红褐色乌苏里貉TYRP1基因的克隆及表达水平分析

Cloning and Expression Level Study on TYRP1 Gene in Reddish-brown Wusuli Raccoon Dog

实验旨在探讨红褐色乌苏里貉和野生型乌苏里貉TYRP1基因的核酸序列以及表达水平差异。本实验克隆了红褐色和野生型乌苏里貉TYRP1基因CDS序列,并利用实时定量PCR技术对TYRP1基因在2种毛色貉中的mRNA表达水平进行了分析。结果表明:2种貉TYRP1基因CDS序列全长1 614 bp,包括完整的开放阅读框,共编码537个氨基酸;红褐色乌苏里貉TYRP1基因编码序列与狗、猪、绵羊、小鼠、人、牛、猫、马、虎鲸和黑猩猩核酸序列高度同源,同源性分别为99.07%、90.56%、89.64%、83.22%、88.90%、89.39%、90.87%、88.63%、90.25%和88.65%,与野生型乌苏里貉序列完全相同。利用SPSS软件进行独立样本t检验,结果表明TYRP1基因在2种貉皮肤组织中均可稳定表达,利用F=(1+E)-△△Ct计算可知TYRP1基因在红褐色乌苏里貉中的表达量是野生型乌苏里貉的0.93倍,但差异不显著(P>0.05)。该实验为TYRP1基因的功能分析及红褐色乌苏里貉毛色突变分子遗传机制的探讨提供了一定的理论基础。

In order to explore the differences of nucleic acid sequences and expression level of TYRP1 gene between reddish-brown Wusuli raccoon dog and wild-type Wusuli raccoon dog,the CDS sequences of TYRP1 gene in the two kinds of raccoon dog were cloned.The expression level of TYRP1 gene was compared in the two kinds of raccoon dog.The results showed that the CDS sequence was 1 614 bp in length which contained the entire open-reading frame encoding 537 amino acid.Alignment analysis indicated that the nucleotide sequence showed a high homology with the TYRP1 sequences of canis familiaris （99.07％）,sus scrofa （90.56％）,Ovis aries （89.64％）,mus musculus （83.22％）,homo sapiens （88.90％）,bos taurus （89.39％）,felis catus （90.87％）,equus caballus （88.63 ％）,orcinus orca （90.25％）and pan troglodytes （88.65％）.A complete match was found in the two kinds of raccoon dog.Based on the independent-samples T test in SPSS software,the results showed that TYRP1 gene was stably expressed in of skin tissue of reddish-brown Wusuli raccoon dog and the expression quantity of TYRP1 gene in reddish-brown Wusuli raccoon dog was 0.93 times of that of wild-type Wusuli raccoon dog （F =（1 ＋E）-△△ct）.However,there was no significant difference between them（P＞0.05）.This provides theoretical basis for the functional analysis of TYRP1 gene and mutation mechanism of hair color in reddish-brown Wusuli raccoon dog.