| Cytochrome P450c17（Cytochrome P450c17,CYP17A1）and cytochrome P450aromatase（cytochrome P450arom,CYP19A1）play an important role in follicular development,ovulation,secretion and synthesis of reproductive hormones.Cytochrome P450c17（Cytochrome P450c17,CYP17A1）and cytochrome P450aromatase（cytochrome P450arom,CYP19A1）also affect the reproductive performance of animals.Mining genes related to lambing traits in Qianbei Ma goat and screening molecular markers for multiple lambs will help promote the breeding of multiple litter lines in Qianbei Ma goat.In this study,CYP17A1 and CYP19A1 were used as the target genes,and the multi-lamb Qianbei Ma goat ewes（more than 3litters）were used as the research objects.PCR direct sequencing technology was used to explore the association between CYP17A1 and CYP19A1 genes polymorphisms and lambing traits.RT-q PCR technology was used to detect the m RNA levels of CYP17A1and CYP19A1 genes in the gonadal axis of Qianbei Ma goat.IHC was used to detect the localization and expression of CYP17A1 and CYP19A1 proteins in ovarian tissue.The cultured granulosa cells were identified by cell immunofluorescence.Subsequently,the constructed eukaryotic expression vectors and RNA interference vectors of CYP17A1 and CYP19A1 genes were transfected into ovarian granulosa cells to explore the effect of each recombinant plasmid on granulosa cell proliferation,estrogen The effects of alcohol and progesterone secretion and gene expression on related functional indicators were revealed,the genetic effects of CYP17A1 and CYP19A1 genes in Qianbei Ma goat were revealed,and the molecular mechanism of CYP17A1 and CYP19A1 genes regulating goat lambing traits was analyzed.The results of the study are as follows:1.Seven SNPs were detected in CYP17A1,g.59G>C,g.128G>A,g.129A>G,g.164T>C,g.2045G>A,g.4387G>A,g.4456C>There are two genotypes in T;except for the g.59G>C site,which did not deviate from Hardy-Weinberg equilibrium（P<0.01）,the rest of the loci did not deviate significantly from Hardy-Weinberg equilibrium（P<0.01）.The m RNA secondary structure and minimum free energy were changed,and there was no correlation between each mutation site and the lambing number of Qianbei Ma goat.Seven SNPs were detected in CYP19A1,and g.48818C>A was a moderate polymorphism and deviates from Hardy-Weinberg equilibrium（P<0.01）.The mutation of this site also caused changes in m RNA secondary structure and libertarianism.CYP19A1 gene g.48818C>A locus AA type was significantly correlated with litter size（P<0.05）.2.RT-qPCR results showed that CYP17A1 and CYP19A1 genes were expressed in the gonadal axis of single-and multiple-lamb Qianbei Ma goat,and their expression in multiple-lamb ovary tissues was higher than that in other tissues（P<0.01）.IHC results showed that CYP17A1 and CYP19A1 proteins were expressed in all levels of follicles and granulosa cells of Qianbei Ma goat.It is speculated that CYP17A1 and CYP19A1 genes mainly play corresponding functions in ovary and indirectly affect their reproductive performance.3.After pEGFP-N3-CYP17A1/CYP19A1 eukaryotic expression plasmid and sh-CYP17A1/CYP19A1 silencing expression plasmid were transfected into follicular granulosa cells,it could significantly up-regulate and down-regulate the expression of CYP17A1 and CYP19A1 at the transcriptional level in granulosa cells（P<0.01）.,CCK8 results showed that the overexpression of CYP17A1,CYP19A1 can promote the proliferation of granulosa cells,and promote the expression of Cyclin E,PCNA,BMRP-IB,INBHA and FSHR,while silencing the expression can inhibit cell proliferation and inhibit Cyclin E,PCNA in cells,BMRP-IB,INBHA and FSHR expression.In addition,overexpression of CYP19A1 and CYP17A1 can promote the secretion of steroid hormones E2and P4（P>0.05）,and overexpression of CYP17A1can significantly inhibit the expression of steroid synthesis hormone-related enzymes STAR（P<0.01）,CYP11A1,and 3βSHD（P<0.05）,interfering with CYP17A1 can inhibit the secretion of steroid hormones E2and P4（P>0.05）,and significantly promote the expression of steroid synthesis hormone-related enzymes STAR,CYP11A1（P<0.01）and 3βSHD（P<0.05）.Overexpression of CYP19A1 could significantly inhibit the expression of steroid hormone-related enzymes STAR,CYP11A1,and 3βSHD（P<0.01）,and interfering with CYP19A1 could significantly promote the expression of steroid hormone synthase（P<0.01）.In conclusion,CYP17A1 and CYP19A1 may regulate cell proliferation and hormone secretion by changing the expression of Cyclin E,PCNA,STAR,CYP11A1,3βSHD and other genes,and indirectly affect follicle development by affecting the expression of BMRP-IB,INBHA and FSHR,and then affect the follicle development.Reproductive performance of goats. |
