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根据小麦EF手钙离子绑定蛋白(TaCab1)基因序列,利用WMD3软件设计特异的人工miRNA(amiRNA),构建VIGS沉默载体。利用amiRNA-VIGS体系,对小麦的TaCab1基因的功能进行了初步分析。利用Northern blot和实时定量PCR技术分别检测了amiRNA的积累及TaCab1的沉默效率,并利用显微观察技术统计条锈菌侵染小麦后的组织学差异。结果表明,amiRNA可以得到有效的积累,其靶标基因TaCab1可以得到有效的沉默。从表型上看,小麦叶片上条锈菌夏孢子的产孢量也在一定程度上有所降低。组织学观察发现当TaCab1被沉默后,寄主细胞的坏死面积在侵染后期明显增大,条锈菌的菌丝分枝数也明显增多,但菌丝长度明显变短。
According to the sequence of TaCab1 gene of wheat EF, a specific artificial miRNA (amiRNA) was designed by using WMD3 software to construct VIGS silencing vector. Using the amiRNA-VIGS system, the function of TaCab1 gene in wheat was analyzed. Northern blot and real-time quantitative PCR were used to detect the accumulation of amiRNA and the silencing efficiency of TaCab1 respectively. The microscopic observation was used to calculate the histological difference of stripe rust infected wheat. The results showed that amiRNA could be effectively accumulated and its target gene TaCab1 could be effectively silenced. Phenotypically, the amount of sporulation of the spore of the stripe rust on wheat leaves also decreased to a certain extent. Histological observation showed that when TaCab1 was silenced, the necrotic area of host cells increased significantly at the late stage of infection, and the number of mycelial branches significantly increased, but the length of mycelium was significantly shorter.