Excavation And Preliminary Verification Of Shell Color Related MicroRNAs In Manila Clam Ruditapes Philippinarum

The shell color and pattern of the Ruditapes philippinarum are abundant and exhibits complex polymorphisms.The studies have shown that the growth and stress resistance of different shell color clams are significant,and the shell color can be inherited to the offspring stably.Therefore,by studying the correlation of the molecular mechanism of shell color,the mechanism of shell color formation can be elucidated.On the one hand,it can provide favorable evidence for explaining the scientific problem of shellfish shell color formation mechanism,on the other hand,it also provides an important theoretical basis for directional breeding of clam shell color.The microRNA database of white clams,orange clams and zebra clams was constructed by high-throughput sequencing,which obtained 14,488,888 and 24,471,865(white clam);18,993,694 and 14,545,366(orange clam);25,453,550 and 9,519,203(zebra clam)clean reads of three samples.The specific sequences 1,614,570 and 1,849,627(white clam);2,208,239 and 1,125,993(orange clam);1,923,361 and 891,058(zebra clam)were obtained by comparison with the reference genome and each database.Through the comparison and annotated classification with Rfam Library,GeneBank Library,miRBase Library,RepBase Library and Exon,introns,we can get 1058 kinds of microRNAs that are already in the database.A total of 3,478 new microRNAs were predicted using mireap software.The analysis showed that the number of differentially expressed microRNAs was 10,104 and 52 for white and orange,white and zebra,orange and zebra.Target gene analysis shows that the differential expression of microRNAs target genes involved the regulation of multiple signaling pathways : Dopaminergic synapse signaling pathway,Hedgehog signaling pathway and Wnt signaling pathway.Nine microRNAs(miR-216,miR-71,miR-34,miR-193,miR-7-5P,miR-183,miR-137,novel miR-265,miR-219)that significant differences in expression detected by cervical loop real-time PCR,and found that the change of expression is basically consistent with the sequencing results.Among the differentially expressed microRNAs,miR-137 has been confirmed to be related to the body color of animals.In order to study miR-137 effect on the shellcolor of Philippine clam.In this study,injecting synthetic miR-137 agomir and antagomir into clam by means of in vitro injection.The expressions of target gene MITF and TYR of mir-137 predicted were detected by quantitative Real-time PCR.The results showed that the relative expression of target gene MITF in white clam was reduced by injecting agomir,and the relative expression of target gene MITF was increased by injecting antagomir.It was shown that the miR-137 of clams may affect the formation of shell color by signaling pathways,further affect melanin formation.Microphthalmia-associated transcription factor,MITF regulate the melanin formation of animals and affect the body color,plays an important role in the formation of shell color.In this study,a MITF gene(RpMITF)cDNA of philippine was amplified by RACE technology,and a full-length 2412 bp sequence was obtained which including the open reading frame of 1050 BP(ORF),encoding 350 amino acids,and the 3 ’ end non-coding area(3 ’-UTR)length was 1362 BP.Through RNAhybrid analysis,it was found that the range from locus 2069 to locus 2093 of the MITF 3 ’end sequence can be combined with miR-137,indicating that this gene may be one of the target genes of miR-137.Phenoloxidase(PO)is one of the important enzymes in most shellfish innate immune defense system,and participates in the formation process of melanin in the body,this biochemical reaction process plays a decisive role in the process of immune and melanism of the body,therefore,there is a certain relationship between melanin content and immune ability in invertebrates.In order to further confirm the correlation between the melanin formation process and immunity of clams,we studied the changes of phenoloxidase activity in the mantle and hemolymph tissues of three shell color Philippine clams.The results showed that after LPS injection,3 hours after the stimulation of the PO activity of the blood lymphocytes of the orange clam,and 12 hours after the stimulation of the blood lymphocyte activity of the zebra clam and white clam reached the maximum(P < 0.05);the PO activity in the mantle of orange clams and zebra clams is 6 hours after stimulation,and the PO in the mantle of white clams reaches a maximum 24 hours after stimulation(P < 0.05).After injection of PGN,the PO activity of blood lymphocytes of orange clam showed a decreasing process,reaching the lowest value at 24h(P < 0.05),zebra clam reached the maximum at 12 h after stimulation and 6h after stimulation of the white clam(P < 0.05).The PO activity in the mantle tissue of the orange clam reached a maximum at 12 h after injection and was significantly higher than 0 h(P < 0.05).The difference of phenoloxidase activity in different shell color clams after being stimulated by pathogens may be one of the important reasons for the different stress resistance of different clams.The results preliminarily confirmed the color formation and immune process of clam shell color.There is a correlation between them,which can provide theoretical support for the shell color breeding of the Philippine clam.