Differential Expression Of Chicken ERα In Embryonic Tissues And Its Promoter Activity Analysis

Avian can act as an important model for other vertebrate, its sex differentiation and gonadal development has been the hotspot of researchs on avian developmental biology. It was reported that aromatase which is the key enzyme in synthesis of estrogen could induce male embyro converted into feminize, it is suggested that estrogens play a key role in gonadal development in earilier chicken embyro. In this research, expression differences of alternative splicing variants regulated by different promoters in different tissues during embryonic development periods were identified, 5 ’upstream region of ERα was analyzed and different deletion fragments were cloned and recombined into vectors to find activity differences of multiple promoters. Real-time quantitative PCR method was used to obtain quantitative analysis on expression of alternative splicing variants of ERα; the dual luciferase reporter gene system was used to detect promoter activities of different deletion fragments. Expression levels of spliceosome B, C and D in male and female chick tissues from five periods detected by real-time quantitative PCR. It is confirmed that expression of spliceosome B, C and D are found in all detected tissues. The expression of spliceosome B, C and D are most active in both gonads, the expression levels in female gonad is higher than male. According to the higher level in both gonads than other tissues of three spliceosome, we detected the relative expression level of three spliceosome in female and male gonads. It is identified that the expression trendency of spliceosome B, C and D in both gonads are basically identical. In addition, some elements are present to down-regulate the expression of three alternative splicing variants in E14.5 male and female embryo. The relative expression level is spliceosome C>D>B. Deletion fragments which recombined into vectors of pGL3-basic plasmids were transfected into three kinds of cells and activities were detected. Promoter activities were unfound in transfection experiment of PK15 cells, but conversely, promoter activities differences were detected in CEF cells and DF1 cells.The activities differences in CEF cells are as follows: D>B>C>A12>A1>A2. The activities differences in DF1 cells are as follows: D>A12>A2>C>B>A1, promoter D is the most active one than other promoters, and the activities of recombination vector A12(contain promoter A1 and A2) is higher than individual promoter A1 or A2. It is convinced that recombinants of pGL3-basic plasmids are cell-specified. Additionally, promoter activitie differences of ERα may be influenced by different transcription factors in gene regulatory pathways or interaction among promoters.