Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most impo...Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.展开更多
目的观察大鼠脑缺血再灌注后海马CA1区神经元Caspase-3的表达及细胞凋亡指数的动态变化。方法将大鼠随机分为假手术组、模型组。模型组又分为脑缺血再灌注后0,0.5,2,6,24,72,120 h 7个亚组。采用4-VO阻断法制备大鼠脑缺血再灌注模型,用...目的观察大鼠脑缺血再灌注后海马CA1区神经元Caspase-3的表达及细胞凋亡指数的动态变化。方法将大鼠随机分为假手术组、模型组。模型组又分为脑缺血再灌注后0,0.5,2,6,24,72,120 h 7个亚组。采用4-VO阻断法制备大鼠脑缺血再灌注模型,用免疫组织化学染色法及原位细胞凋亡检测法(TUNEL染色)分别观察大脑海马CA1区神经元Caspase-3的表达及细胞凋亡指数。结果假手术组海马CA1区神经元Caspase-3蛋白有少量表达。和假手术组相比,模型组大鼠在脑缺血再灌注后0 h、0.5 h海马CA1区神经元Caspase-3表达无明显变化(P>0.05),再灌注后2 h开始升高(P<0.05)、24 h达高峰(P<0.05),之后逐渐下降,再灌注后120 h仍高于假手术组(P<0.05)。细胞凋亡指数的变化趋势与Caspase-3的表达变化相一致。结论脑缺血再灌注可诱导凋亡相关蛋白Caspase-3的表达,进而导致细胞凋亡。细胞凋亡在脑缺血再灌流损伤中呈动态过程,是神经细胞死亡的重要形式。展开更多
基金supported by the Genomics Initiative of Agriculture and Agri-Food Canada。
文摘Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.
