猪苓Ⅱ型核糖体失活蛋白A链基因的克隆以及原核表达

Molecular cloning and prokaryotic expression of a type Ⅱ ribosome inactivating protein from Polyporus umbellatus

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 ribosome inactivating protein（RIP） gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame c DNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 k Da and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that Pu RIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile,the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this Pu RIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise,we successfully constructed the p ET15b-Pu RIP plasmid,produced and purified the His-Pu RIP fusion protein,which would provide the basic material for polyclonal antibody preparation and gene function research.