Study On Transcriptomes Of Different Shell Color Strains Of Manila Clam Ruditapes Philippinarum And Shell Color Related Genes Cloning And Expression Anaysis

Ruditapes philippinarum is one of the most commercially important marine shellfish in China.Shell color was used as an important trait for selection breeding of shellfish species.To investigate the molecular mechanisms underlying shell color formation and expression are significant for the shellfish genetics and breeding.In this study,we used high throughput sequencing technology to analyze thetranscriptomes of four shell color strains of R.philippinarum to identify the genes associated with shell color polymorphism.The results showed that 492,780,414 raw reads were obtained by sequencing,and 456,080,414 clean reads were obtained by screening,which was assembled into 480,552,960 transcripts and annotated in 7 databases.A total of 18 shell color related genes were selected and validated by real-time polymerase chain reaction(qPCR),ptyr,mitf1,mitf2,mitf4,adc3 and ras were involved in the melanin synthesis pathway.In R.philippinarum,the melanin synthesis pathway and melanin biosynthesis related genes play important roles in pigmentation of the four shell strains of R.philippinarum,which was closely related to color formation on shell surface.The genes heph1 involved in porphyrin and chlorophyll metabolism binding calcium signaling pathway.We infer that this pathway associated with orange shell color of R.philippinarum.Meanwhile,we used next-generation sequencing technology to analyze the transcriptomes in two different shell color strains of R.philippinarum juveniles before and after shell color formation.The results showed that 215,232,470 raw reads were obtained by sequencing,and 169,573,742 clean reads were screened to get 212,107,615 transcripts.We selected 18 genes associated with shell color formation in the juveniles of Manila clam for qPCR validation.Gene annotations show that heph and fth1 play an important role in the formation of orange clam,which are involved in the transport of iron ions in the metabolism of chlorophyll,suggesting that porphyrin and chlorophyll metabolism pathway plays an important role in the formation of background color in orange strain of R.philippinarum.Nnt gene as a melanocytic inhibitor as a nicotinamide was also found in this study.The formation of the surface stripe of the shellfish is more complex than the background color,and both eprs and got2 are down-regulated genes,acting together in the pathway "biosynthesis of secondary metabolites"(ko01110).This pathway maybe promotes the formation of stripes.In order to further explore the key enzyme for melanin synthesis,tyrosinase(tyr)gene by rapid amplification of cDNA ends(RACE)cloning technology was conducted and the full-length 2552 bp was obtained.Using the real-time quantitative PCR,the expression profiles of tyr genes in four different shell color stains and different tissues of R.philippinarum were performed.The results showed that the expression of tyrosinase genes were different in the mantle of four different shell color strains of R.philippinarum.High expression level of tyr2,tyr3,tyr6 and tyr9 were observed in the white zebra strain,which was significantly different with zebra strain and white strain(P < 0.05).The expression of tyr2,tyr6 and tyr10 in the orange strain was the highest compared to other strains,and it was presumed to be related to orange strain.The expression of tyr11 was the highest in zebra strain,significantly different with white strain(P < 0.05),which was related to the background color formation.The relationship between tyrosinase gene and melanin formation was investigated in this study and the results obtained here proved that tyrosinase gene was related to the formation of melanin.Moreover,RNA interference technology was applied to verify the tyr gene silencing in 24 h.The present study provides a reference and useful information for further research on the genetic mechanism and function of R.philippinarum.Meanwhile,we investigated the expression of mitf in different tissues and different color strains of R.philippinarum by real-time quantitative PCR.The expression of mitf1,mitf3,mitf4,mitf6 and mitf7 were higher in the orange strain,and significantly different with zebra strain(P < 0.05).The mitf1,mitf2,mitf3,mitf5 genes were highly expressed in white zerba strain,and significantly different with zebra clam and the white clam(P < 0.05).All genes have a low expression level in zebra strain.It was concluded that the mitf genes were closely related to the formation of melanin pigment.Moreover,RNA interference was utilized to explore the function of mitf gene,the result show that mitf gene silencing was found in 48 h.This work provides valuable information for mitf functional research and molecular breeding in the future.