目的克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列。进一步推测这两个基因编码的蛋白,并分析其特征。方法从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA。利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段。然后对已知片段进行3’ RACE和5’RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列。利用相关软件对获得序列进行翻译和分析。结果首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列。该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9%,与小鼠的相似度为82.7%。TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8%,与小鼠的相似度为86.6%。该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2%,与小鼠的相似度为81.9%。TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2%,与小鼠的一致度为84.4%。结论本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征。
Objective To isolate and obtain the complete sequence of Trp1 and Trp2 genes from white hair black eyes(WHBE) rabbit iris, which represent the second and third members of the tyrosinase-related gene family, and to identify the sequences of protein TRP1 and TRP2.MethodsThe WHBE rabbit iris whole mRNA was reversely transcripted to cDNA. The cDNA was isolated from mouse, rat and human using homologous primers. The fragments and complete sequences of the cDNA were obtained by PCR and RACE. ResultsThe WHBE rabbit coding region was sequenced completely to yield 1604 bp full length and shared 87.9% identity with the human sequence and 82.7% identity with the mouse sequence. The Trp1 mature protein sequence had 513 amino acids and shared 89.8% identity with the human sequence and 86.6% identity with the mouse sequence. The WHBE rabbit Trp2 coding region was sequenced completely to yield 1554 bp full length and showed 83.2% identity with the human sequence and 81.9% identity with the mouse sequence. The WHBE rabbit Trp2 mature protein sequence had 494 amino acids and shared 84.2% identity with the human sequence and 84.4% identity with the mouse sequence. ConclusionsThe protein sequences of TRP1and TRP2 obtained from WHBE rabbit iris in this study show a high homology to TYR and show structural characteristics of the tyrosinase-related protein family.
朱亮,蔡月琴,屠珏,陈民利.白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析[J].中国实验动物学报,2012,(2):43~48.
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