| Yufen 1 is a native egg-laying chicken breed,which was led by Henan Agricultural University,and jointed together with Henan Sangao Agricultural and Animal Husbandry Co.Ltd.and General Department of Henan Animal Husbandry.Line H was fast plume,which was characterized as early maturing,high egg production and carrying the sex-linkage gene of Columbia feather.The genes and proteins related to phenotypic variation of feather color traits in H Line of yufen 1 egg-laying chicken were identified,and the causes of phenotypic variation of feather color in yufen 1 were analyzed from the perspective of gene expression and post-transcriptional regulation,so as to reveal the molecular genetic mechanism of feather color formation and provide a basis for further breeding and promotion of yufen 1 egg-laying chicken.In vitro tyrosine treatment of chicken melanocytes was used to validate and analyze the selected color-related genes.The main results of this study are as follows: 1.Transcriptome analysis of the expression of feather color-related genes in feather regeneration of the line H of yufen 1 egg-laying chickenThe 20 W chickens of yufen no.1 layer were plucked,after 14 days,3 chickens were selected and the dorsal hair follicles(group A,black and white striated feather color on the dorsal side of the neck)and ventral hair follicles(group B,white feather on the ventral side of the neck)were collected for RNA-seq.21,306 genes were identified by high-throughput transcriptome sequencing(RNA-seq)using hair follicles of different neck parts of the line H of yufen 1 egg-laying chicken(group A was the color of black and white stripes on the dorsal side of the neck and group B was the color of white feathers on the ventral side of the neck).Of these,597 are new genes.After the analysis between group A and group B,there were a total of 209 DEGs,including 83 up-regulated DEGs and 126 down-regulated DEGs.Differentially expressed genes involved in pigmentation mainly enriched pathways involved include Melanogenesis,MAPK pathway,c AMP,PI3K-Akt,c GMP-PKG,Adrenergic signaling in cardiomyocytes and Calcium signaling pathways.The DEGs enriched in Melanogenesis pathway are ASIP、KITLG、PVALB,FZD10,WNT7 B,WNT9A,WNT9 B,WNT11,LOC396531,CAMK2 A and CAMK2 B.The DEGs associated with pigmentation were found to be SLC45A2,RAB38 and MED23.The expressions of SLC45A2,MED23,PVALB,WNT7 B,WNT11 and FZD10 were significant difference between the two groups by q RT-PCR(P<0.05).2.Proteomic analysis of the expression of feather color-related proteins in feather regeneration of the line H of yufen 1 egg-laying chickeniTRAQ high-throughput sequencing was performed on the same hair follicle tissue which artificial feather removal process after feather regeneration as the transcriptome to find the DEPs related to the feather color of line H of yufen 1 egg-laying chicken.A total of 382 DEPs were identified,including 160 up-regulated DEPs and 222 down-regulated DEPs.In up-regulated DEPs,RAB23 was found to be related to the synthesis of melanin,and four DEPs,including DVL3,KRAS,MXRA8 and LOC107052863,were enriched in Melanogenesis pathway.In down-regulated DEPs,GNAQ is related to the synthesis of melanin.In this experiment,GNAQ,PVALB,PLCB1,CAMK2 A and CAMK2 B were found to be enriched in pigment synthesis in Melanogenesis pathway.3.Combined transcriptome and proteome to analyze the effect on feather color related genes/proteins in feather regeneration of the line H of yufen 1 egg-laying chickenCorrelation analysis of 382 DEPs and 209 DEPs revealed that 49 genes had significant differences in m RNA and protein levels.Among them,PVALB,CAMK2 A and CAMK2 B gene was significantly different in m RNA level and protein level.DEGs and DEPs were enriched in Melanogenesis,calcium,c GMP-PKG and adrenergic in cardiomyocytes signaling pathway,these pathways are associated with pigmentation.Five DEPs(PLCB1,LOC107052863,PVALB,CAMK2 A,CAMK2B)and 11 DEGs(ASIP,KITLG,PVALB,FZD10,WNT7 B,WNT9A,WNT9 B,WNT11,LOC396531,CAMK2 A,CAMK2B)are enriched in Melanogenesis pathway.4.Effect of adding tyrosine in dietary on the expression of feather color related genes in line H of yufen 1 egg-laying chickenCompared with the control group,the addition of different levels of tyrosine(0.4%,0.6%,0.8% and 1.0%)in the diet could increase the content of tyrosinase in serum of the line H of yufen 1 egg-laying chiken.Compared with the control group,Line H chicken feathers with 0.8% and 1.0% tyrosine were significantly darker in color(P<0.05).These results indicated that adding tyrosine into the diet could increase the content