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采用RT-PCR技术从中国野生猪苓菌核(Polyporus umbellatus)中克隆得到一个Ⅱ型核糖体失活蛋白(typeⅡribosome inactivating protein,RIP)A链基因,该基因c DNA包含的完整开放阅读框为873 bp,编码的氨基酸为290个,分子质量为32.33 k Da,理论等电点为5.58。氨基酸序列分析表明,该基因编码的蛋白具有RICIN超家族蛋白的保守结构域。氨基酸序列多重比对及系统发育树结果显示,猪苓RIP与硬柄小皮伞(Marasmius oreades)亲缘关系最近。实时荧光定量PCR分析表明,这个基因在不同的菌核部位都有表达,且在蜜环菌侵染的菌核部位表达显著上调,提示该基因可能参与了猪苓响应生物胁迫过程。此外,利用基因重组技术构建p ET15b-Pu RIP原核表达载体,获得了高质量的His-Pu RIP融合蛋白,为多克隆抗体的制备提供材料基础,为研究基因功能奠定基础。
A type Ⅱ ribozyme inactivating protein (RIP) A chain gene was cloned from Chinese Polyporus umbellatus by RT-PCR. The complete open reading frame of this gene contains 873 bp, encoding 290 amino acids with molecular mass of 32.33 k Da and theoretical isoelectric point of 5.58. Amino acid sequence analysis showed that the gene encoded by this gene has the conserved domain of RICIN superfamily protein. Multiple alignment of amino acid sequences and phylogenetic tree results showed that Polyporus umbellatus RIP was closest to Marasmius oreades. Real-time quantitative PCR analysis showed that the gene was expressed in different sclerotia, and the expression was significantly up-regulated in the fungus nucleus infected by Armillaria mellea, suggesting that the gene may be involved in the process of Polyporus umbellatus in response to biotic stress. In addition, we constructed the prokaryotic expression vector p ET15b-Pu using gene recombination technology and obtained high quality His-Pu RIP fusion protein, which provided the material basis for the preparation of polyclonal antibody and laid the foundation for the study of gene function.