| It is well known that Insulin-like Growth Factors(IGFs)are widely involved in gonadal development and reproduction in teleost fish.igf3 is expressed in many tissues of teleost fish,especially in gonads,But the expression pattern and transcriptional regulation mechanism of igf3,whether igf3 can affect sex determination and differentiation of fish are still unclear.In this study,We used Epinephelus coioides as the research object,and firstly used real-time quantitative PCR(RT-PCR)to detect the expression of sex-differentiation and reproductive-related genes after IGF3 stimulation.To study the regulation of igf3 on sex-related factors in grouper,and explore the mechanism of igf3 on reproductive regulation and its function on ovarian physiological function;for explorring the reproduction of igf3 on the grouper Regulation and its mechanism of action on ovarian physiological function,we studied on transcriptional activity regulation of igf3 promoter.These can help us inquire into the mechanism of sex-related genes and understand the functional effects of igf3 in gender differentiation.Through this experiment,we can effectively regulate the reproductive and sex differentiation of the grouper,and artificially induce the mature ovary of the grouper,solve the problem that the sexual maturity cycle is long and the ratio of male to female is unbalanced,and improve the reproductive efficiency which is conducive to expansion.The breeding and production of the grouper,and provides preliminary material preparation for the actual production of highly active oxytocin.The main research results of this thesis are as follows:1.The ovarian primary cells of the grouper were incubated in vitro,and the cells were stimulated with three different concentrations of IGF3 recombinant protein at 10 n M,100 n M and 1000 n M.After 6 hours,taken samples to prepare templates and detected the expression levels of sex-related genes foxl2,dax1,cyp11a1,cyp19a1 a and cyp19a1 b by RT-PCR.It was found that IGF3 promoted the expression of foxl2,dax1,cyp11a1,cyp19a1 aand cyp19a1b.IGF3 had the most significant effect on the expression of dax1 and cyp19a1 b.Three concentrations of IGF3 at 10 n M,100 n M and 1000 n M significantly increased dax1 and cyp19a1 b expression in a concentration-dependent manner.10 n M of IGF3 can significantly up-regulate their expression,Both 100 n M and 1000 n M IGF3 polypeptides up-regulated the expression of dax1 and cyp19a1 b more significantly.IGF3 polypeptide had a significant effect on the expression of foxl2 and cyp19a1 a.10n M IGF3 slightly up-regulated the expression of foxl2 and cyp19a1 a,but did not show significant difference.The stimulation of IGF3 at 100 n M significantly up-regulated the expression of foxl2 and cyp19a1 a,whereas effect of 1000 n M IGF3 on the expression of both is not as significant as 100 n M.With the concentration of IGF3 stimulated,cyp11a1 also showed a trend of increasing concentration,but the expression of it was not significant by stimulating of 10 n M and 100 n M IGF3 polypeptide,its expression level was only significant at 1000 n M.The experimental results show that igf3 has a regulatory effect on genes related to sex differentiation.2.The primary ovarian cells of the grouper were incubated in vitro,and the primary cells were stimulated with three different concentrations of IGF3 recombinant protein(10n M,100 n M and 1000 n M.After 6 hours,the template was prepared and used in RT-PCR to detect the expression of reproductive-related gene lhr1(receptor of lh)and fshr(receptor of fsh)The results showed that 10 n M IGF3 recombinant protein could significantly up-regulate the expression of lhr1.The expression of lhr1 was further increased under the action of 100 n M IGF3 polypeptide,and the expression was highest under the stimulation of 1000 n M IGF3.The up-regulation of IGF3 recombinant protein on lhr1 was observed concentration-dependent effect.however,none of the three concentrations of IGF3 recombinant protein affected the expression of fshr.This result indicates that igf3 can regulate the expression of reproductive related genes in the gonads.3.We obtained the igf3 promoter sequence of the grouper from the genomic database,designed igf3 primers and cloned the igf3 promoter,and constructed the p GL3.0-igf3-promoter luciferase reporter vector.Then we according to the ORF sequence of the sf1,dmrt1,dax1 and foxl2 genes of the grouper,screened the positive clones and constructed the pc DNA4.0-sf1,pc DNA4.0-dmrt1,pc DNA4.0-dax1,pc DNA4.0-foxl2 expression gene vectors.By transfecting fish GS cells,the igf3 promoter was combined with sex-related factors,and detected luciferase activity on a microplate reader to study the transcriptional activity regulation mechanism of igf3 promoter.The results showed that when transfected with foxl2,dax1,dmrt1 or sf1 respectively,the igf3 promoter was directly activated by sf1,and the activity enhancement of igf3 promoter by 200 ng and 400 ng of sf1 was extremely significant,And with the increase of sf1 dose,the stronger the transcriptional activation of sf1 on igf3.neither foxl2 nor dax1 affect igf3 promoter activity at doses of 200 ng or 400 ng.The igf3 promoter activity was directly inhibited by dmrt1 and was dose-dependent.200 ng of dmrt1 can significantly inhibited igf3 promoter activity,400 ng of dmrt1 can significantly inhibited igf3 promoter activity.When fox1 is co-transfected with sf1 or co-transfected with dax1 and sf1,the activation of igf3 promoter by sf1 can be enhanced.In particular,foxl2 can significantly enhance the activity of igf3 promoter activated by sf1.Conversely,co-transfection of dmrt1 with sf1 significantly inhibited the activation of igf3 promoter by sf1.The results of the experiment indicated that the igf3 promoter was regulated by sex-related genes,suggesting that igf3 is involved in the sex differentiation of the grouper.4.The results showed that igf3 could regulate the expression of genes related to reproductive and sex differentiation of grouper,while the genes related to sex differentiation could also affect the promoter activity of igf3.It is speculated that igf3 has a certain effect on the reproduction of grouper.And involved in the process of gonadal differentiation of the grouper. |
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