of tyrosinase in serum,and promote the deposition of melanin in feathers of the line H of yufen1 egg-laying chicken.Using high-throughput sequencing technology,RNA-seq analysis was performed on the dorsal hair follicles(TB)and ventral hair follicles(TF)of h-line chickens in the treated group and the CB and CF in the control group respectively.A total of 18,360 genes were identified and 775 new genes were predicted.Analysis between CB and TB groups revealed 659 DEGs,including 253 up-regulated DEGs and 406 down-regulated DEGs.Analysis between CF and TF groups revealed a total of 501 DEGs,including 250 up-regulated DEGs and 251 down-regulated DEGs.The pathways associated with pigment deposition in which the DEGs are mainly enriched include: Melanogenesis,MAPK pathway,c AMP pathway,p I3K-Akt pathway,CGMP-PKG signaling pathway,Adrenergic signaling in cardiomyocytes,Wnt signaling pathway,m TOR signaling pathway,Jak-STAT signaling pathway and Calcium signaling pathway.DEGs“EDNRB2,GNAQ,WNT3,WNT11,PRKCB,WNT16,CALM and PVALB ” in the Melanogenesis pathway.After q RT-PCR,EDNRB2,GNAQ,WNT3,WNT11,PRKCB and PVALB were significantly different in the CB and TB groups,and GNAQ and WNT3 were significantly different in the CF and TF groups.5.Spatiotemporal expression analysis of candidate genes related to feather color in line H of yufen 1 egg-laying chickenRNA-seq and iTRAQ sequencing were performed on the hair follicles of line H of yufen 1 egg-laying.The temporal and spatial expression analysis of candidate genes SLC45A2,MED23,EDNRB2,PRKCB,GNAQ,WNT3,WNT7 B,WNT11,FZD10 and PVALB in line H of yufen 1 egg-laying chicken aged 0 to 12 weeks was found SLC45A2 absolute expression was highest,and the relative expression was highest at 8 weeks.The relative expression of MED23 also reached the highest at 8 week.By analyzing the temporal and spatial expression profiles of hair follicles in line H of yufen 1 egg-laying chicken with different growth times,it was found that the expressions of EDNRB2 and PRKCB reached the highest value at 10 W,and WNT7 B,WNT11 and FZD10 reached the highest value at 8W.The experiment confirmed that SLC45A2,MED23,EDNRB2,PRKCB,WNT7 B,WNT11 and FZD10 genes were related to the feather color formation of the line H of yufen 1 egg-laying chicken.6.Effects of tyrosine treatment on melanin synthesis related genes in chicken melanocytesTo test whether candidate genes SLC45A2,MED23,EDNRB2,PRKCB,FZD10,WNT11 and WNT7 B are involved in the synthesis of melanin,chicken melanocytes were treated with different concentrations of tyrosine,we found that at 10-6 mol/L,m RNA expressions of EDNRB2 and PRKCB genes in cultured chicken melanocytes were the highest,and there were significant differences from treatment groups(0 mol/L),10-9 mol/L,10-8 mol/L and 10-7 mol/L(P<0.05),at 10-8 mol/L,SLC45A2 gene expression was significantly increased,which was significantly different from the control group(0 mol/L),10-9 mol/L,10-7 mol/L and 10-6 mol/L;at 10-7 mol/L,the expression of MED23 gene was the highest,which was significantly different from the control group(0mol/L),10-9 mol/L,10-8 mol/L and 10-6 mol/L.It has been proved that the expression of melanin-related genes in chicken melanocytes can be changed by adding tyrosine in vitro.The experiment proved that SLC45A2,EDNRB2,PRKCB and MED23 genes were related to the synthesis of melanin,which may be related to synthesis of melanin in melanocytes.The regulation of plumage is complex and is controlled by multiple genes.The line H Columbia plumage color of yufen 1 egg-laying chicken was a sex-linkage trait.We screened out the differentially expressed genes related to feather color in the hair follicle growth stage and the genes related to pigment synthesis in the black part of the feather under the condition of tyrosine treatment through two groups of treatment experiments(plucking treatment and tyrosine addition treatment).SLC45A2,MED23,EDNRB2,PRKCB,WNT7 B,WNT11 and FZD10 genes consistent with moult and feather color stability were determined according to moult characteristics at different growth and development stages of chicken feathers.Finally,functional validation of these genes was performed on tyrosine treated melanocytes,confirming that SLC45A2,EDNRB2,PRKCB and MED23 genes were involved in melanin synthesis and were associated with feather replacement and feather color stabilization.Based on the comprehensive analysis,we believe that SLC45A2,EDNRB2,PRKCB and MED23 may be related to the color regulation of the line H of yufen 1 egg-laying chicken. |
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