Identification And Regulation Mechanism Of Candidate Genes For Melanin Deposition In Skin Of Xichuan Black Bone Chicken

The Xichuan black bone chicken as a rare local chicken species is mainly distributed in Xichuan County,Henan Province,China.This variety has the typical five black characteristics?i.e.beak,skin,bones,legs and meat five parts of the body?,and it is an important genetic resource in black bone chickens.With the attention of "green food" and "black food",black bone chicken has become the first choice for green and healthy food,making the market demand for meat,egg and medicinal trinity in the Xichuan black bone chicken higher and higher.When consumers buy black-bone chickens,they generally observe the skin color of black-bone chickens as a basis for purchase.It is known that skin color and melanin content of black bone chicken are closely related to their product value and medicinal effects.The formation of melanin deposition in black bone chicken is catalyzed by tyrosinase.In black bone chicken,there may be a coordinated regulation mechanism between tyrosine metabolism,gene and melanin deposition,regulating skin tissue melanin deposition.High-throughput sequencing technology and exogenous additives are used to screen key genes regulating skin melanin deposition,also inside body and outside body experiments are carried out to study the regulation of nutrient level expression of selected genes,exploring the relationship between nutrient,gene expression and melanin deposition.It lays a foundation for improving the molecular mechanism of pigmentation formation in poultry,and also provides more research targets and ideas for the study of nutrient additives in the actual production to control the back color of Xichuan black bone chicken.The main results are as follows:Part ? Screening candidate genes related to skin color of Xichuan black bone chicken based on whole genome resequencingFive Xichuan black bone chickens were whole genome resequencing,we also downloaded the public available sequence data of 24 individuals.Principal component and population structure analysis showed that Xichuan black bone chicken is in a distinct clade apart from eight other breeds.Linkage disequilibrium analysis showed that the selection intensity of Xichuan black bone chickens is higher than for other chicken breeds.We focused on the selection signals of black chickens.Fst analysis identified 1,469 genes were selected and pathway annotations were performed on these genes.The results showed that these selected genes were mainly enriched in the Neuroactive Ligand-Receptor Interaction Pathway,cAMP Signaling Pathway,Purine Metabolism,Glyceride Metabolism,Wnt Signaling Pathway.EDN3,SLM02,ATP5e and LOC101747896 in the 10.74Mb-10.89Mb range of chromosome 20 were the strongest selection.LOC101747896 is a new long noncoding RNA located near EDN3,SLM02 and ATP5e.Phylogenetic tree analysis of the EDN3 and LOC101747896 genes indicated that the skin-related chicken?Silky fowl chicken,Dongxiang blue-eggshell chicken and Xichuan black bone chicken?were clustered on one large branch.It further indicates that LOC101747896 may have an effect on melanin deposition.Part ? Screening candidate genes related to skin color of Xichuan black bone chicken based on transcriptome sequencingUsing NR-type color difference meter to detect the color of the yellow skin and black skin of the Xichuan black bone chicken,the L value of the balck skin is lower than that of the yellow skin;the HE staining of the yellow skin and the black dorsal skin shows that the basal layer and hair follicle of the black dorsal skin has a large amount of elanin particles.The transcriptome was sequenced on the skin of 1 day old yellow and black dorsal skin Xichuan black bone chickens,and 6 mRNA libraries of different dorsal skins of Xichuan black bone chicken were constructed.17,148 mRNAs were screened,and 16,608 mRNAs were found in yellow skin and black skin expression.345 and 195 mRNAs were specifically expressed in the yellow skin and the balck skin.Differential expression analysis of mRNAs in the skin tissues of yellow and black dorsal skin chicken.The results showed that there were significant differences in the expression levels of 171?147 up-regulated and 22-down-regulated?mRNAs in the tissue of yellow skin and the black skin?Fold-change ? 2,and P-value?0.05?.The CNC,CPAT,CPC and Pfam analysis software were used to screen lncRNAs on the skin of Xichuan black bone chicken,and 1,089 lncRNAs were obtained.Among them,56 IncRNAs were differentially expressed?Fold-change? 