摘要
目的为棉花抗黄萎病基因工程育种提供新的基因源。方法以海岛棉为材料,利用同源序列法和RACE技术克隆海岛棉中SAR途径的主要抗病信号元件NPR1(noneexpresserofPRgene)的全长cDNA序列。结果推导的氨基酸序列与已知NPR1的同源性较低(39%～57%),但在功能区的同源性较高(79.2%),GbNPR1蛋白中含有BTB和锚蛋白重复序列结构域,且在起始密码子上游存在2个W框。在拟南芥中,A.tNPR1起始密码子上游有3个W框,对诱导NPR1的表达和激活NPR1介导的植物防卫反应至关重要,锚蛋白重复序列则是NPR1实现其功能必不可少的。构建了组成型植物表达载体,通过农杆菌介导法导入烟草,转基因植株经PCR和Southern杂交检测,表明目的基因已整合到烟草基因组中。离体叶片接种法对转基因烟草进行抗病性鉴定,表明对烟草赤星病菌(Alternariaalternata)的抗性有明显提高。转基因烟草经T1代遗传分析发现,基因以单拷贝插入。结论本研究得到的基因为海岛棉中NPR1的同源基因。
[ Objective ] The purpose of this study is to find a new gene resource for cotton Verticillium wilt-resistance genetic engineering. [Method] G barbadense vat. 7124 was used as a starting material to clone the full-length cDNA of NPR1 （none expresser of PR gene）- GbNPR1 which is a key regulator in SA （salicylic acid）-mediated systemic acquired resistance （SAR） by homologous cloning and RACE techniques. [Result] In the GbNPR1 molecule, there are two ＂W- boxes＂ in the upstream of ATG that are necessary for both induction of NPR1 transcription and NPRl-mediated activation of plant defense responses. The deduced amino acid sequence of GbNPR1 has low homology （39%-57%） to the other known NPRI proteins; however, they have higher homology （79.2%） in the functional domain. GbNPR1 contains the BTB and ankyrin repeat domain that is known to be the molecular basis for NPR1 function ir ,bidopsis. Plant expression vector harboring GbNPR1 gene was constructed and transferred into Nicotiana tabacum var. NC89 via Agrobaterium-mediated gene transfer. PCR and Southern-blot analysis indicated that the gene has been integrated into tobacco genome. In vitro leaf-disease challenge test of transgenic plants by inoculation of Alternaria alternata demonstrated that the transgenic plants over expressing GbNPRI shown enhanced resistance compared with the non-transgenic plants. Genetic analysis of transgenic Ti indicated that inserted gene is one copy. [ Conclusion ] All of the results indicated that the obtained gene is the homologous gene of NPR1 gene in G barbadense.
出处
《中国农业科学》
CAS
CSCD
北大核心
2006年第5期886-894,共9页
Scientia Agricultura Sinica
基金
国家"863"计划资助项目(2004AA212103)
关键词
海岛棉
NPR1基因
转基因烟草
赤星病
抗病性
Gossypium barbadense
NPR1 gene
Transgenic tobacco
Alternaria alternata
Disease resistance
