Yu Deshui, Liao Libing, Zhang Yi, Xu Kedong, Zhang Ju, Liu Kun, Li Xiaoli, Tan Guangxuan, Zheng Jurui, He Yong, Xu Changling, Zhao Jinjin, Fu Beibei, Xie Jiaxing, Mao Jie, Li Chengwei
Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou 466001, China; Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou 466001, China.
Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou 466001, China; College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, China.
Plasmid. 2018 Jun;98:52-55. doi: 10.1016/j.plasmid.2018.09.002. Epub 2018 Sep 8.
RNA interference (RNAi), based on hairpin RNA (hpRNA) expression, plays an important role in functional analysis of plant genes. Traditional methods for making RNAi constructs usually involve multiple time-consuming cloning steps. We have developed a Gateway-compatible binary vector for RNAi-mediated gene knockdown in plants from pCAMBIA2301 and pHANNIBAL vectors. The new plant RNAi binary vector, named pCAMBIA2301-GW-RNAi, has two inverted repeated Gateway cassettes driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. This enables site-specific recombination at two sites by one Gateway LR reaction without restriction enzymes and ligases. The pCAMBIA2301-GW-RNAi vector's effectiveness was evaluated by Agrobacterium-mediated transient co-expression assays of overexpression and silencing constructs of HvCEBiP in Nicotiana benthamiana followed by western blot analysis. Obtained results show that the developed RNAi vector successfully knocked down 35S-driven expression of HvCEBiP, as expression levels of the encoded HvCEBiP protein were significantly reduced.
基于发夹RNA（hpRNA）表达的RNA干扰（RNAi）在植物基因功能分析中发挥着重要作用。传统的构建RNAi载体的方法通常涉及多个耗时的克隆步骤。我们从pCAMBIA2301和pHANNIBAL载体开发了一种用于植物中RNAi介导的基因敲低的Gateway兼容二元载体。这种名为pCAMBIA2301-GW-RNAi的新型植物RNAi二元载体有两个由花椰菜花叶病毒35S（CaMV 35S）启动子驱动的反向重复Gateway盒。这使得通过一次Gateway LR反应在两个位点进行位点特异性重组，无需限制性内切酶和连接酶。通过在本氏烟草中对HvCEBiP的过表达和沉默构建体进行农杆菌介导的瞬时共表达分析，随后进行蛋白质印迹分析，评估了pCAMBIA2301-GW-RNAi载体的有效性。获得的结果表明，开发的RNAi载体成功敲低了35S驱动的HvCEBiP表达，因为编码的HvCEBiP蛋白的表达水平显著降低。