2,and P-value?0.05?.We mainly focused on the melanogenesis pathway.In the gene expression analysis,the expression of six genes?DCT,TYR,EDNRB2,MClR,cAMK and KIT genes?in the black tissue was significantly higher than that in the yellow tissue?P<0.05?.We constructed a functional lncRNA-mRNA co-expression network.interaction analysis revealed that the known melanin genes differentially expressed on chromosome 20,such as EDN3,SLMO2 and ATP5e,were located near TCONS₀0054154 and the expression pattern was positively correlated TCONS 00054154 may have the key role in the melanin deposition in black bone chicken skin and providing a theoretical basis for studying the role of IncRNA in chicken pigmentation from transcriptome levels.Whole genome resequencing of black bone chickens was selected from 1,469 genes and transcriptome sequence analysis of 171 gene-related differential expressions of yellow skin and black skin,indicating that 13 genes were shared,including EDN3,SLM02,ATP5e and LOC101747896 genes,further explanation that these genes may be involved in pigmentation.Part ? Study on biological function of non-coding RNA LMEPBLAST sequence alignment of LOC101747896 and TCONS54154 using the NCBI database,LOC101747896 and TCONS54154 were both on chromosome 20,and transcript variant X5 sequences of TCONS54154 and LOC101747896 were 100%similar.Bioinformatics analysis LMEP is located on chromosome 20,and the RACE assay yields a full-length of 3093 nt of LMEP sequence which containing six exons.Prediction of coding ability showed that it was non-coding RNA and accorded with the characteristics of IncRNA.Melanocytes of Xichuan black bone chicken were isolated and cultured in vitro by protease ? and trypsin digestion.The morphological observation of the cells showed that the melanocytes were triangular,fusiform or dendritic.The results of melanocyte growth curve showed that the growth of melanocytes was slow in the early stage,and it increased exponentially after 7 days and the proliferation ability of melanocytes decreased after 10 days.The isolated melanocytes were identified by dopa staining,cell immunofluorescence staining and transmission electron microscopy.The results showed that the melanocytes of Xichuan black bone chicken were successfully isolated.Subcellular localization LMEP is mainly located in the cytoplasm.After treatment of melanocytes with different concentrations of tyrosine,the expression of LMEP increased significantly with the increase of tyrosine concentration?P<0.05?.The expression levels of LMEP in 20-week-old skin tissue treated with different concentrations of tyrosine showed that the expression level of LMEP increased with the increase of tyrosine concentration compared with the control group,but the difference was not significant?P>0.05?.After over-expression or interference with LMEP,the intracellular tyrosinase content was measured.Compared with the control group,the intracellular tyrosinase content was significantly increased after overexpression of LMEP?P<0.05?.After interfered with LMEP,intracellular The tyrosinase content decreased?P<0.05?.It is further explained that LMEP may play an important role in the pigmentation process and is the focus of subsequent research.Part ? Effect of exogenous addition of tyrosine on LMEP gene expression and melanin depositionCell level testing showed that adding different concentrations of tyrosine?0,10-9,10-8,10-7,10-6 mol/L?could affect the morphology and content of tyrosinase in melanocytes for 72 hours.However,when adding 10-6 mol/L,the number of cells increased significantly,the cell obviously became larger,and the content of tyrosinase was the highest.When the concentration was 10-9 mol/L,melanocytes could proliferate significantly?P<0.05?,and decreased with the increase of tyrosine,but higher than the control group,which indicating that tyrosine can promote the proliferation of melanocytes,but there is a dose-dependent effect.Cell level was used to detect the expression of pigment-related genes in different treatment groups.The results showed that different concentrations of tyrosine had significant effects on the expression of pigment-related genes compared with the control group?P<0.05?,further indicating exogenous Nutritional additives can affect the expression of pigment-related genesSix groups of Xichuan black bone chickens aged 0-20 weeks were fed with different levels of tyrosine supplementation.They were divided into control group?0%?and treatment group ????0.2%,0.4%,0.6%,0.8%,1.0%?.The tyrosine content in serum and skin and skin color of 20-week-old Xichuan black bone chicken were significantly affected by the addition of 0.8%tyrosine in vivo?P<0.05?.Compared with the control group,the expression of pigmentation-related genes increased in 20-week-old dorsal skin tissue,but the difference was not significant?P>0.05?.Indicating that pigmentation was caused by multiple factors,and more investigated were needed for subsequent researchTo sum up,integrated genome resequencing and transcriptome sequencing,screening for genes related to pigment synthesis,and verifying at the cellular and individual levels,LMEP can increase the content of tyrosinase.Different additions of tyrosine can promote the increase of LEMP expression,and there is feedback between LMEP and tyrosine.The study adds exogenous nutrients to affect gene expression.To provide a scientific basis for the optimum amount of tyrosine in the actual production and the rational use of tyrosine in the diet of Xichuan black bone chicken.It also deepens the understanding of the molecular regulation mechanism of melanin deposition in black bone chickens,and provides a basis for nutrient regulation and genetic breeding related to melanin deposition in Xichuan black bone chicken.