Identification of cis-acting elements and trans-acting factors regulating the glycoprotein vitellogenin receptor: Unraveling the transcriptional regulatory network of vitellogenesis in the Pacific white shrimp Penaeus vannamei.

Vitellogenin (Vg), a major yolk protein macromolecule, serves as a key nutrient source for oocyte maturation during vitellogenesis and ovarian development in oviparous animals. Beyond Vg synthesis, efficient uptake and accumulation of exogenous Vg by developing oocytes are critical for successful ovarian development. This process is mediated by the vitellogenin receptor (VgR), which is a membrane-bound glycoprotein VgR exhibits strict ovary-specific expression in crustaceans. However, the molecular mechanisms governing its tissue-specific transcriptional regulation during ovarian development remain poorly defined. In this study, we cloned a 679 bp VgR promoter fragment from the Pacific white shrimp (Penaeus vannamei), a globally dominant aquaculture crustacean, using genome walking. Dual-luciferase reporter (DLR) assays precisely mapped the core high-transcriptional-activity region to -273 to +62 bp relative to the transcription start site (TSS), which was used as bait for yeast one-hybrid (Y1H) screening. A high-quality ovarian Y1H cDNA library was constructed via Gateway technology, and nine candidate trans-acting factors, including two functionally unannotated novel proteins, were identified through bait interaction, bioinformatic prediction, ovarian expression profiling, and DLR validation. Notably, this was the first study to experimentally confirm that these nine factors directly participate in VgR transcriptional regulation in P. vannamei. Collectively, our findings unraveled the cis-acting elements and trans-acting factors underlying VgR (a key reproductive glycoprotein) regulation, providing critical insights into the species-specific transcriptional mechanism of VgR during crustacean ovarian development. These results offered potential molecular targets for optimizing P. vannamei reproductive performance and advancing sustainable selective breeding strategies for aquacultural crustaceans, aligning with global aquatic food security goals.