.The research contents are as follows:1.Identification and molecular characterization of igf3 gene in turbotThe igf3 gene of Scophthalmus maximus was cloned by molecular cloning technology.The CDS region(864 bp)of the gene encoded 287 amino acids,of which the content of serine was the highest;The analysis of physical and chemical properties showed that its molecular weight,hydrophilic coefficient,theoretical isoelectric point and instability coefficient were 29.95 k Da,-0.502,9.33 and 58.36 respectively.The secondary structure of Igf3 contained one insulin growth factor domain and two low complex domains,and Igf3 had a relatively similar tertiary structure with other members of IGF family(Igf1 and Igf2).However,Igf3 was different from the acidity and hydrophobicity of Igf1 and Igf2,and it was an unstable basic hydrophilic protein.Comparing the amino acid sequence and phylogenetic evolution of Igf3 in various organisms,it was found that Igf3 in turbot had low similarity with other fish and was partially conserved in the evolution of bony fish.The highest homology with Igf3 was Xiphias gladius(79.25%),while the amino acid homology with zebrafish was only18.46%.The three genes of IGF family in different species were clustered into three independent branches.Igf3 had low homology with Igf1 and Igf2,which were 14.15%and14.18% respectively.2.Expression and functional analysis of igf3 gene in male and female gonads of turbotThe results of real-time fluorescence quantitative PCR showed that the igf3 gene of turbot was expressed in gonads,its highest expression appeared in testis,and almost no expression was detected in heart,liver,brain,muscle,kidney,gill,stomach,and other tissues.The results of RT-PCR were similar.The results of in situ hybridization showed that igf3 was highly expressed in primary spermatocytes and interstitial fibroblasts in testis,and mostly expressed in oocytes,granulosa cells and vesicular cells in ovary,suggesting that igf3 gene plays an important role in the early gonadal development of turbot.In order to explore the correlation between igf3 and gender related genes,the expression of gender related genes in gonads was detected by quantitative PCR.The results showed that sf1,sox9 and dazl were highly expressed in testis,and foxl2,wnt4 and piwil2 were highly expressed in ovary.After the primary cells of turbot were interfered with the final concentration of 75 nM siRNA for 36 h,the expression of igf3 was significantly inhibited,and the expression of sf1 in testis and ovary had no significant changes,while the expression of sox9 and dazl in testis decreased significantly,and the expression of foxl2,wnt4 and piwil2 in ovary were also down regulated.It is proved that igf3 gene can directly or indirectly regulate the expression of gender related genes,and its effect in testis is obvious in ovary,which is of great significance for gender differentiation and gonadal development.3.Prokaryotic expression and bioactivity detection of Igf3In order to further explore the growth promoting effect of igf3 as a growth factor in turbot,the prokaryotic expression vector pET-32a-IGFⅢ of turbot igf3 gene was successfully constructed: primers containing enzyme digestion sites were designed,turbot igf3 cDNA was cloned and connected to pET-32 a vector,and the recombinant plasmid was induced into E.coli BL21(DE3)to produce his labeled fusion protein,After SDSPAGE identification,30 k Da purified protein was obtained.The cell proliferation induction experiment of primary gonadal cells of turbot in vitro proved that the recombinant Igf3 protein had the biological activity of promoting cell proliferation in testis and ovarian cells,and the acquisition of this protein also laid the foundation for the preparation of efficient growth promoting additives.After incubation with recombinant protein Igf3,the expression of G1 phase related factor cyclin D in primary gonadal cells was significantly up-regulated,while the expression of S and G2 phase related factor cyclin A was not significantly up-regulated,which proved that Igf3 had an effect on cell cycle regulation and cell growth process.