| Hyriopsis cumingii,as a unique pearl mussel of freshwater waters in China,produced more than70% of the total freshwater pearls in China.Color is one of the main factors affecting the quality of pearls,which can not only improve the value of pearls,but also enrich the types of pearl products.Carotenoids are a kind of lipid-soluble natural colorants.Existing studies showed that carotenoids metabolism significantly affected the color of aquatic animals.Studies also found that pearl color was closely related to carotenoids.Most animals lacked enzymes associated with carotenoid synthesis,so they could only take them from food.The metabolism of carotenoids in shellfish is a complex process.The metabolic mechanism of absorption,transport and cleavage is regulated by multi-level factors,and involves the participation of many key genes.In this study,several genes related to carotenoid metabolism,including HcGSTP1,HcBCO1 and HcBCO2,were screened from the membrane transcriptome library,and their functions were verified by taking white Mussels(H.cumingii with white inner shell color),purple Mussels(H.cumingii with purple inner shell color)and golden Mussels(H.cumingii with golden inner shell color)as the research objects to explore their roles in carotenoid metabolism and the influence of shell and pearl color.1.H.cumingii HcGSTP1 gene cloning,expression,and functional validationCarotenoids are important natural pigments,and it has been found that carotenoid metabolism is significantly related to shell color.Glutathione thiotransferase Pi(GSTP1)is a carotenoid-binding protein,which plays an important role in transportation of carotenoids.In order to elucidate the role of HcGSTP1 gene in carotenoid transport and explore the correlation between its expression and shell color,we cloned and identified the c DNA full-length sequence of HcGSTP1 gene in H.cumingii with a 618 bp-long open reading frame(ORF),encoding 205 amino acids,and it contained a GST-N-pi domain and a GST-C-Pi domain.The expression level of the HcGSTP1 gene in the hepatopancreas and axetopods of purple mussels was substantially greater than that of white mussels(P<0.01),and it was significantly higher in the marginal and central membranes of purple mussels(P<0.05).In situ hybridization of mantle tissue revealed a high positive signal of the HcGSTP1 gene in the outer fold,dorsal membrane area,peritoneal region,junction between the outer fold and the middle fold,and several middle folds.After injection of dsRNA interference chain,the interference rate of HcGSTP1 gene expression in the mantle reached 83.74%(P<0.05),and the total carotenoids content(TCC)in the mantle decreased by 30.12%(P<0.05).These results preliminarily confirmed that HcGSTP1 gene plays an important role in carotenoid transport in H.cumingii,which may affect the color of shells or pearls,and provided molecular basis for further understanding of the mechanism of carotenoid transport and color formation of shells and pearls in H.cumingii.2、Cloning and expression analysis of HcBCO1 and HcBCO2 genes in H.cumingiiBCO1 and BCO2 are key enzymes in animal carotenoids metabolism(cleavage),and their accumulation will affect the body color.In order to clarify the function of HcBCO1 and HcBCO2 genes in carotenoid degradation in H.cumingii,the correlation between these genes and shell color was investigated.In this study,we cloned HcBCO1 and HcBCO2 genes from H.cumingii,the c DNA full-length sequences were 1831 and 1987 bp,and the open reading frame(ORF)were 1560 and1987 bp,respectively encoding 519 and 529 amino acids(Gen Bank entry number: MW171289),and all of which contained a RPE65 domain.Gene expression analysis found that the expression level of HcBCO1 was the highest in the tissues of white H.cumingii,and the expression level of HCBCO1 was significantly different from that of the corresponding tissues of golden and purple H.cumingii(hepatopancreas,edge mantle,adductor muscle,central mantle,and foot)(P<0.05).While the expression level of HcBCO2 in the adductor muscle,central mantle,edge mantle and gill of white Mussels was significantly higher than that of purple Mussels and golden Mussels(P<0.05),and the expression level of HcBCO2 in the tissues above in golden Mussels was the lowest.The results of in situ hybridization revealed that the HcBCO1 signal was distributed over the whole outer fold,the middle fold,the intersection of some middle fold and inner fold,and the peritoneal area.Significant HcBCO2 signals were seen in the mantle’s outer fold,the peritoneal area,the intersection between the outer and middle folds,and several middle folds.3、Studies on carotene degradation of HcBCO1 and HcBCO2 genes in H.cumingiiThe protease encoded by β-carotene-15’,15’-oxygenase(BCO1)and β-carotene-9’,10’-oxygenase(BCO2)genes can cleave β-carotenoids,and the p AC-BETAipi plasmid can produce β-carotenes in Escherichia coli.Therefore,a pathway to synthesize β-carotenes can be constructed in E.coli by means of genetic engineering,and then the constructed recombinant plasmid can be transformed into a strain that can synthesize β-carotenes,and the function of the gene can be verified by the change of color.In this study,through the prokaryotic expression experiment,it was found that the recombinant plasmids of p GEX-BCO1 and p GEX-BCO2 were transformed into the bacteria containing β-carotenes,and the color changed significantly from orange to light,indicating thatβ-carotenes were degraded to a certain extent;In addition,the degree of degradation of carotenoids by p GEX-BCO1 was higher than that by p GEX-BCO2.After the interference silencing of HcBCO1 and HcBCO2 genes,the expression levels of HcBCO1 and HcBCO2 genes in the experimental group were significantly decreased(P<0.05)compared with the blank control group and the negative control group,and decreased by 78.62% and 75.50%,respectively.In addition,the total carotenoids content(TCC)of mantle was increased by 64.75% and 57.80%(P<0.05),respectively.In conclusion,HcBCO1 and HcBCO2 genes had the function of cleaving β-carotenes,and HcBCO1 gene had a stronger ability to degrade carotenoids than HcBCO2 gene,which preliminatively confirmed that HcBCO1 and HcBCO2 genes played an important role in the process of carotenoid cleavage in H.cumingii,and thus may play an important role in the color formation mechanism of shells and pearls of H.cumingii. |
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