Study On Cloning And Expression Level Of MC1R Gene In Individuals Of Different Plumage Color In Korea Quail

There are more than30locis about genetic of feather color in Quail, andrelationship between plumage color phenotype and genotypeis a complex. Wild graysignaling protein (ASIP) gene, solute carrier family45member2(SLC45A2)gene,tyrosine-related protein1(TYRP1)gene, melanin glucocorticoid receptor1(of MC1R)gene, within the cortex of the body (EDNRB2) gene is the candidate genes of the quailfeather color, these genesthe expression product are involved in the regulation ofmelanin synthesis and transport process. The melanocortin receptor1(of MC1R) wascoded by the extended loci of coat color and played an important role from animalbody color.In this study, the MC1R gene mutations were found by the methods sequencingthe mixed samples of DNA. The gene distribution of mutations were studied in thefour kinds of plumage color (Maroon, yellow feathers, white feather, black feather)quail populations by PCR-SSCP and A1u1-RFLP. To reveal the relationship betweenthe MC1R gene and the traits of plumage color in quail. Experiments show that,2mutant locis werefound inMC1R genes of quail by DNA sequencing method: the A/G mutation, which resulted in the encoded protein IIe58Val mutation, the site wasanalyzed by using PCR-SSCP; Another mutation site is C/T mutations, but it did notlead to the encoded proteins mutation, he site was analyzed by using A1u1-RFLP.Eventually found the A/G mutation site in4plumage quail groups had no significantdifference (P>0.05), but C/Tmutation site in4plumage quail groups had significantdifference(P <0.05). This study did not find the Glu92Lys mutation in the Korea quail,which was reported in the group of Japanese quail, indicating that the mechanisms ofmutations are different between Korea black feather quail and Japanese black featherquail, And black feather mutations of Korea quail may be also relevant with other genemutations.This experiment, cloned Korean quail MC1R gene, full-length sequencing results after biological software stitching, The fragment size of coding region was942bp,only exon1, encoding314amino acids in Korea quail MC1R gene. Comparison usingBLAST and found that the MC1R gene sequence of this fragment published inGenbank. Quail were highly homologous, as high as98%. Four plumage color Koreaquail DNA as a template, PCR reaction using the SeqMan program and Chromassoftware analysis found that a SNP (A-212-G) was located in the MC1R gene exon212bp position in four kinds of plumage quail, found second SNP (C-577-T) in the577bp position, only Maroon no obvious variation, the other three kinds of plumagecolor was mutation.qRT-PCR technology was used for the determination of the expression in the skintissue of the MC1R gene when the four plumage color in quail embryos in12days. theexpression of MC1R gene are obvious differences in four plumage color quail skintissue, the expression of the gene of Maroon quail skin tissue was significantly higherthan the expression level in the black feather quail skin (Maroon> yellow feather>white feather> black Feather).The protein structure of MClR gene were Predicted in the maroon and blackfeathersquail, the prediction results protein secondary structure of MClR the Koreanquail showed that, The7α-helix,10β-sheet,3transmembrane α-helix were formatedof in Maroon quail MClR protein. The mutants Black Feather quail MClR proteinstructure due to the composition and sequence of the amino acid change, resulting inprotein conformational change, thus affecting its function. Only formed five a-helix5β-fold,3three transmembrane a-helix in the protein structure MClR of mutant blackfeather quail. According to the Kyte-Doolittle hydrophilic amino acid standard,Maroon quail MClR hydrophilic region of the protein (≥0)that we canseeThe segmentof amino acids is5-12,26-33,112-127,142-163,214-236, and302-312. the mutantblack feather quail MClR, hydrophilic regions (≥0) is the section5-12,26-33,85-89,93-101,165-170and230-235amino acids. Maroon quail MClR amino acids surfaceexposed area in accordance with the the Emini principle, for7-10,26-30,112-124,142-162, and218-229, the protein surface may be located in the region of218-229,and302-310. Predicted in the polypeptide chain based on the amino acid chargedamino acid for the positively charged area for7-11,28-32,112-127,138-157,159-168,185-189,209-238,255-259,267-271and300-311segments;13-17,52-56,75-78,91-95,103-108of amino acids as the negative charge region, and288-296segment; blackFeather the quail MClR surface, the exposed area of7-10,26-30,112-124,142-162 and218-229section of amino acids, which most likely is located in the protein surfacearea of amino acids142-162and218-229segment. According to the amino acidcharged situation, the predicted amino acid polypeptide chain to the positively chargedarea7-10,27-31,108-113,145-157section; amino acids for the negative charge region13-17,32-43,52-5675-85,86-90,99-103and157-165segments.