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采用功能筛选策略从以PVX病毒载体建立的棉疫病菌(Phytophthora boehmeriae)cDNA文库中克隆了能诱导烟草产生过敏反应的激发子基因(片段).以农杆菌Mog101为宿主、利用PVX病毒表达载体构建了含有6800个单克隆的棉疫病菌cDNA文库,采用牙签接种本氏烟(Nicotiana benthamiana),从4100个克隆中筛选到12个能引起烟草过敏反应的阳性克隆.对上述阳性克隆进行序列测定和分析,结果显示,其中7个与已知的基因具有较高的同源性,其中PBC43编码一个特异的elicitin蛋白;PBC163编码一个Rab蛋白,与大豆疫霉的RAB同源性较高;PBC241编码抗增殖蛋白(prohibitin);PBN62编码一个热激蛋白60(HSP60).还有5个克隆在公共数据库中没有同源基因,提示可能是该病原菌特异的激发子基因.上述结果表明,利用病毒表达载体构建cDNA文库,用功能筛选策略可从棉疫病菌全基因组范围内筛选诱导烟草产生过敏反应的激发子基因,该方法可望为其他植物病原菌激发子基因的克隆提供技术平台.
A functional screening strategy was used to clone an elicitor gene (fragment) capable of inducing anaphylactic reaction in tobacco from a cDNA library of the PVX virus vector (Phytophthora boehmeriae). The Agrobacterium tumefaciens Mog101 was used as a host and constructed using the PVX virus expression vector A cDNA library containing 6,800 monoclonal anti-Phytophthora infestans was obtained, and Nicotiana benthamiana was inoculated with a toothpick to screen 12 positive clones that could cause anaphylactic reaction in tobacco from 4100 clones. The above positive clones were sequenced and The results showed that seven of them shared high homology with the known genes, of which PBC43 encoded a specific elicitin protein. PBC163 encoded a Rab protein with high homology to RAB of P. sojae. PBC241 encoding PBN62 encodes a heat shock protein 60 (HSP60) and five clones have no homologous genes in the public database, suggesting that this gene may be a specific elicitor of the pathogen.The above results indicate that using virus expression The vector was used to construct a cDNA library. The functional screening strategy was used to screen the whole genome of the infectious disease virus to induce the elicitor group Cloning, which is expected to stimulate the genes for other plant pathogens provide the technology platform.