Effects Of Overexpression And SiRNA Interference Of FABP4 Gene On Hormone Secretion In Goose Follicular Granulosa Cells

Fatty acid binding protein,also known as fatty acid binding protein 4?FABP4?or ap2,is a member of the fatty acid binding protein?FABPs?family.It is a member of the fatty acid binding protein?FABPs?family.It can combine a variety of hydrophobic compounds,widely distributed in fat and ovarian tissues,and participate in the synthesis and metabolism of fatty acids.It plays an important role in the deposition of fatty acids in eggs of poultry.The fatty acid nutrition deposition of eggs during laying process improves the laying performance.This makes FABP4 research in poultry breeding become a hot topic gradually.Huoyan goose was regarded as a model animal with its excellent laying performance.The effect of FABP4 on the secretion of reproductive hormone in the ovarian granulosa cells of Eyan goose was investigated in this experiment.In this study,the eukaryotic expression vector of FABP4 gene was constructed.The total RNA was extracted from the ovarian tissue of the Huoyan goose,and the target fragment of the FABP4 gene was obtained by the reverse transcription and PCR amplification.After connection and transformation,the clone carrier pMD18T-FABP4 was constructed.After double enzyme digestion,the FABP4 gene fragment was connected to the eukaryotic expression vector pcDNA3.1,and the pcDNA3.1-FABP4 overexpression vector was constructed.Then,the ovarian granulosa cells were isolated and cultured,and the cells were used for the interference of FABP4 gene and the over expression transfection test.The cells were divided into four groups:blank,negative control,FABP4-siRNA interference and pcDNA3.1-FABP4overexpression.The effect of FABP4 on estradiol?E₂?and progesterone?P₄?was tested by enzyme linked immunosorbent assay after transfection,and the effects of FABP4 on the expression gene CYP11A1 and CYP19A1 related with synthesis and secretion of goose ovarian hormone were investigated in mRNA and protein expression levels.The results showed:?1?The eukaryotic expression vector pcDNA3.1-FABP4 was successfully constructed.?2?The follicle granulosa cells of the Huoyan goose were successfully separated and cultured in vitro.?3?The FABP4-siRNA interference primers and pcDNA3.1-FABP4 overexpression vectors were successfully transfected into the follicle granulosa cells of the Huoyan goose.?4?The detection of E₂ and P₄content in the cell supernatant found that the RNA interference group was significantly lower than that of the blank group and the negative control group?P<0.05?.The overexpression group was significantly higher than the blank group and the negative control group?P<0.05?.?5?Protein and mRNA levels detected two genes of CYP11A1 and CYP19A1.The results showed that the RNA interference group was significantly lower than that of the control group?P<0.05?,and the overexpression group was significantly higher than the control group?P<0.05?.The results showed that FABP4 could affect the secretion of E₂ and P₄ from granulosa cells of primary cultured goose follicles by affecting the expression of CYP11A1 and CYP19A1.