Functional analysis of retinal-binding protein in the visual system of the nocturnal marine shellfish Pacific abalone...

The Pacific abalone (Haliotis discus hannai) is a nocturnal species that inhabits marine benthic ecosystems, characterized by its aversion to light and preference for darkness during daylight hours, with movement and feeding predominantly occurring at night. However, the morphology and structure of its visual organs and the mechanisms underlying its perception of cyclical light variations are unknown. This study analyzed the Retinal-binding protein (RALBP) gene to elucidate its role in the abalone's visual system. Using the rapid amplification of cDNA ends technique, the RALBP gene was cloned, revealing an open reading frame (ORF) of 1188 bp encoding 396 amino acids. A recombinant expression vector was subsequently constructed to produce a recombinant protein with a molecular weight of 45.3 kDa. This purified recombinant protein was used as an antigen to generate a polyclonal antibody, with enzyme-linked immunosorbent assay (ELISA) results indicating an antibody potency of ≥512 K. Immunofluorescence assays demonstrated strong RALBP signals in the pigment epithelial and photoreceptor cell layers of the retina, with no detection in other retinal layers. To evaluate circadian expression patterns of RALBP under different light spectra, including full-spectrum natural light, short-wavelength blue light (450 nm), and long-wavelength orange light (560 nm), western blotting and the Cosinor program were utilized. The results revealed significant cosine rhythmic expression of RALBP under all light conditions (P < 0.05), with higher expression levels during the day compared to the night (P < 0.05). This is the first study that provides insights into the morphological and structural characteristics of the abalone retina and demonstrates that the abalone's ability to detect cyclical light changes is determined by the diurnal expression of RALBP. These findings establish a theoretical basis for understanding the evolution of visual organs and circadian regulation mechanisms in marine shellfish.