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自裂殖酵母中克隆得到pac1基因,与GenBank中相关的核苷酸序列有99.3%的相似性.编码蛋白质序列分析表明,其具有RNaseⅢ结构域和双链RNA结合结构域.体外活性测定表明,以pET-5a系统在大肠杆菌中表达的pac1产物能够降解dsRNA.将pac1导入双元载体pBI121,以培养7~10d的小麦幼胚为受体材料,利用农杆菌株LBA4404对小麦品种陇鉴127进行转化,获得了41株G418抗性植株,经点杂交(dotblot)、RT-PCR和nptⅡELISA检测证实,其中25株整合有外源基因并能正常表达.对这25株转基因小麦进行大麦黄矮病毒的抗性鉴定表明,有12株表现低度抗性,表现为低接毒量时无症状,接毒量提高时发病且严重;有12株表现中度抗性,表现为低接毒量时无症状,接毒量提高时局部有不严重症状;有1株表现高度抗性,两种情况下均无症状.抗性实验结果表明了pac1介导的抗性具有剂量效应的特点.
The pac1 gene was cloned from the fission yeast and had a similarity of 99.3% with the related nucleotide sequences in GenBank.The sequence analysis of the encoded protein showed that it has the RNase Ⅲ domain and the double-stranded RNA binding domain.According to the in vitro activity assay, The pac1 product expressed in E.coli of pET-5a system can degrade dsRNA.The pac1 gene was introduced into the binary vector pBI121 to cultivate wheat embryos 7 ~ 10d for the receptor material, using Agrobacterium tumefaciens strain LBA4404 for the wheat variety Longjian 127 41 strains of G418-resistant plants were obtained and confirmed by Dotblot, RT-PCR and nptⅡISA, of which 25 were integrated with foreign genes and expressed normally.These 25 transgenic plants were yellow-dwarfed Virus resistance identification showed that 12 strains showed low resistance, which showed asymptomatic symptom with low dose, increased incidence with severe dose, and 12 strains showed moderate resistance with low dose Asymptomatic symptoms were elevated when the dose was increased, and one was highly resistant and asymptomatic in both cases. The results of the resistance experiments showed that pac1-mediated resistance has a dose-response profile.