Cloning and expression of glutathione S-transferase pi gene and analysis of carotenoid transport function in Hyriopsis cumingii

Hyriopsis cumingii, a pearl mussel endemic to China's freshwater waters, produces more than 70% of the country's freshwater pearls. However, the low quality of pearls is a crucial issue, among which color is one of the main factors affecting pearl quality. Carotenoids are the class of fat-soluble natural colorants, and many studies have shown that carotenoids metabolism significantly affects the color of aquatic objects, and pearl color is closely related to carotenoids. Carotenoids cannot be synthesized directly in most animals and must be ingested from food. The metabolism of carotenoids in shellfish is a complex process, and the mechanism of absorption, transport and cleavage in the body is regulated by multi-level and multi-level factors, involving many key genes. Carotenoids are important natural pigments, and it has been found that carotenoids metabolism is significantly related to shell color of shellfish. Glutathione S-transferase Pi (GSTP1), a carotenoid-binding protein, was used to elucidate the function of the HcGSTP1 gene in carotenoids transportation in H. cumingii and to explore the correlation between the expression of HcGSTP1 gene and shell color of H. cumingii. HcGSTP1 gene was cloned and identified in this study, its sequence characteristics and evolution were analyzed. The expression and localization of HcGSTP1 gene in H. cumingii were detected by qRT-PCR and in situ hybridization technology. The function and mechanism of HcGSTP1 gene were preliminarily analyzed by RNAi technology. The results showed that the full-length cDNA sequence of HcGSTP1 gene in H. cumingii was 1 317 bp, of which opening reading frame (OFR) was 618 bp, encoding 205 amino acids and containing a GST-N-pi domain and a GST-C-Pi domain. The results of qRT-PCR showed that the expression level of HcGSTP1 gene in hepatopancreas and axetopods of purple mussel was significantly higher than that in the corresponding tissues of white mussel (P<0.01), and the expression level in the marginal membrane and central membrane of purple mussel was significantly higher than that in the corresponding tissues of white mussel (P<0.05). The results of in situ hybridization (ISH) showed that the positive signals appeared in the outer fold, dorsal mantle, ventral mantle, partial middle fold, and junction of outer fold and middle fold of pallial mantle. RNAi technology results showed that the interference rate of HcGSTP1 gene expression in the fringe mantle reached 83.74% (P<0.05), and the total carotenoids content (TCC) in the fringe mantle was reduced by 30.12% (P<0.05). These experimental results preliminarily validated the vital function of HcGSTP1 gene on carotenoids transportation in H. cumingii. Furthermore, it might affect the color of shells and pearls, and would provided molecular basis for further understanding of the mechanism of carotenoids transport and color formation of shells and pearls in H. cumingii.