马铃薯抗晚疫病基因RB、R3a过表达及R3a特异性识别关键结构域的确定  

Overexpression of the Potato Late Blight Resistance Genes RB and R3a and Identification of Domains Required for Recognition Specificity of R3a

作　　者：梁春波[1,2] 陈伊里[1] 李颖[2] 黄三文[2] 

机构地区：[1]东北农业大学农学院,哈尔滨150030 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《园艺学报》2011年第1期87-94,共8页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30671319)

摘　　要：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 potato genes RB and R3a encode coiled-coil,nucleotide binding site,leucine-rich repeat（CC-NBS-LRR）proteins and confer specific recognition to AvrB and Avr3a effectors of the late blight pathogen Phytophthora infestans,manifested as a hypersensitive response（HR）at the initial site of infection.In this study,overexpression of RB and R3a were constructed by cloning coding domains to the vector which contains CaMV35S promoter.Chimerical constructs of domain swapping between R3a and its homolog I2GA1 in CC,NBS and LRR were generated by overlap extension polymerase chain reaction.The recognition capability of the overexpressing and domain swapping constructs to AvrB and Avr3a were tested by Agrobacterium transient transformation assay（ATTA）.The results showed that the expression of RB was much higher and HR appeared almost one day earlier under the control of CaMV35S promoter than the native one.For R3a,there was no difference of expression and HR observed under the control of these two kinds of promoter.Domain swapping revealed that the recognition specificity of R3a is mostly determined by the domain of LRR.

关 键 词：马铃薯 晚疫病 重叠延伸PCR 结构域交换 瞬时表达 

分 类 号：S532[农业科学—作物学]