Study Of Structure And Evolution And Its Regulating Mechanism Of Agouti Gene In Mink

The coat color is a key character of mink,which is an economical animal.Agouti gene regulates pigmentation producing switching from eumelanin to phaeomelanin and forming colorful coat.However there are few researches about the genetic of mink coat color.Agouti gene,as the locus for the wild coat color,and other genes for pigment synthesis in mink are on the progress.Black mink,coffee mink and white mink were used for the clone,character of structure and evolution of gene of Agouti.Also RNA-Seq technology was used for skin of black mink and coffee mink.5’ UTR of DCT and Tyrp1 gene was cloned and the promoter activity was detected.It was aimed to find the genetic base for coat color of mink and to support the breeding exercises for the colorful coat of mink.Agouti gene of mink long for 25 478 bp contained the whole coding region(KP981640)was cloned by PCR amplification and sequencing.It was found that it contained 53.9 % transposons elements,34.5 % palindrome structure,tandem repeats and SSR and so on,which formed the complicated secondary folding structure.Intron 3 of Agouti gene of mink contained a SINE insertion mutation long for 601 bp.This locus was in Hardy-Weinberg equilibrium in coffee mink and white mink,but not in black mink.The insertion genotype frequency in black mink was lower than coffee mink and white mink(P < 0.05).The SINE deletion was the superior genotype.The promoter activity was found in SINE insertion mutation fragment long for 1605 bp in forward direction(P < 0.05),while the deletion mutation fragment long for 998 bp in both directions had no promoter activity(P > 0.05),either the 1605 bp fragment in reverse direction.The SINE long for 601 bp was found to be transcribed in transcriptome data as a transcript long for 973 bp,while the transcript of Agouti gene was not found.The genetic diversity and evolution was analyzed of 112 Agouti gene sequences and 172 ASIP amino acid sequences belonging to 38 species and 51 mammals respectively.The highest genetic diversity(0.142 and 0.249,θη)and evolution rate(0.230 and 0.294,Dxy),and the lowest conservation(0.590 and 0.582,C)were found in Teleostei and Neognathae.The lowest genetic diversity(0.010～0.017,θη)and evolution rate(0.012～0.017,Dxy),and the highest conservation(0.940～0.982,C)were found in the high Haplorrhini.Agouti gene mainly evolved under negative selection during the differentiation of 51 mammals.It evolved under negative selection in Neognathae,and 2 negative selection amino acid sites were found in ASIP.However positive selection and 2 positive selection amino acid sites were found in Cetartiodactyla.Positive selection and negative selection were also found in Muroidea and Haplorrhini,respectively.18 conservative sites were found in 131 ASIP sequences of 51 mammal-species spaning from mouse to human.The signal peptide had the high evolution rate(1.084)and the loop region had the high conservation(88.31 %).The transcriptome data of skin were analyzed for the black and coffee mink of 1 month and 6 month.8145 Unigenes were expressed differently in mink of 6 month,of which 2006 Unigenes were up-expressed in coffee mink and 6139 Unigenes were down-expressed.The genes that expressed differently were enriched in Notch pathway and had the higher ratio to the background genes,also were enriched in the two Go items of keratin filament and intermediate filament and the enrichment ratio was higher than 0.6.The items of melanin biosynthetic process,developmental pigmentation and melanosome membrane were enriched in mink of 1 month,while the enrichment of hair follicle development was significantly lower than the others.Mink of 6 months was also enriched in melanin biosynthetic process as mink of 1 month,but it was enriched in hair cell differentiation and hair follicle development items,and the enrichment of melanosome item was significantly lower than the others.MYO5 A,Tyrp1,PMEL,MC1 R,SLC45A2,TYR and OCA2 were differently expressed in black and coffee mink skin.5’UTR region of DCT gene of mink long for 8203 bp was sequenced(KY412850-KY412852).36 SNPs and 1 transposon element long for 204 bp were found in it.The transposon,named can-SINE special in Carnivora,was inferred to come from Soboliphyme baturini and intrude into genome after the differentiation of Caniformia.The 32 bp element located-392～-361 upstream of transcription start site,the proximal region structure and other cis-elements concurrent played a role in promoter activity,while CpG islands located-797～-688 and GC box located-636～-608 silenced DCT gene transcriptions.The TATA boxes located-49～-39 and-31～-24 regulated the basic transcription of mink Tyrp1.The enhancer located-277～-60 improved the transcription of Tyrp1.The transcription activity of Tyrp1 was significantly higher in 293 T cells than in A375 cells.It was concluded that the SINE inserted in intron 3 with promoter activity resulted in transcript silence of Agouti gene of mink.The polymorphism and differentiation of Agouti gene was correlated with the evolution of species,also the richness of coat color.Positive selection or negative selection was found in Agouti gene to adapt to the nature or artificial breeding,but it was mainly in negative selection.The differential expression genes of Black mink and coffee mink of both 1 month and 6 month were enriched in Go item of pigment biosynthesis.The regulation elements in downstream of the locus of-392 cooperated to initiate the transcript of DCT gene,while the CpG islands and GC box in locus of-797～-608 inhibited the transcript of DCT.The region downsteam of-60 of Tyrp1 was sufficient to intiate the transcript of Tyrp1.