摘要
【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882bp,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。
【Objective】Ribosome inactivating proteins （RIPs） are a group of important proteins that inhibit the growth of plant pathogens and development of insects. Cloning the RIPs genes is very important for research their resistant function and applications.【Method】A novel DNA fragment coding 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【.Result】The full length of CassinⅠwas 882 bp. Compared with two 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 （TCS）, respectively, and there was no significant homology compared with other sequences of RIPs in GenBank. Result of multiple alignment about TCS, β-luffin and CassinⅠshown that 9 amino acid residues forming the active site of the TCS were conserved in CassinⅠ except that 2 residues was different. All the RIP active sites of CassinⅠ sequence were contained in P1/P2 amplification region.【Conclusion】It is the first report that a novel RIP gene was cloned from the Cassia occidentalis.. The results reported in this paper have laid a foundation for further studies of RIP gene function and development of transgenic plants to control plant diseases and insects.
出处
《中国农业科学》
CAS
CSCD
北大核心
2008年第2期417-423,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金(30370929)
广东省科技攻关项目(C20209)
关键词
望江南
核糖体失活蛋白
基因克隆
特征分析
Cassia occidentalis
Ribosome inactivating protein
Gene cloning
Signature analysis
