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Generation of a promising universal RNAi vector system to control plant pests

  • Yu Pan
  • Yan Dong
  • Ruixue Wang
  • Minsheng Yang
Original Article
  • 15 Downloads

Abstract

RNA interference has great potential value in plant pest control. We used existing plasmids and selected the conservative sequence of the chitinase (Chi) gene, which is highly homologous with many plant pests, to be the target sequence. The first step was to use conventional PCR technology to build the cloning vector pGr398 with two groups of isocaudomers. The target gene forward and reverse sequences were inserted into this carrier through the A-T connection. Then the cloning vector with the target fragment was connected to the vector pBI121 to obtain the RNAi vector. Finally, the two RNAi plant transformation vectors pGr320 (GFP) and pGr326 (Chi) were successfully constructed. These were used to verify the feasibility of the RNAi plant transformation vector system. Using this vector system, we imported two RNAi plant transformation vectors into Agrobacterium to obtain the transformation vector (G0 and G3). We successfully obtained transgenic tobacco lines using the Agrobacterium-mediated leaf disc method. By feeding tobacco to cotton bollworm larvae, we successfully reduced expression of the Chi gene in the larvae and achieved the RNAi effect to some extent, demonstrating the feasibility of our system. We successfully created a simple universal RNAi plant transformation vector system that can improve the construction efficiency of RNAi vectors and promote widespread use of RNAi technology in plants.

Keywords

RNA interference Vector construction RNAi vector Transgenic tobacco 

Abbreviations

Amp

Ampicillin

Cef

Cefotaxime sodium

Chi

Chitinase

CK

Control

CTAB

Cetyltrimethyl ammonium bromide

DPS

Data processing system

dsRNA

Double-stranded RNA

GFP

Green fluorescent protein

Kan

Kanamycin

MS

Murashige and Skoog medium

PCR

Polymerase chain reaction

RNAi

RNA interference

RT-PCR

Reverse transcription PCR

siRNA

Small interfering RNA

WT

Wild-type

Notes

Acknowledgements

This study was supported by the Basic Research Plan Project of Hebei Province (No. 18966801D) and the National Key Program on Transgenic Research of China (No. 2018ZX08020002).

Funding

This study was supported by the Basic Research Plan Project of Hebei Province (No. 18966801D) and the National Key Program on Transgenic Research of China (No. 2018ZX08020002).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Our study does not involve ethics approval and consent to participate.

Consent to publish

All authors read and approved the final manuscript.

Supplementary material

11738_2019_2810_MOESM1_ESM.tif (2 mb)
Fig. S1 The nucleotide sequences of Chi gene share high identity in many Lepidopteran insects. (From up to down, these are Lymantria dispar, Phyllonorycter ringoniella, Spodoptera frugiperda, Agrotis ipsilon, Mamestra configurata, Mamestra brassicae, Spodoptera littoralis and Helicoverpa armigera) (TIF 2017 KB)
11738_2019_2810_MOESM2_ESM.xls (20 kb)
Supplementary material 2 (XLS 20 KB)

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Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019

Authors and Affiliations

  1. 1.Forest Department, Forestry CollegeHebei Agricultural UniversityBaodingPeople’s Republic of China
  2. 2.Hebei Key Laboratory for Tree Genetic Resources and Forest ProtectionBaodingPeople’s Republic of China
  3. 3.Cangzhou Forestry BureauCangzhouPeople’s Republic of China

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