望江南中核糖体失活蛋白新基因CassinⅠ的克隆及特征分析

 【目的】克隆在植物抗病虫过程中具有重要作用的核糖体失活蛋白新基因，为该类蛋白基因功能研究和有效利用创造条件。【方法】通过分析多种不同来源的植物核糖体失活蛋白的氨基酸序列，据其保守区域设计1对植物通用简并引物P1和P2，对药用植物望江南基因组进行PCR扩增，获得一种新的RIP基因片段。通过RACE法，获得了其全长cDNA序列-CassinⅠ。【结果】CassinⅠ基因全长为882 bp，其推导的氨基酸序列与葫芦科两个代表核糖体失活蛋白β-luffin和Trichosanthin（TCS）的相似性分别为58.2%和34.7%，与GenBank中其它核糖体失活蛋白的序列无显著相似性。多重序列比对发现：CassinⅠ有9个氨基酸残基与TCS的N-糖苷酶活性位点的残基完全相同，另外两个残基发生了变化。所有的活性位点都在P1/P2扩增区段内。【结论】首次在望江南中克隆和报道一种新的核糖体失活蛋白基因，为进一步研究该基因的功能、并应用于作物转基因抗病虫害育种研究奠定了基础。

Cloning and Prokaryotic expression of A Novel Genes of Ribosome Inactivating Protein from Cassia occidentalis

【Objective】Ribosome inactivating proteins (RIPs) are group of important proteins that inhibit the growth of plant pathogens and development of insects. Cloning the RIPs genes are very important to research their resistant function and take into applications.【Method】A novel DNA fragment coding for RIP was isolated from Cassia occidentalis by applying a PCR strategy in which a pair of universal degenerated primers deduced from the two blocks with strong conserved amino acid regions in multiple plants was used. Then the full length cDNA (CassinⅠ) of the fragment was obtained by rapid amplification of cDNA ends with RACE method. The cDNA sequence of CassinⅠwas cloned into the pET 30a vector for expression in Escherichia coli strain BL21(DE3) pLyss .【Results】The full length of CassinⅠwas 882 bp. Compared with the corresponding regions of 2 kinds of typical RIP from Cucurbitaceae, the homologies of the deduced amino acid sequences of CassinⅠshared 58.2% and 34.7% with that of β-luffin and Trichosanthin respectively, and there was no significant homology compared with other sequences of RIPs in Genbank. The fusion protein with a molecular weight of about 33kDa was expressed when the bacteria harboring the recombinant pET 30a vector was induced on 42℃.【Conclusion】To our knowledge, this is the first report that a novel RIP gene was cloned from the Cassia occidentalis.. The results reported in this paper sets up the foundation for further studies of RIP gene function and development of transgenic plants to control plant diseases and insects.