Study On SNP And Its Expression In Skin Tissue Of Rex Rabbit

Objectives: Rex rabbit is a species cultivated by breeding of rabbit mutant individuals.Because of the excellent fur quality,particularly its rich color,Rex rabbit is deeply loved by consumers.A large number of studies have shown that coat color of Rex rabbit is determined by pigments in the body,mainly by tyrosine-derived pigments(melanin and its derivatives).The coat color is affected by the ratio and relative quantity between brown melanin and melanin.The spatiotemporal expression of the pigment also affects the shade and pattern formation of the mammalian coat color.This experiment explored the relationship between the genes involved in melanin formation(MITF,TYR,TYRP1,TYRP2,SILV,and mouse P genes)and the color of the Rex rabbit.Methods: The mutational characteristics and polymorphisms of MITF,TYR,TYRP1,TYRP2,SILV and P genes in different breeds(lines)of Rex rabbits were analyzed by gene sequence amplification and sequencing.The relative expression of the above 6 genes in the skin of Rex rabbits with different fur colors(white,black and blue)was detected by RT-PCR and Q-PCR methods at mRNA level.The polymorphisms of the hair color-related genes of Rex rabbits were combined with the relative expression levels of the above genes in skin tissues.The relationship between the target genes and the coat color of Rex rabbits was analyzed to provide a theoretic basis for the detection of the regulation mechanism of Rex rabbit hair color formation.Results:1.Polymorphism analysis(1)Two polymorphic loci were found in exon 2 of TYR gene: SNP1(885 G > A)and SNP2(886 A > C).SNP1 is a synonymous mutation,and SNP2 is a sense mutation.These two mutation resulted in the mutation of Amino Acid No.284 proline to threonine(CCC ? ACC).The chi-square test did not find that the two mutant loci genotypes were associated with the color of the rex rabbit.(2)There was one polymorphic site in exon 2 of P gene,namely SNP(670 A>G).This is a synonymy mutation and did not change the amino acid sequence.(3)Three polymorphic loci were found in exon 2 of TYRP1 gene: SNP1(570 A > G),SNP2(581 T>A),and SNP3(582 T insertion).The TYRP1 gene coding region 570 bp A>G made Tryptophan No.186 a terminator.The T >A mutation found at the 581 bp region of the TYRP1 gene coding region resulted in the transforming of No.190 from tyrosine(Tyrosine)to Phenylalanine.The insertion of the base T at the 582 bp coding region of the TYRP1 gene caused changes in the amino acid sequence of 191 and beyond.The chi-square test showed no correlation between the hair color of the Rex rabbit and the 570 bp mutation in the second exon of the TYRP1 gene(P>0.05).The 581 bp and 582 bp mutant genotypes of the second exon of TYRP1 gene were significantly correlated with white,black and cyan blue(P<0.01).2.Organization expression analysis(1)MITF,TYR,TYRP1,TYRP2,SILV and mouse P were all expressed in the skin tissues of Rex rabbits.(2)The mRNA relative expression of MITF gene was the highest in the skin tissues of black Rex rabbits,and the lowest in the skin tissues of white Rex rabbits.In addition,mRNA expression of MITF gene in skin tissues of black rex rabbits was significantly different from that of white and blue-violet rex rabbits(P<0.01).The mRNA expression of MITF gene in the skin of white rex rabbit was significantly different from that of blue-violet rex rabbit(0.01<P<0.05).(3)The relative expression of P gene mRNA was the highest in the skin tissues of blueviolet Rex rabbits,and the lowest in white Rex rabbits.In addition,the mRNA expression of P gene in the skin tissues of blue-violet Rex rabbits was significantly different from that of white Rex rabbits(0.01<P<0.05),but not significantly different from that of black Rex rabbits(P>0.05).There was no significant difference in mRNA expression of P gene between blueviolet rex Rabbit and white Rex rabbit.(4)The relative mRNA expression of TYR gene was the highest in the skin tissues of blue-blue Rex rabbits,and the lowest in white Rex rabbits.In addition,the mRNA expression of TYR gene in the skin tissues of blue-violet rex rabbits was significantly different from that of white rex rabbits(0.01<P<0.05),but not significantly different from that of black rex rabbits.There was no significant difference in mRNA expression of TYR gene between blue-violet Rex rabbits and white Rex rabbits(P>0.05).(5)The relative expression of TYRP1 mRNA in the skin of black Rex rabbits was the highest,while in the skin of white Rex rabbits it was the lowest.The m RNA expression of TYRP1 gene in skin tissues of black rex rabbits was significantly different from that of white and blue-violet rex rabbits(P<0.01).There was no significant difference between white rex rabbits and blue-violet rex rabbits(P>0.05).(6)The relative mRNA expression of SILV gene in the skin of black Rex rabbits was the highest,while in the skin of white Rex rabbits it was the lowest.The mRNA expression of SILV gene in skin tissues of black rex rabbits was significantly different from that of white and blue-violet rex rabbits(P<0.01).There was no significant difference between white rex rabbit and blue-violet rex rabbit(P>0.05).(7)The relative expression of TYRP2 mRNA was the highest in the skin of black Rex rabbits,and the lowest in the skin of white Rex rabbits.The mRNA expression of TYRP2 gene in skin tissues of black rex rabbits was significantly different from that of white and blueviolet rex rabbits(P<0.01),and was significantly different(P<0.01)between white Rex rabbits and blue-violet Rex rabbits.Conclusion:Six genes related to fur color may be involved in the formation and regulation of fur color.The specific influence mechanism needs further research;TYR,TYRP2 and P genes with key mutation sites may be the key factors in the fur color formation process of rex rabbits,and can be used as candidate genes to study the fur color formation mechanism.