Mechanism Of Slc7a11 In The Melanin Deposition Of Rex Rabbit Fur

Cystine/glutamate translotacor belongs to solute carrier family 7 member 11,encoding cystine/glutamate transporter xCT and affecting the ratio of eumelanin to phaeomelanin by controling production of phaeomelanin,which lead to changes in animal hair color and skin color.In our previous study,it was found that the expression levels of Slc7a11 differed significantly in the skin of rex rabbit with different coat colors,so we postulate that Slc7a11 gene is likely to be involved in the process of pigmentation.For further understanding the mechanism of molecular regulation in the process of pigment deposition.This research separated and identified melanocytes first.real-time PCR was used to detect the expression pattern of Slc7all in the skin of rex rabbit with different coat colors.Dual luciferase assay and EMSA wes performed to regulatory effect of transcription factor FOU2F1 on trans-activation of Slc7all promoter.The result of this study lays the foundation expanding the function of Slc7a11 gene in the production of melain and in-depth analysis of pigmentation mechanism.Main results for this study are as follows:1.Separating and identify melanocytes for the first time,all these provide experimental materials for the research of project.The back skin was harvested from black rex rabbits and rabbit skin melanocytes were obtained by the two step digestion method,which showed typical characteristics with multi-dendritic or bipolar shape,thin and long dendritic cells and special outgrowth.Dopa staining were found in the melanocytes present brownor or black particle.and the positive immunocytochemical signals were found for S-100,TYR,TYRP1 in melanocytes.All the above supported melanogenic traits of melanocytes,compared with control group,the positive staining for s-100 was brown in the cutoplasm,dendrite of melanocytes,The positive staining for TYR was claybank in the nucleus and TYRP1 was pale claybank in the melanocytes,Positive results are successfully isolated and identify rabbit melanocytes.2.Clone Slc7a11 gene sequence using RACE system.The rex rabbit of Slc7a11 cDNA sequence includes 31 bp 5’-UTR,1509 bp ORE and 132 bp 3’-UTR(contains ploy[A]tail),committed sequence to GenBank(GenBank number:KY971639.1).The localization of Slc7a11 gene in skin was tested by immunohistochemistry and the results revealed that it expressed in epidermis,follicles,hair root sheach of rabbit skin.The dye frome light to deep and is expressed substentively in skin.real-time PCR was used to detect the mRNA expression levels of Slc7a11 was highest in yellowish-brown skin,that is 3.7 times the white skin;Slc7a 11 protein is analysed in different hair colour skin tissu by Wes system,the resultes reveal that it can express in all skin tissue,however,the expression levels of dark brown and yellowish-brown skin tissue wes highest than that in other coat colour.These results showed that Slc7a11 gene was associated with coat color formation.3.In order to analyse the function mechamism of Slc7a11 in melanogenesis.Using RNAi jamming technique and overexpression vector of Slc7a11 gene was constructed and transfected into melanophore.mRNA and protein expression level of MITF,TYR gene ect were detected by real-time PCR and Wes systerm.The results showe that,when Slc7a11 gene was over-expressed or repressed,the expression level of coat color related genes were significant change,the mRNA was significantly and positively corrected with the protein expression(P<0.05).which is in line with expression of Slc7a11 gene.Examined melanins synthesis in transferred melanocytes by microplate reader show that when Slc7a11 was overexpression or repression of gene expression,melanin content were increased or reducted compared with control group.We can conclude that Slc7all gene has a effect on the expression of TYR,MITF pigmentation-associated genes and influence melanogenesis.4.For the purpose of identification of the regulation mechansim of Slc7a11 promoter,2500 bp promoter region of Slc7a11 before the initiation codon was cloned,Firstly,potential transcription-factors in Slc7all promoter were predicted and ten series of deletion plasmids was constructed for transfecting RAB-9 cells,The result of dual-luciferase experiment showed that there was one core region in Slc7all promoter,-769bp to-619bp.The results of predition showed that transcription factor POU2F1 binding sites existing in that region.The Slc7all promoter activity increased considerably after site-specificmutagenesis assay(P<0.01).The results explains that FOU2F1 effect of inhibition the Slc7all promoter activity.The result was further confirmed that Competition EMSA experiment was further confirmed to the POU2F1 protein in combination with Slc7all promoter regions,-713～-703bp,regulating the activity of Slc7a11 ptomoter by competition EMSA experiment.