Functional Verification Of The Key Genes HcMPPED2 And Hc-GSK3β In Melanin Synthesis Pathway Of Hyriopsis Cumingii

Hyriopsis cumingii plays an irreplaceable role in China freshwater pearl industry.It is the highest output of freshwater pearls in China.With the rapid economic development,consumers’ demand for high-quality pearls is also increasing.The criteria for evaluating pearl quality include size,color,shape,luster,surface finish and pearl thickness.Studies have shown that the synthesis and metabolism of melanin can affect the inner shell color of pearls,which in turn affects the color of pearls.The research subjects of this study were the white mussels and the purple mussels.We found the MPPED2 gene with significantly different expression in the fringe mantle tissue of the white and purple mussels,while the GSK3β gene was in the melanin metabolism pathway.MPPED2 gene and GSK3β gene were obtained from the pearl sac transcriptome of H.cumingii.The roles of the MPPED2 and GSK3β genes in the melanin synthesis of H.cumingii and their relationship with the inner shell color and nacre color were explored.1.Cloning and functional verification of HcMPPED2 gene in H.cumingii.The full-length c DNA sequence of HcMPPED2 gene of H.cumingii was cloned by race technology,with a total of 1481 bp,including 909 bp long open reading frame(ORF),encoding 302 amino acids.In multiple amino acid sequence alignment,it had the highest sequence similarity(37.7%)with the mppede2 gene of gigatopelta aegis.The real-time fluorescence quantitative results showed that there were significant differences in the expression in the hepatopancreas,adductor muscle,and middle mantle of white and purple mussels(P < 0.05).There was significant difference in the expression of foot and fringe mantle(P < 0.01).The expression level of white mussels in fringe mantle was significantly higher than purple mussels(P < 0.01).The results of in situ hybridization(ISH)showed that a stronger specific m RNA signal was detected in the dorsal mantle epithelial cells of the fringe mantle,suggesting that HcMPPED2 may be involved in nacre formation.After 72 h of ds RNA interference treatment,the relative expression of HcMPPED2 in fringe mantle of mussels was significantly decreased(P < 0.05),and the interference rate of the interference chain on the HcMPPED2 gene reached 82.23%.Compared with the PBS group,the melanin content of the i RNA group was significantly increased 37.38%(P < 0.05).Compared with the GFP group,the PBS group significantly increased by 20.14%(P < 0.05).2.Cloning and expression analysis of Hc-GSK3β Gene in H.cumingii.In this study,the full-length c DNA of Hc-GSK3β gene was obtained by RACE with a total length of 1867 bp,including an ORF of 1261 bp encoding 420 amino acids,and a conserved S＿TKc domain in the ORF.Real-time fluorescence quantitative analysis showed that Hc-GSK3β gene was expressed in hepatopancreas,gill,foot,adductor muscle,middle mantle,and fringe mantle.There were significant differences in hepatopancreas and gill(P < 0.05).The difference was extremely significant in the foot,adductor muscle and fringe mantle,and the expression level of purple mussels in fringe mantle was higher than that in white mussels(P < 0.01).While,there was no difference in the expression in the middle mantle(P > 0.05).The results of in situ hybridization(ISH)showed that there were positive signals in the outer fold(OF),middle fold(MF),inner fold(IF),dorsal mantle(DM),and ventral mantle(VM)of the fringe mantle in H.cumingii.The signal expression in the outer fold is stronger.The expression of the positive signal has an increasingly strong trend from the epithelium of the dorsal mantle and the ventral mantle to the outer fold.No signal was found in the negative control group.3.Correlation analysis of SNP loci screening and parameters of inner shell color traits of Hc-GSK3β gene in H.cumingii.After screening,a total of 6 SNP sites were obtained in Hc-GSK3β genome sequence;C+185A site is moderately polymorphic.G+153A,T+249C,T+255G,T+341G and T+374C sites is low polymorphism.There was no difference in the b parameter of all loci(P > 0.05).C+185A,T+374C have no difference in the four inner shell color-related parameters(L,a,b,d E)(P > 0.05).G+153A,T+249C and T+374C were significant differences in L,a,and d E parameters(P < 0.05).Above expriment results have shown that the Hc-GSK3β gene has a certain correlation with the color of the nacre and inner shell of in H.cumingii.It helps further to understand the formation mechanism of inner shell color and pearl color of H.cumingii.