Hpfrl, a homological sequence of hrf1, was cloned from a japonica cultivar R109 by PCR analysis. The homologies of hpfr1 compared with hrfl are 92% and inferred homologies of amino acid from hpfrl compared with that from hrfl are 74.8%. Hpfrl gene was ligated into expression vector pET30a(+) which has T7 promoter to construct recombinant plasmid pHOSJ4. The function of this protein is similar to harpin_(Xoo) and causes HR in non-host plant. The seguences of hpfrl and hrf1 are the same from the initiation code ATG to 225 bp site. The sequences from ATG to 225 bp site were cloned and constructed expression vector pHOSJ4-225. The crude recombinant proteins also cause HR in Xanthi tobacco. Sprays of the proteins onto Xanthi tobacco induce the resistance of tobacco to TMV remarkably. The contrast analyses of LSD indicate that the protein extraction of BL21 (DE3)/pHOSJ4 and BL21 (DE3)/pHOSJ4-225 can dramatically enhance tobacco resistance to TMV.%利用PCR技术从粳稻R109中克隆到与hrf1同源的序列,命名为hpfr1,与hrf1基因的同源性为92%.推导的氨基酸同源性为74.8%.hpfr1基因连接到含T7启动子的高表达载体pET30a(+)上构建重组质粒pHOSJ4,并转化宿主菌BL21(DE3)产生表达菌株BL21(DE3)/pHOSJ4.hpfr1与hrf1基因在起始密码子ATG至225个碱基处完全相同.将ATG至225碱基的序列克隆,并构建表达载体pHOSJ4-225,表达产物注射烟草能引起过敏反应.BL21(DE3)/pHOSJ4和BL21(DE3)/pHOSJ4-225的蛋白抽提物诱导烟草抗烟草花叶病毒病均达极显著水平,浓度为60μl/ml时防效最显著,分别为96.35%、95.4%,与harpinXoo防效(95.9%)相近.
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