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通过将马铃薯抗晚疫病基因RB、R3a编码区克隆到具有组成型CaMV35S强启动子的双元表达载体上,结合农杆菌介导的瞬时表达,分析过表达条件下RB-AvrB,R3a-Avr3a的特异性识别情况。并利用重叠延伸PCR实现R3a与同源序列I2GA1在卷曲螺旋(CC)、核酸结合位点(NBS)和富含亮氨酸重复序列(LRR)三个结构域的互换,根据过敏性反应(hypersensitive response,HR)是否被阻断分析各个结构域对特异性识别的影响,确定特异性识别的关键结构域。结果表明:CaMV35S启动子驱动的RB基因在表达量上较自身启动子有明显提高,使HR反应加快;R3a的表达量和特异性识别Avr3a诱导产生HR反应的速度在两者之间均无明显差别。另外,R3a与I2GA1在LRR区域序列发生交换后,识别Avr3a诱导产生HR反应的能力也发生了交换,即R3a特异性识别Avr3a的关键序列位于LRR结构域内。
The RB and R3a coding regions of potato late blight resistance genes were cloned into the binary expression vector with the constitutive CaMV35S strong promoter, and the expression of RB-AvrB, R3a-Avr3a Specific recognition. And the overlap of R3a with the homologous sequence I2GA1 in three domains of the coiled coil (CC), nucleic acid binding site (NBS) and leucine-rich repeat (LRR) was achieved by overlap extension PCR. According to the allergic reaction ( hypersensitive response (HR)) was blocked. The effect of each domain on the specific recognition was analyzed to determine the key domains for specific recognition. The results showed that the expression of RB gene driven by CaMV35S promoter was significantly higher than that of self-promoter and the HR reaction was accelerated. The expression of R3a and the rate of specific induction of HR reaction induced by Avr3a were not significantly different difference. In addition, the exchange of R3a with I2GA1 in the LRR region sequence also recognized the ability of Avr3a to induce HR responses, ie the key sequence of R3a that specifically recognizes Avr3a is located within the LRR domain.