牦牛MC1R、MITF和Agouti基因序列特征及在皮肤组织中的表达

发布时间：2018-01-19 02:13

本文关键词： 牦牛毛色荧光定量PCR 表达量　出处：《甘肃农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：牦牛毛色影响毛的利用经济价值。本研究以天祝白牦牛和甘南牦牛为研究对象,通过基因克隆、DNA混合池测序、荧光定量PCR等方法。克隆牦牛MC1R、MITF及Agouti基因CDS区序列,进行生物信息学分析。对调控毛色候选MITF、Agouti基因的编码区与牦牛毛色性状的相关性进行分析。通过实时荧光定量PCR技术,分析MC1R、MITF及Agouti基因在不同毛色牦牛的皮肤组织相对表达量。研究结果如下:(1)克隆获得了牦牛MC1R基因、MITF基因及Agouti基因完整编码区序列(CDS)。MC1R基因编码区(CDS)序列长度为1021 bp,其编码317个氨基酸,该蛋白无信号肽,但有七个跨膜区。MITF基因编码区(CDS)序列长度为1460 bp。其编码413个氨基酸,MITF-M蛋白存在10个氨基酸构成的M域,即MLEMLEYNHY。该氨基酸序列无信号肽,且不具有跨膜结构域。MITF-M有25个磷酸化位点。Agouti基因是一条长593 bp的DNA序列。该序列编码133个氨基酸,ASIP有一个22个氨基酸的信号肽,且没有跨膜结构域。ASIP是一种膜外蛋白。该基因的序列在第6-129位氨基酸之间具有完整的DNA binding ASIP功能域。ASIP有5个磷酸化位点。MC1R、MITF-M和ASIP均是亲水性蛋白。(2)利用DNA混合池、直接测序法对牦牛MITF基因和Agouti基因序列的编码区进行检测分析。所得结果显示,在牦牛MITF基因和Agouti基因序列编码区并无突变位点。表明牦牛MITF基因和Agouti基因编码区序列不存在与毛色相关联的功能突变。(3)实时荧光定量PCR检测分析得到MC1R基因、MITF基因的仅在皮肤组织中特异性表达。Agouti基因在卵巢、肌肉组织及皮肤组织中均有表达,且其皮肤组织中表达最高。Agouti基因m RNA表达量在黑色毛皮肤组织和白色毛皮组织中差异不显著。MC1R基因m RNA相对表达量在纯黑色毛皮组织中显著高于纯白色毛皮组织。说明皮肤组织中MC1R基因m RNA的高表达可能利于黑色素的沉着。MITF基因m RNA相对表达量在纯黑色毛皮组织中显著高于白色毛皮组织。说明牦牛白色被毛与黑色被毛性状可能与MITF基因在皮肤组织中m RNA的表达量高低有关联。
[Abstract]:In this study, Tianzhu White Yak and Gannan Yak were selected as the research objects, and the DNA mixed pool was sequenced by gene cloning. Fluorescence quantitative PCR and other methods were used to clone the CDS region of MC1RN MITF and Agouti gene of Yak, and to analyze the bioinformatics. The candidate MITF for controlling coat color was analyzed. The correlation between the coding region of Agouti gene and the hair color traits of yak was analyzed, and MC1R was analyzed by real-time fluorescence quantitative PCR. The relative expression of MITF and Agouti genes in the skin of different hairy yaks was studied. The results were as follows: 1) the yak MC1R gene was obtained by cloning. The length of the complete coding region of MITF gene and Agouti gene was 1021 BP, encoding 317 amino acids. The protein has no signal peptide, but has seven transmembrane regions. The length of CDSs of MITF gene coding region is 1460 BP, which encodes 413 amino acids. There are 10 amino acids in the M domain of MITF-M protein, that is, MLEMLEYNHY. the amino acid sequence has no signal peptide. Moreover, there are 25 phosphorylation sites. Agouti gene is a 593bp DNA sequence with no transmembrane domain .MITF-M, which encodes 133 amino acids. ASIP has a signal peptide of 22 amino acids. There is no transmembrane domain. ASIP is an extracellular protein. The sequence of the gene has complete DNA binding between the 6-129 amino acids. ASIP functional domain. ASIP has 5 phosphorylation sites. MC1R. MITF-M and ASIP are both hydrophilic proteins. The coding regions of MITF gene and Agouti gene of yak were detected by direct sequencing. There was no mutation in the coding region of yak MITF gene and Agouti gene, which indicated that there was no functional mutation associated with coat color in the coding region of MITF gene and Agouti gene of yak. 3) MC1R gene was obtained by real-time fluorescence quantitative PCR analysis. The specific expression of MITF gene was only in the skin tissue. The MITF gene was expressed in the ovary, muscle tissue and skin tissue. There was no significant difference in the expression of m RNA between black fur skin and white fur tissue. MC1R gene m. The relative expression of RNA in pure black fur tissue was significantly higher than that in pure white fur tissue, indicating that the high expression of MC1R gene m RNA might be beneficial to melanin deposition. MITF gene m. The relative expression of RNA in pure black fur tissue was significantly higher than that in white fur tissue, indicating that the characters of white coat and black coat of yak might be associated with MITF gene in skin tissue m. The level of RNA expression is related.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.85

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