| Since the early 1980s,China's production of freshwater pearls has been leading the world,and Hyriopsis cumingii is the most important pearl mussel.The pearls produced by H.cumingii are of good quality and colorful.Pearl color has an important influence on its value.The color of pearls is affected by the nacre color of shell of the donor pearl mussel.The exosomes are involved in shell formation.Whether exosomes are involved in shell pearl formation is worthy of attention.In this paper,the white line mussels(shell nacre is white)and purple line mussels(shell nacre is purple)were used as experiment materials which had been under mass selection by our laboratory.The role of exosomes in the color formation of shell nacre was studied by miRNA and proteome sequencing.The main results are as follows1.Extraction,identification and miRNA sequencing analysis of mantle exosomes in white and purple mussel of H.cumingiiFor the first time,the mantle exosomes of white and purple H.cumingii were successfully extracted.The morphology of the exosomes was identified as saucer-like structure by transmission electron microscope.Nanopartical tracking analysis showed that the mean size of white and purple mussel exosomes was 175.2±5.1 nm and 176.1±5.4nm,respectively.High-throughput Illumina sequencing and expression analysis were performed on the white and purple mussel mantle exosomes.By comparison with the miRbase V21 database,136 and 159 miRNAs were obtained from mantle exosomes of white and purple mussels,respectively.Using the standard of |log2(Fold change)|?2,and a p value ?0.05,a total of 54 differentially expressed miRNAs were identified,of which 15 were highly expressed in exosomes of white mussel and 39 highly expressed in exosomes of purple mussel.The differential expression miRNAs that might target some nacre color formation genes were predicted with software,and these miRNAs were verified by qPCR.The results indicated that the expression patterns of those miRNAs were generally consistent with the sequencing results.Therefore,sequencing results were reliable.2.Regulation of mantle exosome miRNA on genes related to nacre color formation in H.cumingii.The software predicted that miR-15b might target hcApo,a gene related to carotenoid metabolism in H.cumingii,which has been shown to involved in nacre color formation.Using double luciferase reporter gene assay,we found that miR-15b can inhibit the expression of hcApo.Thirty-six mussels were divided into three groups,which injected with miR-15b antagomir,antagomir negative control and 1×PBS,and half of mussels in each group were fed with ?-carotene.It was found that miR-15b antagomir can inhibit the expression of miR-15b in hepatopancreas,gill,fringe mantle and middle mantle,while the expression of hcApo increased in these tissues.After mussels were fed with ?-carotene,the tissue expression of hcApo also increased.The hcApo gene expression level was highest under the dual effects of miR-15b antagomir and ?-carotene.These results indicate that miR-15b can negatively regulate the expression of hcApo gene,and thus participate in the formation of the pearl frontal color of H.cumingii.The software predicted that miR-4504 might target HcMitf,a gene related to melain synthesis in H.cumingii,which has been shown to involved in nacre color formation.Using double luciferase reporter gene assay,we found that miR-4504 can inhibit the expression of HcMitf.Twenty-seven mussels were divided into three groups,which injected with miR-4504 antagomir,antagomir negative control and 1 × PBS,respectively.It was found that miR-4504 antagomir can inhibit the expression level of miR-4504 in fringe mantle,middle mantle,gills,foot,hepatopancreas and kidney.Then the expreesion level of HcMitf and its downstream tyrosinase gene HcTyr increased significantly.After injection of miR-4504 antagomir,the tyrosinase activity and melanin content in the fringe mantle and middle mantle of significantly increased.The above research shown that miR-4504 can participate in the melanin synthesis by regulating the HcMitf gene,thereby affecting the formation of shell nacre color of H.cumingii.3.Proteomic analysis of mantle exosome in white and purple mussel of H.cumingii.A total of 4,861 proteins were obtained by TMT(Tandem Mass Tags)quantitative protein sequencing analysis of mantle exosomes of white and purple H.cumingii.Functional annotation analysis of these proteins(GO,COG,KEGG,and domain annotation)found some related proteins that have been reported to be involved in shell formation and nace color formation.Based on the standard of(|log2(Fold change)|?1.2 or?0.83 and p-value?0.05),a total of 758 differentially expressed proteins were identified(396 highly expressed in exosome of purple mussel,and 362 highly expressed in exosome of white mussel).Fourteen differentially expressed proteins were randomly selected for parallel reaction monitoring(PRM)assays,and the results were consistent with the sequencing results.The GO enrichment analysis of differentially expressed proteins found that metal ions(such as Fe,Cu,Zn,Mn,Mg,etc.)combine GO term enriched the most differential proteins,followed by carbohydrate metabolism(polysaccharide)GO term.KEGG enrichment analysis of differential proteins found that metabolism pathway enriched more differential proteins,but the enrichment degree was lower.Although the amount of differential protein enrichment in mineral absorption pathway is not large,the enrichment degree is the highest.This pathway contains three iron metabolism-related proteins.The results of enrichment analysis found that the difference structure of nacre and iron element may lead to the difference of the color of shell nacre.4.Cloning and expression analysis of genes involved in nacre color formation in H.cumingii.According to the results of proteome sequencing analysis,the HcFerritin in H.cumingii was studied from the gene level.First,the HcFerritin gene was cloned,with a gene length of 2,435 bp,encoding 172 amino acids,and containing a eukaryotic HcFerritin domain.The result of q-PCR showd that HcFerritin gene was expressed in all tissues(fringe mantle,middle mantle,adductor muscle,gills,foot,hepatopancreas and blood,and the expression was highest in the gill.Comparative analysis showed that HcFerritin expression level in fringe mantle and middle mantle of purple mussel were significantly higher than that of white mussel.The results of in situ hybridization showed that some strong signals for HcFerritin in the dorsal epithelial cells of the mantle pallial.The iron ion stress experiment indicated that iron ion can induce the expression of HcFerritin gene in H.cumingii.A coproporphyrinogen-? oxidase gene,HcCPOX was cloned and characterized from H.cumingii,which was 1477 bp in length and encoded 397 amino acids,and containing a coprogen-oxidas domain.Tissue expression results showd HcCPOX was expressed in all tissues,and the expression was highest in the gill.Comparative analysis showed that the expression level of HcCPOX in all tissues except the middle mantle in purple mussels were significantly higher than that in white mussels.The results of in situ hybridization showed that some strong signals for HcCPOX in the dorsal epithelial cells of the mantle pallial.Coproporphyrinogen-III oxidase activity was detected on the fringe mantle,gills,foot and blood.It was found that the coproporphyrinogen-III oxidase activity of gill,fringe mantle and foot in purple mussel were significantly higher than that in white mussel.In RNAi assay,the expression levels of HcCPOX in the gills,fringe mantle,foot,and blood were suppressed,while the coproporphyrinogen-? oxidase activity of these tissues were also suppressed. |
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