Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants

Publication date: Available online 8 September 2018Source: PlasmidAuthor(s): Deshui Yu, Libing Liao, Yi Zhang, Kedong Xu, Ju Zhang, Kun Liu, Xiaoli Li, Guangxuan Tan, Jurui Zheng, Yong He, Changling Xu, Jinjin Zhao, Beibei Fu, Jiaxing Xie, Jie Mao, Chengwei LiAbstractRNA 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.
Source: Plasmid - Category: Biotechnology Source Type: research