The Sequencing Of MRNA And LncRNA Reveal The Molecular Regulation Mechanism Of Melanin Deposition In Skin Tissue Of Minxian Black Fur Sheep

Minxian Black Fur(MBF)sheep is one of the endangered local sheep breeds in our country and it was famous for producing black fur in the early period.MBF sheep have prominent black traits,including black hair,skin,eyelids,lips,oral mucosa and tongue,etc.The special phenotype is due to the difference in melanin deposition between MBF sheep and general sheep breeds,and the fundamental reason for the difference in phenotype lies in the difference in gene regulation and genetic basis.In this study,based on the characteristics of MBF sheep in terms of black phenotype,we used MBF sheep as the experimental animal group,and the Small Tail Han(STH)sheep with significant phenotypic difference as the control group.Melanin content and histological morphology was macroscopic inspection and analysis(n=6),and then performed transcriptome and lnc RNA sequencing to explore the regulation mechanism of melanin deposition represented by skin tissue in MBF sheep(n=3),and further detect the concentration of melanin synthesis precursors and key enzymes in the synthesis process(n=6).Focusing on melanin,it comprehensively analyzes the regulation mechanism of melanin deposition in MBF sheep.This study provided a theoretical basis for the cultivation and improvement of characteristic traits of MBF sheep,and then provided assistance for the variety protection of MBF sheep.At the same time,the study constructed and expanded the database data related to MBF sheep’s omics.The main results were as follows:(1)Melanin has an extraordinarily wide distribution in MBF sheep and STH sheep,and the melanin content in each tissue of MBF sheep is significantly higher than that of STH sheep(P < 0.01).The distribution trend of melanin was different in MBF sheep and STH sheep,multiple comparisons results showed that the liver,tongue and skin exhibit higher melanin content,while the bone and fat exhibit lower melanin content in MBF sheep(P < 0.05);the liver and heart exhibit lower melanin content,while the skin,bone and muscle exhibit lower melanin content in STH sheep(P<0.05).(2)The skin tissues of MBF sheep and STH sheep were basically similar in structure,and there were no significant difference of skin epidermal thickness between MBF sheep and STH sheep(P > 0.05),but the skin thickness and dermis thickness of MBF sheep were significantly lower than that of STH sheep(P < 0.01).(3)Melanin staining results showed that the distribution of melanin in the skin tissues of MBF sheep and STH sheep was obviously different,the melanin was mainly distributed in hair follicles and epidermis in the skin tissue of MBF sheep,while the distribution of melanin in the skin tissue of STH sheep was less,and it is mainly distributed in the dermis.(4)The RNA-seq technology was used to detect m RNA and lnc RNA in the skin tissues of MBF sheep and STH sheep,then we obtained a total of 103.18 Gb clean data and the data size of each sample reached 16 Gb.The Q30 scores were greater than 92.62% for each sample and the unique mapped reads ratios ranged from 75.61 to 79.97%.The results showed that the sequencing and mapping results were relatively good.(5)The total number of genes obtained by RNA-seq analysis was 28480,and the number of new genes was 10015,while the number of known genes was 18,465.The number of differentially expressed genes was 133 by quantitative analysis of gene expression,and there were 78 up-regulated genes and 55 down regulated genes.Enrichment analysis of differentially expressed genes showed that the differentially expressed genes were significantly enriched in 37 GO functional terms,including 28 BP terms and 9 CC terms;the differentially expressed genes were significantly enriched in two KEGG signaling pathways,including “Tyrosine metabolism signaling pathway” and “Melanogenesis signaling pathway”.(6)We observed a total of 32693 lnc RNAs and 15961 of these lnc RNAs have46716 target genes.There were 195 differentially expressed lnc RNAs were detected,of which 102 were up-regulated and 93 were down-regulated in MBF sheep.The target genes of differentially expressed lnc RNAs were significantly enriched in two KEGG signaling pathways,including “NF-kappa B signaling pathway” and“Cytokine-cytokine receptor interaction signaling pathway”.(7)After analysis,it was determined that there were nine key genes regulating the melanin deposition of MBF sheep,including TYR,TYPR1,TYPR2(DCT),PMEL,GPR143,MC1 R,OCA2,SLC45A2,MLANA.(8)The concentration of melanin precursor(tyrosine)in skin tissue of MBF sheep were significantly higher than that of STH sheep(P < 0.05),the concentration of three key enzymes(TYR,TYRP1 and TYRP2)in skin tissue of MBF sheep were significantly higher than that of STH sheep(P < 0.01).The concentration of the three key enzymes(TYR,TYRP1 and TYRP2)were consistent with the expression level of the corresponding genes in MBF sheep and STH sheep,and the results further explained that TYR,TYRP1 and TYRP2 play a key role in the melanin deposition process of MBF sheep.Through this research,we initially explored the content and distribution of melanin in MBF sheep and STH sheep,and we also preliminarily determined that the key signaling pathways of regulating melanin deposition in MBF sheep were“Tyrosine Metabolism signaling pathway” and “Melanogenesis signaling pathway”.The key regulatory genes involved melanin deposition in MBF were TYR,TYPR1,TYPR2(DCT),PMEL,GPR143,MC1 R,OCA2,SLC45A2,MLANA.The tyrosine gene family was the most direct gene in the process of melanin synthesis or black pattern regulation in MBF sheep.