Cloning,Functional Verification Of Wnt4 Gene And Correlation Analysis Of SNP With Laying In Black Muscody Duck

Muscovy ducks have the characteristics of fast growth and high remuneration for feed,which is why they are loved by most farmers and have become a characteristic economic industry in some parts of the south of my country.Although Muscovy ducks have excellent characteristics,such as delicious meat and great adaptability,their low fecundity and poor egg-laying performance have always been weak points that need to be urgently resolved.In this study,black Muscovy ducks were used as a research object.Through the cloning and functional verification of the candidate gene Wnt4,which has an impact on fertility,the mechanism of influence of Wnt4 on ovarian development,ovulation and reproductive capacity of Muscovy ducks was explored,and was used for the subsequent genetic crossing and enhancement of trait breeding provide a solid foundation.The research process and the results are as follows:1.RNA was extracted from 7 different tissues(spleen,breast muscle,lung,heart,kidney,liver,ovary)of Peking duck and Muscovy duck in the early stage of laying,and real-time fluorescent quantitative PCR method was used to detect the difference in the expression of Wnt4 gene in the two kinds of ducks.The results showed that there was no significant difference in the expression of Wnt4 gene in spleen,breast muscle,lung,heart,kidney,and liver in Peking duck and black Muscovy duck.However,in ovarian tissues,the expression level of Wnt4 gene in Muscovy ducks is significantly higher than that in Peking ducks,which proves that Wnt4 gene participates in the basic development of Peking ducks and Muscovy ducks,and plays a certain role in the ovarian function of Muscovy ducks.2.Design primers to clone the Wnt4 gene from the ovarian tissue of Muscovy ducks,and use bioinformatics software to predict and analyze the composition and structure of the amino acid sequence of the gene: The full-length sequence of the Wnt4 gene in the ovaries of Muscovy ducks is 1762 bp,and the length of the coding region 1002 bp,encoding 334 amino acids,and the relative molecular mass of the protein is 64 KDa.It contains 3 kinds of protein secondary structures: α-helix,β sheet and β turn.The prediction of signal peptide and transmembrane structure results shows that the protein encoded by this gene has no signal peptide and transmembrane structure region.The analysis of the phylogenetic tree shows that the biological status of this gene is evolutionarily conserved.3.Wnt4 gene function verification and Western Blot results showed that: after the recombinant plasmid carrying Wnt4 gene was transferred into granule cells for over-expression,WB detection of the extracted protein revealed that its protein size fragments were consistent with the predicted results;Real-timefluorescent quantitative PCR detection of steroid-related genes found that after over-expression of Wnt4 gene in Muscovy duck granulosa cells,TGFβ2,FSHR,St AR and CYP11A1 genes were significantly up-regulated;AMH was significantly down-regulated,but FSH expression levels were not significantly different.It shows that Wnt4 gene can regulate the expression of steroid-related genes in black Muscovy ovarian granulosa cells,and promote the proliferation and differentiation of granulosa cells.4.To verify the relationship between Wnt4 gene polymorphism and egg production traits,record the laying data of 1,200 black Muscovy ducks during laying period,collect blood samples from 186 black Muscovy ducks during laying period for PCR,and record the enzyme digestion type result.The results of gene polymorphism analysis are as follows: In the first exon SNP of the gene,three genotypes(TA,AA,TT)were detected,and the number of eggs produced by individuals with AA genotype was significantly higher than that of individuals with TT gene and TA gene.There is no significant difference in the number of eggs between individuals with TT genotype and individuals with TA genotype(P>0.05),which proves that there is no significant correlation between the SNP in the first exon of Wnt4 gene and the egg-laying traits of black duck.This study proved that Wnt4 gene is involved in the basic development of Peking duck and Muscovy duck,and Wnt4 gene plays a certain role in the ovarian development function of Muscovy duck.At the same time,in this study Wnt4 gene was cloned in black Muscovy ducks for the first time,and bioinformatics analysis confirmed that the biological status of this gene was evolutionarily conserved.It was verified that Wnt4 gene can up-regulate TGFβ2,St AR,CYP11A1 and other genes that promote the development of granulosa cells,while down-regulating the AMH gene to promote the proliferation and differentiation of granulosa cells in black Muscovy ducks.At the same time,it can also up-regulate the expression of FSHR in the follicle granulosa cells of Muscovy duck,promote its combination with FSH,and promote the development of follicles.Finally,this study analyzed the association between the SNP of the Wnt4 gene and the egg-laying traits of black Muscovy ducks,and the results showed that the SNP locus of this gene was not significantly related to the egg-laying traits.