| Hyriopsis cumingii is a unique freshwater pearl oyster in China,which can cultivate excellent pearls.There are many factors that affect the quality of pearls,of which color is one of the most important reasons,and melanin is one of the reasons that affect the formation of pearl colors.In this study,the genes Creb and PKC related to the pearl layer color of mussel were screened from the transcriptional Library of laboratory.The relationship between the two genes in the melanin synthesis process and the color of pearl layer of mussel was studied.1.Cloning a Creb gene and anayly in Hyriopsis cumingiiThe full length of the HcCreb(Gen Bank accession No.MT816340)gene was obtained by 3’and 5’RACE cloning and through the publicly available sequences.The HcCreb gene sequence is 1463 bp in total,of which the 5’-UTR was 15 bp,the 3’-UTR was 344 bp,The cds region has a total length of 1104bp,which encodes a total of 367amino acids.Its mature protein has a molecular weight of 119.63kda.The isoelectric point calculated was 4.43.HcCreb was expressed in both purple and white mussels,and there was a significant difference in expression between adductor muscle(p<0.01)and mantle tissue(p<0.05).Other tissues did not show significant differences(except for gill tissue),and in general,the gene expression level of purple H.cumingii is higher than that of white H.cumingii.Among the two colors of H.cumingii mussel,the gene expression level of gill tissue is the highest,followed by the mantle.A strong and specific m RNA signal was detected in epithelial cells on the dorsal side of the mantle,indicating that HcCreb may be involved in nacre formation.After arbutin treatment,the expression of HcCreb decreased significantly.By further testing the changes in mantle melanin content it was found that the melanin content after after arbutin treatment was significantly lower than other group(p<0.05).It is speculated that HcCreb gene may plays a certain role,in the process of melanin synthesis and nacre color formation.2.Cloning and expression analysis of protein kinase C HcPKC gene of H.cumingiiThe full length of PKC(Gen Bank accession No.MW241548)gene of H.cumingii was obtained by 3’and 5’race cloning and sequencing.The full length of the HcPKC gene is 2134 bp,including 12 bp in the 5’region and 1246 bp in the 3’region.There is an ORF region of 876 bp,which encodes a total of 291 amino acids.Its mature protein has a molecular weight of 117.04kda and a theoretical isoelectric point of 4.73.The results of fluorescence quantitative analysis showed that the expression of HcPKC in various tissues of purple mussel was higher than that of white mussel,and there was significant difference in marginal membrane.In purple mussel,the highest expression was in marginal membrane,with significant difference,in white mussel,the highest expression was in adductor muscle,with no significant difference among tissues.Strong and specific m RNA signals were detected in the dorsal epithelial cells of the mantle pallial layer,indicating that HcPKC may be involved in nacre formation.3.SNP screening of HcPKC gene and the association with nacre color trait in H.cumingiiThe full length of HcPKC gene was obtained and primers were designed in exon.Five SNPs were found after sequencing.Through the correlation analysis between these polymorphic loci and pearl layer color traits of H.cumingii,it was found that the A+332G locus of HcPKC gene was moderate polymorphic,and the other four loci were low polymorphic.There was a strong linkage disequilibrium between A+87T,G+145T,G+217T,A+328G and A+332G(D’>0.75,R~2>0.33).A total of 24 haplotypes were obtained.Among them,The frequency of T1 in the white H.cumingii group was higher than that of the purple group. |
