大鲵Agouti基因的克隆、表达及多态性分析

刺鼠信号蛋白(Agouti)是哺乳动物和鸟类黑色素合成过程中的重要调控因子,影响动物的体色(毛色)。为研究Agouti在两栖动物体色形成过程中的作用,本研究利用PCR技术扩增得到大鲵Andrias davidianus的Agouti基因部分cDNA序列并进行了相关的生物信息学分析,进一步使用实时荧光定量PCR检测了大鲵Agouti基因在皮肤、肝脏等10个组织和器官中的表达情况,并检测了4种不同体色大鲵皮肤组织中Agouti基因的表达量。同时采用直接测序法,比较了不同体色大鲵Agouti基因编码区的序列差异。结果显示,大鲵Agouti基因cDNA序列长1 068 bp,开放阅读框399 bp,编码132个氨基酸残基。蛋白质同源性分析表明,大鲵Agouti蛋白具有与其他物种一致的保守Agouti结构域,其蛋白质序列与两栖爬行类序列相似性较高,与哺乳动物和鸟类相似性较低。系统进化分析显示,大鲵Agouti基因与高山倭蛙Nanorana parkeri、美国短吻鳄Alligator mississippiensis、中华鳖Pelodiscus sinensis等物种的亲缘关系较近。实时荧光定量PCR分析表明,Agouti基因mRNA在大鲵不同组织中均有表达,皮肤中的表达量最高。在4种不同体色大鲵皮肤组织中,黄色皮肤中的Agouti基因表达量高于其他体色。不同体色大鲵Agouti基因编码区序列一致。大鲵Agouti基因独特的序列特征及其表达的组织特异性暗示了其在两栖动物体色形成过程中可能具有与其他物种不同的调控机制。这些结果为进一步研究Agouti在大鲵体色形成过程中的作用提供了基础资料。

Cloning and Polymorphism of Agouti Gene and Its Expression in Different Skin Color Phenotypes in Andrias davidianus

Agouti protein is one of key regulatory factors involving in melanin biosynthesis of mammals and birds and plays a role in animal skin color formation. To investigate the relationship between Agouti gene and skin color formation of amphibians, the Agouti gene of giant salamander (Andrias davidianus) was cloned by PCR followed by sequence characterization using bioinformatics tools. Meanwhile, the expression patterns of Agouti gene in 10 different tissues and in 4 types of skin color phenotypes were detected by quantitative real-time PCR. Furthermore, nucleotide mutation sites of Agouti gene were explored by direct DNA sequencing. The obtained Agouti cDNA was 1 068 bp in length and contained a 399 bp open reading frame encoding a peptide of 132 amino acids. Sequence analysis predicted that Agouti of A. davidianus had a conserved Agouti domain. Multiple sequence alignment showed that Agouti of A. davidianus had high amino acids similarity with amphibians and reptiles, but had low similarity with mammals and birds. These results were further confirmed by phylogenetic tree analysis. Quantitative expression analysis showed that Agouti mRNA expressed in all the tested tissues of A. davidianus, and among of which, the maximum expression level was detected in skin. Besides, the expression of Agouti gene in yellow skin was significantly higher than that in other colors of skins. Polymorphism analysis revealed that the cDNA sequences of Agouti genes in A. davidianus were totally identical among different skin color phenotypes. The specific sequence characteristics and expression pattern of Agouti gene in A. davidianus suggested that there might be different regulatory mechanisms of this gene in skin color formation compared with other species. These findings provide helpful information for further studying the role of Agouti in skin color formation of A. davidianus.