Identification And Functional Analysis Of Pigment Related Genes HcMitf And HcUros In Hyriopsis Cumingii

Hyriopsis cumingii is the most important freshwater pearl mussel in China and has high economic value.The color of pearl directly affects the pearl value.Previous studies have shown that the color of the pearl is related to the melanin synthesis.At the same time,it is also found that the presence of porphyrin in different color pearls is one of the important substances affecting the pearl color.In this study,the laboratory-constructed purple and white line mussels were selected as the research objects,and microphthalmia-associated transcription factor gene and uroporphyrinogen ? synthetase gene were identified from the transcriptome,detecting the relationship between these two genes and the color of the pearl nacre.The main findings are as follows: 1.Cloning and expression analysis of HcMitfThe HcMitf gene was obtained by molecular cloning technology and was 2664 bp in length,containing a 1332 bp ORF encoding 443 amino acids.The amino acid sequence of the HcMitf gene contains a helix-loop-helical domain(bHLH)with no signal peptide and GenBank accession number was MH349354.The HcMitf was found to widespread tissue distribution but expression was higher in purple mussels than in white mussels,mostly in mantle,liver,kidney,gill,and foot with the exception of the adductor mussel(P<0.01).In situ hybridization results indicated that HcMitf was expressed in the dorsal epithelial cells of the mantle pallial.2.The function of the HcMitfUsing RNA interference,the expression of HcMitf was reduced by 78%(P < 0.01)and expression of HcTyr was also significantly suppressed and consequently total melanin content was decreased(P < 0.05).After implantation,the expression of HcMitf and HcTyr reached a peak at 6h and returned to normal at 96 h.Subsequently,the expression level of HcMitf was significantly increased at 14 d compared with the normal value,while the HcTyr expression level was significantly different at 35 d.Linear correlation coefficient(R= 0.919)suggested that the expression vs.time curves of these two genes are correlated.3.Cloning and expression analysis of HcUrosThe HcUros gene was obtained by molecular cloning technology and was 1448 bp in length,containing a 858 bp ORF encoding 443 amino acids(GenBank accession no.MK599473).The amino acid sequence of the HcUros gene contains a HemD domain with no signal peptide.The result of q-PCR showed that HcUros was found to widespread tissue distribution but expression was higher in purple mussels than in white mussels,mostly in haemolymph and mantle.In situ hybridization results indicated that the HcUros gene had a positive hybridization signal in the epithelial cells of the mantle pallial.4.The function of the HcUrosFirstly,the regulation relationship between HcUros gene and downstream HcPpo gene in porphyrin synthesis pathway was explored by RNAi technology.By injecting the dsRNA of HcUros,the results showed that the expression levels of HcUros gene in the negative control group did not change significantly compared with the blank control group,but the expression level of the experimental group was rapidly decreased by 62.5%.(p < 0.01),indicating that theexpression of HcUroswas successfully inhibited,and the downstream gene HcPpo expression was also significantly reduced.Secondly,the insert surgery was used to explore the expression of HcUrosduring pearl formation.After implantation,the expression of HcUros decreased significantly at 3h and then obviously increased to 48 h and 96 h,and returned to normal at 7d.Finally,the expression reached the highest at 28 d.