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

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 Li

doi:10.1016/j.plasmid.2018.09.002

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

Plasmid. 2018 Jun:98:52-55. doi: 10.1016/j.plasmid.2018.09.002. Epub 2018 Sep 8.

Authors

Deshui Yu¹, Libing Liao², Yi Zhang³, Kedong Xu¹, Ju Zhang¹, Kun Liu¹, Xiaoli Li¹, Guangxuan Tan¹, Jurui Zheng⁴, Yong He⁴, Changling Xu⁴, Jinjin Zhao⁴, Beibei Fu⁴, Jiaxing Xie⁴, Jie Mao⁴, Chengwei Li⁵

Affiliations

¹ 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.
² Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou 466001, China; College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, China.
³ 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; College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.
⁴ 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; College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, China.
⁵ 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 Engineering Research Center of Crop Genome Editing, Henan Institute of Science and Technology, Xinxiang 453003, China. Electronic address: lichengweiwau@hotmail.com.

PMID: 30201136
DOI: 10.1016/j.plasmid.2018.09.002

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Agrobacterium / genetics*
Gene Knockdown Techniques / methods*
Genes, Plant*
Genetic Vectors*
Nicotiana / genetics*
Nicotiana / microbiology
Plasmids / genetics*
RNA Interference*