Characterization and Function Analysis of β, β-carotene-9′, 10′-oxygenase 2 (BCDO2) Gene in Carotenoid Metabolism of the Red Shell Hard Clam (Meretrix meretrix)

Lulu Fu,Heming Shi,Wenfang Dai,Yinghui Dong,Hanhan Yao,Zhihua Lin,Yongbo Bao

doi:10.3389/fmars.2021.746026

Abstract

The relationship between carotenoid and shellfish shell color has gained increasing attention. β, β-carotene-9′,10′-oxygenase 2 (BCDO2) is a key enzyme in animal carotenoid metabolism, and its accumulation affects the change in body color, as demonstrated in mammals, birds, and fish. However, it is unclear whether BCDO2 is involved in the formation of the red shell color of clam. To explore the molecular structure and biological function of BCDO2 gene in the process of carotenoids accumulation, in this study, the BCDO2 from hard clam Meretrix meretrix (designated as Mm-BCDO2) was cloned and characterized, and the single-nucleotide polymorphisms (SNPs) associated with shell color were detected. The results of qRT-PCR indicated that Mm-BCDO2 gene was expressed in all six tested tissues, and the expression of mantle was significantly higher than other tissues (P &lt; 0.05). The association analysis identified 20 SNPs in the exons of Mm-BCDO2, among which three loci (i.e., c.984A &gt; C, c.1148C &gt; T, and c.1187A &gt; T) were remarkably related (P &lt; 0.05) to the shell color of clam. The western blot analysis revealed that the expression level of Mm-BCDO2 in the mantle of red shell clams was stronger than that of white shell clams (P &lt; 0.05). Further, the immunofluorescence analysis indicated that the single-layer columnar cells at the edge of the mantle were the major sites for the Mm-BCDO2 secretion. This study explored the potential impacts of BCDO2 gene on the shell color of M. meretrix, which provided a theoretical basis for a better understanding of the important role of BCDO2 in carotenoid metabolism.

Highlights

Carotenoids are a class of fat-soluble pigments, which can be classified into two groups: the xanthophylls and the carotenes (Wu et al, 2016)
Based on the amino acid sequence encoded by the BCDO2 gene, the protein molecular weight was predicted to be 60.06 kDa, corresponding isoelectric point = 5.83, including a RPE65 domain (11–308, 356–517 aa)
Protein structure prediction showed that Mm-BCDO2 protein composed of 9 α helices, 38 β-sheets, and a large number of random coils and had the typical characteristics of RPE65 protein family with a cleft and a right-handed twisted anti-parallel β-barrel (Figure 1B)

Summary

Introduction

Carotenoids are a class of fat-soluble pigments, which can be classified into two groups: the xanthophylls (oxygenated-contained group) and the carotenes (hydrocarbons without oxygenated group) (Wu et al, 2016). The zeaxanthin, lutein, and astaxanthin have a positive effect on antioxidant and light filtering by preventing vision loss and eye diseases (Li et al, 2017), protecting the eyesight to a certain extent (Felix et al, 2011). Carotenoids and their derivatives are involved in the coloration of feathers, skin, and shells, which play a signal role in mate selection, social competition, and species recognition (Blount and Mcgraw, 2008). Studies have found that the expression level of BCDO2 affects the body color of animals (Eriksson et al, 2008). Genetic disruption (low-temperature expression and detergent choice) of BCDO2 can significantly affect binding and turnover rates of the recombinant enzymes with various xanthophyll substrates and increase the concentration of dietary pigment in plasma and blood of the mice (Babino et al, 2015)

Methods

Results

Conclusion