Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish  

作　　者：Jiali Ying Yan Wang Liang Xu Tiaojiao Qin Kai Xia Peng Zhang Yinbo Ma Keyun Zhang Lun Wang Junhui Dong Lianxue Fan Yuelin Zhu Liwang Liu 

机构地区：[1]National Key Laboratory of Crop Genetics&Germplasm Enhancement and Utilization/Key Laboratory of Horticultural Crop Biology and Genetic Improvement(East China)of Ministry of Agriculture and Rural Affairs/College of Horticulture,Nanjing Agricultural University,Nanjing210095,China [2]College of Horticulture and Landscape Architecture,Yangzhou University,Yangzhou 225009,China [3]College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China

出　　处：《Journal of Integrative Agriculture》2024年第5期1557-1567,共11页农业科学学报（英文版）

基　　金：This work was supported by Jiangsu Seed Industry Revitalization Project,China[JBGS(2021)071];Fundamental Research Funds for the Central Universities,China(YDZX2023019);the National Natural Science Foundation of China(32172579);the earmarked fund for Jiangsu Agricultural Industry Technology System,China[JATS(2023)421];the Jiangsu Postgraduate Scientific Research Innovation Plan,China(KYCX21_0610-2021);the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).

摘　　要：Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

关 键 词：Raphanus sativus L. VIGS CRISPR/Cas9 Agrobacterium rhizogenes A.tumefaciens RsPDS 

分 类 号：S631.1[农业科学—蔬菜学]