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Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Published on: May 27, 2016
Functional Insights into Bpcreb1 in Bellamya purificata: Expression Dynamics and regulatory Roles during gonadal
Yijin Ren1, Xin Jin1, Jiaying Song1
1Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China; National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China; Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China.
Abstract:
Cyclic AMP response element-binding protein 1 (CREB1), a conserved bZIP transcription factor, is crucial for animal reproduction. Therefore, this study investigated its role in gonadal development of the freshwater snail Bellamya purificata using qRT-PCR, ISH, RNAi, and DNA methylation analysis. Results revealed that the creb1 CDS (867 bp, 288 aa) features a highly conserved BRLZ domain. Significantly, tissue expression showed markedly higher creb1 transcript levels in testes versus ovaries (p < 0.01). ISH localized enriched mRNA in ovarian oocytes/follicular walls and testicular spermatogonia/sperm. Functionally, efficient RNAi (max. 71.98% silencing) caused acute downregulation of sex genes: female β-catenin, cyp17a1 and sox9 expression fell 27%-34%, while male expression dropped 58%-77%. Critically, prolonged interference induced testicular defects, namely reduced mature sperm and increased spermatogonia, implicating Bpcreb1 in spermatogenesis-potentially via tssk and sox9 regulation. Furthermore, epigenetic analysis found consistently low Bpcreb1 promoter methylation (<5%) in both sexes, yet RNAi did not alter this hypomethylation, hinting at non-methylation regulatory mechanisms. In summary, creb1 demonstrably regulates gonadal development in B. purificata, with a pivotal role in spermatogenesis. Consequently, these findings provide a theoretical foundation for studying its gonadal development and monosex breeding strategies.
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