水貂酪氨酸酶(TYR)基因克隆、SNPs筛查及其皮肤组织mRNA差异表达分析

旨在克隆和分析水貂酪氨酸酶（tyrosinase，TYR）基因编码区，揭示该基因编码区SNPs及其皮肤组织mRNA差异表达规律与水貂毛色表型的关系。本研究采集7月龄雄性金州黑水貂、名威银蓝水貂和红眼白水貂共计301个样本的血液，利用聚合酶链式反应（PCR）方法对水貂TYR基因5个外显子进行分段克隆，并拼接获得其完整编码区序列（coding sequence，CDS）；采用PCR产物直接测序技术筛查3种毛色水貂外显子1、4和5中的SNPs；采集9只7月龄雄性水貂（黑、灰与白色被毛各3只）背中部皮肤组织，通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）技术检测TYR基因mRNA在3种毛色水貂皮肤组织中的表达水平。结果表明，克隆的水貂TYR基因序列长2 391 bp，由5个外显子组成，编码区长1 596 bp，编码的531个氨基酸中包括信号肽（18个氨基酸）和成熟肽（513个氨基酸），该序列已上传GenBank（KJ716783）。SNPs筛查发现，3种毛色水貂TYR基因外显子4和5不存在突变位点，外显子1发现2处变异：c.441G>A和c.138T>A，c.441G>A为同义突变且仅存在于金州黑水貂群体。qRT-PCR分析显示，TYR基因mRNA在3种毛色水貂皮肤组织中均表达，且在金州黑水貂中的表达极显著高于银蓝水貂和红眼白水貂（P<0.01），在银蓝水貂中的表达显著高于红眼白水貂（P<0.05）。研究结果提示，TYR基因c.138T>A位点及其皮肤组织mRNA差异表达水平与水貂毛色显著关联。

Cloning,SNPs Screening and mRNA Differential Expression Analysis of TYR Gene in Skin of Mink(Neovison vison)

The aim of this study was to clone and analyze the complete coding region sequence of TYR gene, further reveal the relationship between SNPs and its mRNA differential expression level in skin and coat color phenotype in mink (Neovison vison). A total of 301 blood samples of 7-month-old male minks (Jinzhou black, Mingwei silverblue and Red eye white) were collected, and the polymerase chain reaction (PCR) method was used to clone 5 exons of the mink TYR gene in sections and the complete coding sequence (CDS) was spliced. The PCR products were directly sequenced to screen SNPs in exon 1, 4 and 5 of TYR gene in minks with three kinds of coat color phenotypes. The back skin tissues of 9 7-month-old male minks (3 black, gray and white coats each) were collected, and quantitative real-time PCR (qRT-PCR) technology was used to detect TYR mRNA expression levels in skin of minks with 3 coat colors. The results showed that the mink TYR gene sequence was 2 391 bp in length, containing 5 complete exons, the full-length coding region was 1 596 bp, which encoded 531 amino acids containing signal peptide(18 amino acids) and mature peptide(513 amino acids). The sequence had been submitted GenBank database and its accession number was KJ716783. SNPs analysis showed that there was no mutation site in exon 4 and 5 of TYR gene, and two mutations were found in exon 1, c.441G>A and c.138T>A. However, c.441G>A was synonymous mutation and existed only in Jinzhou black mink population. The qRT-PCR results showed that TYR mRNA was expressed in the skin of all the three kinds of coat color types of minks, and its expression level in the Jinzhou black mink was extremely significantly higher than that in Mingwei silverblue and Red eye white mink (P<0.01). The TYR mRNA expression level in Mingwei silverblue mink was significantly higher than that in Red eye white mink (P<0.05). The results of present study indicated that the c.138T>A locus and mRNA expression levels of TYR gene might be associated with the coat color phenotype of mink.