Functional Identification And Polymorphism Of AMH Gene In Black Muscovy Duck

Anti-mullerian hormone(AMH)is a glycoprotein secreted by the testicular Sertoli cells and the female ovary’s granulosa cells which are only expressed in the gonadal gland.AMH is a ligand member of the Transforming growth factor beta superfamily(TGFβsuperfamily).In female animals,AMH is secreted by ovarian granulosa cells and acts on primordium follicles to prevent premature depletion of ovarian reserves.AMH gene has been studied extensively in mammals but seldom in poultry.In this study,the eukaryotic expression vector of AMH gene in black muscovy ducks was constructed and transfected into Muscovy ovary granulocytes for functional validation.The association between single nucleotide polymorphisms of AMH gene and egg production and body weight data was analyzed.The results were as follows:1.To investigate the expression pattern of AMH in the ovaries of Muscovy ducks,Quantitative real-time PCR(q RT-PCR)was used to detect the expression of AMH and its anti-mullerian hormone receptor type 2(AMHR2)in the ovaries and different levels of follicles of high and low birth Muscovy ducks.We found that the expression of AMH/AMHR2 in the ovaries of low-yielding Muscovy ducks was significantly higher than that of high-yielding Muscovy ducks.With the development of follicles,the expression level of AMH decreased gradually.AMH expression was highest in large white follicles and lowest in grade follicles.It is preliminarily concluded that AMH plays an inhibitory role in the development of follicles.2.In this study,we successfully cloned the CDS region sequence of AMH gene in black muscody duck.The sequence was 1833 bp long and encoded 610 amino acids.It was a secreted protein with three secondary structures and signal peptides.The eukaryotic overexpression vector was constructed,which laid a foundation for further study of the effect of AMH on Muscovy ducks granulocyte cells.3.To verify the function of AMH in Muscovy ovarian Granulosa cells,we transfected the AMH overexpression vector into Muscovy ovarian Granulosa cells.The expressions of AMH and its related genes were detected by the fluorescence quantitative method.The results showed that compared with the control group,the expression of AMHR2,ALK2,ALK3,and ALK6 of AMH receptors were significantly increased in the treatment group.But the expression of FSHR was not significantly different,which proved that AMH could play a role in granulosa cells through AMHR2,ALK2,ALK3,and ALK6 receptors.There was no significant difference in the expression of apoptosis-related genes caspase-3,Myc,and Bcl-2 between the experimental and control groups.The expression levels of proliferation-related genes CDK2 and PCNA in AMH treatment group were lower than those in blank control group,indicating that AMH inhibited the proliferation of granulosa cells.4.To study the relationship between single nucleotide polymorphisms of AMH gene and laying and body weight traits,this study randomly selected 186 cage-reared Muscovy ducks.The SNPs of AMH gene were screened by restriction enzyme(PCR-RLFP),and we analyzed the correlation between AMH gene and the traits of laying and body weight.And we finally screened three sites g.1731G>A、g.2044T>C、g.3531C>T,among them,the AG of g.1731G>A is the dominant genotype,and the correlation analysis results show that the number of eggs of the AG genotype in the g.1731G>A mutation site is significantly higher than that of the AA type(P<0.05),and g.1731G>A is not significantly related to body weight(P>0.05);CC of g.2044T>C is the dominant genotype,the number of laying eggs of CC genotype was significantly higher than that of TT and TC(P<0.05),and the weight of CC genotype was significantly higher than that of TT(P<0.05);g.3531C>T is not correlated with egg number and body weight(P>0.05).In summary,the single nucleotide polymorphism of AMH in this study has an impact on egg production.g.1731G>A and g.2044T>C can provide references genetic markers for breeding excellent Muscovy duck breeds.In conclusion,this study studied the expression pattern of AMH in high and low parturnal MMuscovy ducks and different levels of follicles.The AMH gene of black Muscovy duck was cloned,and the AMH overexpression vector was constructed successfully.The function of AMH was verified at the cellular level,and the association between AMH gene polymorphism and production performance was analyzed.This study revealed the mechanism of AMH in Muscovy ovarian Granulosa cells,provided a theoretical basis for further research,and provided a reference genetic marker for breeding high-quality Black Muscovy duck varieties.