Study On Melanin Deposition And Hair Color Gene Screening Of Raccoon Dogs(Nyctereutes Procyonoides)

The raccoon dog(Nyctereutes procyonoides)is one of the valuable fur animals in canine.In the long-term breeding and management,the wild-type raccoon dog is brown,it has mutates into white,black,and red/brown raccoon dog successively.However,white raccoon dog cultivation quantity is less,the number of black and red/brown raccoon dog increased year by year,but the coat color changes mechanism is not fully understood,this study selected the same tire time 4 months four color outline each 24 raccoons dog,venous blood was collected to determine tyrosine enzyme activity,selected with the same tyres time his mother all three of four kinds of colour raccoon dog,collected by hair melanin distribution of microscopic observation,and determination of eumelanin and total melanin content of the guard hair and fluff,at the same time,collection of skin coat transcriptome sequencing and analysis of the transcription of this structure and analyze the differences in expression,comment out significantly differentially expressed genes related to melanin and fluorescence quantitative PCR verification,in the end,Online software based on machine learning and artificial neural network was used to analyze the primary,secondary and tertiary structures of KIT genes and encoded proteins.The results are as follows:(1)Through white,black,red/brown,and wild raccoon dog guard hair and on the microscopic observation found that the black particles of the hair of white raccoon dog were the least.The black particles of the hair of red/brown raccoon dog formed a dense black area in the fur and the pulp of the hair.The content of total melanin in the hair of white,red/brown,wild and black raccoon dog increased in turn by spectrophotometry.The content of total melanin in the hair of wild raccoon dog was significantly higher than that of white and red/brown(P<0.01),and significantly lower than that of black(P<0.01).The determination of total melanin content in the coat was in good agreement with the microscopic observation.The results showed that the tyrosinase activity of black and wild raccoon was extremely significantly higher than that of white raccoon(P<0.01),and that of red/brown raccoon dog was significantly higher than that of white(P<0.05),but there was no significant difference in tyrosinase activity between black,wild,and red/brown raccoon dog.(2)Illumina Hiseq2000 high-throughput sequencing platform was used for transcriptome sequencing of skin of the raccoon dog,the 12 samples produced about 35 Gb of Clean Data.From the skin of white,red/brown,black,and wild raccoon dog,155,222 Unigene,155,928 Unigene,122,710 Unigene,and 155,457 Unigene were obtained.All Unigene were annotated in NR,Swiss-PROt,Nt,GO,COG and KEGG databases,among which 108,988 Unigene were annotated and 64,387 coding protein frames(CDS)were annotated.Among them,61,845 CDS were compared to the protein library,but only 2,542 CDS were predicted.There are a total of 41,522 microsatellite loci.Single nucleotide polymorphism analysis showed that there were more base conversions than substitutions.DESeq2 packet of R was used to analyze differentially expressed genes in skin transcriptome of raccoon dog(|log2(foldchange)|>1,FDR≤0.05).The results showed that there were 27 differentially expressed genes between black and wild raccoon dog,among which 19 genes were up-regulated and 8 genes were down-regulated.There were 11 differentially expressed genes between red/brown and wild raccoon dog,among which 8 genes were up-regulated and 3 genes were down-regulated.There were443 differentially expressed genes between white and red/brown raccoon dog,among which 298 genes were up-regulated and 145 genes were down-regulated.According to GO and KEGG analysis,Loc101179511,MRPL54 and BAHD1 were found to be the genes related to the production of melanin in the differentially expressed genes of black and wild raccoon dog,which may increase the synthesis of actin,promote the transport of melanosome,and make the content of melanin in the coat of black raccoon dog more than that of wild.Among the differentially expressed genes of red/brown and wild raccoon dog,the genes related to melanin production are LOC482436 and IGFBP6.It may be that the differentially expressed genes affect amino acid metabolism and degradation,and then affect the synthesis of some enzymes in the melanin production process,thus reducing the melanin synthesis of red/brown raccoon dog.Among the differentially expressed genes between white and red/brown raccoon dog,there are 22 genes related to melanin production,among which TYR and TYRP1 directly affect the melanin production content,and MCIR may affect the type of melanin production.7 genes were randomly selected for q RT-PCR verification,and the results were consistent with transcript.(3)the raccoon dog KIT gene is 2,919 bp,encoding 972 amino acids,protein belongs to acidic water type,random curl as the main secondary structure,there are two areas across the membrane,membrane outer signal peptide,contains 21 sugar-based loci,60 phosphorylation sites,four conservative domain structure,closely with five protein,phylogenetic tree built "consistent with the classification of biology.In conclusion,the coat color difference between black and red/brown raccoon dog has nothing to do with tyrosinase activity.However,the white raccoon is low tyrosinase activity.The MC1 R gene maybe related to the type of melanin produced by the raccoon dog,and the TYR gene is related to the amount of melanin produced by raccoon dog.The structure of the KIT gene and the encoding protein of raccoon dog have species-specific properties.