| Ruditapes philippinarum is one of the most important Marine shellfish in China.Shell color is an important genetic trait.Understanding its genetic mechanism can provide theoretical basis for germplasm improvement and seed production.In this paper,the extraction and determination of melanin content of clam with different shell color were carried out,and miRNA sequencing was carried out on the mantle tissue of Manilaclam with different shell color.The key miRNA related to shell color was screened out: lgi-miR-2d,and a preliminary study was conducted on lgi-miR-2d and its target gene mitf.The main results are as follows:1.In this study,the melanin was extracted by hydrochloric acid hydrolysis from the shells of R.philippinarum with four shell colors,and the melanin was quantified by tissue colorimetry,and the activity of enzymes related to melanin synthesis in the mantles of R.philippinarum with different shell colors was detected by ELISA.The results showed that the content of melanin in black clams was the highest and that in white clams was the lowest.UV scanning of the extracted initial product of melanin showed that the variation trend of its absorbance value was similar to that reported in other shellfish,and it was a typical absorption spectrum of melanin.Therefore,it was preliminarily confirmed that the extracted substance was melanin.The results of tissue colorimetry showed that the content of melanin in the outer membrane was the highest in black clams and the lowest in white clams.The activity of enzymes related to melanin synthesis was detected by ELISA in the mantle tissues of R.philippinarum with different shell colors.The activity of enzymes related to melanin synthesis was different in the mantle tissues of R.philippinarum with different shell colors.2.We used high-throughput sequencing and whole-transcriptome analysis to study the molecular mechanism of shell color formation and regulation,and constructed small RNA libraries from the mantle tissues of four strains of clams.The results showed that lgi-miR-2d and tgu-miR-133-3p negatively regulated mitf,herc2 and other melanin-related genes,respectively,which may be involved in the formation of melanin by activating the melanin synthesis pathway.In addition,aaemiR-71-5p and dme-miR-7-5p may be involved in shell formation by targeting shell formation related genes such as calmodulin and imsp3 in calcium signaling pathway.The candidate miRNAs and target genes of different shell color groups were verified by fluorescence quantitative PCR.The results showed that miR-2,miR-7,miR-71 and miR-133 may be involved in shell melanogenesisby regulating target genes such as mitf,calmodulin and herc2,respectively.3.Bioinformatics prediction method was used to analyze the relationship between lgi-miR-2d and mitf genes of other homologous species.The results showed that lgi-miR-2d seed sequence had a targeted regulatory relationship with the 3'UTR region of mitf gene of most shellfish.In vivo functional experiments verified that after injection of lgi-miR-2d antagomir,the relative expression of lgi-miR-2d in the positive treatment group showed a significant decrease(P < 0.05)compared with the negative control group and the blank control group,and the expression levels of target genes mitf,tyr,po and other shell color related genes in the post-injection treatment group showed a significant increase,but there was no significant change in the expression levels of the negative control group and the blank control group.Therefore,we speculated that lgi-miR-2d may be involved in the regulation of melanin production of R.philippinarum through the targeted regulation of Rpmitf.We further proved that lgi-miR-2d had a targeted negative regulatory relationship with Rpmitf by using a dual-luciferase reporter assay experiment.4.The cloning and expression characteristics of Rpmitf,the target gene of lgimiR-2d,were analyzed by gene cloning and q PCR,and its function was verified by RNA interference technology.The results showed that Rpmitf was 1878 bp in length,with an open reading frame of 1365 bp and encoding 454 amino acids.The 5'untranslated region(5'-UTR)and 3' untranslated region(3'-UTR)are 85 bp and 428 bp in length,respectively.The predicted molecular weight of Rpmitf is 50.95 k Da and the isoelectric point(p I)is 5.38.Sequence analysis showed that Rpmitf contained two conserved characteristic domains,MITF?TFEB?C?3?N and HLH,without transmembrane structure and signal peptide.According to the results of homology analysis,the amino acid sequence of Rpmitf had the highest homology with the mitf sequence of M.meretrix.The relative expression analysis of Rpmitf gene showed that Rpmitf was expressed in the inner membrane,outer membrane,foot,gill,digestive gland,adductor muscle and siphon of R.philippinarum.Among them,the relative expression level of Rpmitf in the inner of mantle was significantly higher than that in other six tissues.Among the five strains of R.philippinarum,the expression of Rpmitf in the inner mantle of black clams was the highest,followed by "White zebra clams" and "Zebra clams 2",and the expression of Rpmitf in white clams and orange clams was the lowest.After the ds RNA of Rpmitf was injected,the results showed that compared with the blank control group,the expression levels of melanin-related genes such as mitf and tyr did not change significantly in the negative control group,but significantly decreased in the positive treatment group,indicating that the ds RNARpmitf successfully inhibited the expression of mitf and downstream genes.In situ hybridization experiment showed that Rpmitf had positive signals in both the inner and outer mantle regions,but the signal in the inner mantle region was stronger than that in the outer mantle region. |
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