关键词: 望江南, 核糖体失活蛋白, 基因克隆, 特征分析

Abstract: 【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.

Key words: Cassia occidentalis, Ribosome inactivating protein, gene cloning, Prokaryotic expression