绵羊YAP1基因全长cDNA克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Full-Length cDNA Sequence of YAP1 Gene in Sheep

【目的】克隆绵羊YAP1基因cDNA并分析其序列及其蛋白的遗传特性及其在不同组织中不同程度的表达情况。【方法】以湖羊为研究对象,利用RT-PCR和RACE技术获得绵羊YAP1序列,并结合生物信息学方法对绵羊YAP1的序列进行深入分析。【结果】从湖羊背最长肌中克隆YAP1基因的全长cDNA序列;利用生物信息学技术,对绵羊YAP1基因与其它物种的同源性,蛋白质序列的跨膜区,亚细胞定位,亲水性,潜在信号肽剪切位点,糖基化位点,磷酸化位点,编码产物进行功能预测分析,及其编码蛋白的二级结构,三级结构等进行分析。【结论】克隆获得绵羊YAP1基因序列全长为1 712 bp,包括1 212 bp的编码区序列,编码403个氨基酸。同源性分析发现,绵羊YAP1核苷酸序列与推测氨基酸序列与灰仓鼠的同源性最高,分别达到78.77%和92.51%,而与人、黑猩猩的一致性差异较小。氨基酸序列分析发现该基因编码蛋白为水溶性蛋白,相对分子量为44079.0Da,理论等电点为4.91,无信号肽序列和跨膜区;亚细胞定位主要在细胞核,不属于分泌蛋白;预测含有33个磷酸化位点,7个糖基化位点,具有两个WW结构域,二级结构以无规则卷曲为主,三级结构显示,YAP1结构域区构成弯曲状螺旋结构。预测YAP1蛋白主要在调节功能过程中有着关键作用。以上研究为进一步探讨YAP1基因在肌肉生长过程中的功能研究奠定了遗传信息基础。

[Objective] There was no report on YAP1 gene and its protein in the past days, this study was carried out in order to clone the YAP1 cDNA in sheep and provide a basic foundation for the study of YAP1 genetic characters, as well as analyze its tissue different level expression patterns. [Method] Hu sheep was regarded as the research object, and the full-length eDNA sequence of the YAP 1 gene was acquired from the longissimus dorsi muscle by RT-PCR and RACE, and the sequence was analyzed in depth by bioinformaties method. [Result] The full-length eDNA sequence of the YAP1 gene was successfully obtained from Hu sheep longissimus dorsi muscle. Bioinformaties technology was successfully used to analyze the YAPI homology among different species, transmembrane region of YAP1 sequence, subeellular localization, hydrophilic, potential signal peptide cleavage sites, glycosylation sites, phosphorylationloeus, product function prediction analyisis, YAP1 secondary structure and teriary structure. [Conclusion] The full-length of YAP1 gene was 1 712bp, including 1 212bp coding sequence encoding 403 amino acids. The homology analysis found that the nucleotide sequence and amino acid sequence of sheep YAP1 gene share the highest 78.77% and 92.51% identity with the Cricetulus YAP1 while there was no large difference between Human and Chimpanzee. The amino acid sequence analysis revealed that the YAP 1 gene of sheep encoded water-soluble protein and its relative molecular weight was 44079.0 Da, isoelectric point was 4.91, was a kind of hydrophilic non-transmembrane protein. Subcellular localization of YAP1 was in the mucleus, and it did not belong to the secreted protein. The YAP1 protein contained 33 phosphorylation sites, 7 glycosylation sites and 2 WW domain. The secondary structure of YAP1 was mainly composed of random coil. The tertiary structure of domain area of YAP1 protein showed a forniciform helix structure. Forecast YAP1 had a key role in regulatory functions process. The above studies have laid an information foundation for further study of the YAP1 gene function in the sheep muscle development process.