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

Jiali Ying ¹Yan Wang ¹Liang Xu ¹Tiaojiao Qin ¹Kai Xia ¹Peng Zhang ¹Yinbo Ma ²Keyun Zhang ³Lun Wang ²Junhui Dong ¹Lianxue Fan ¹Yuelin Zhu ¹Liwang Liu⁴

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作者信息

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,Nanjing 210095,China
2. College of Horticulture and Landscape Architecture,Yangzhou University,Yangzhou 225009,China
3. College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China
4. 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,Nanjing 210095,China;College of Horticulture and Landscape Architecture,Yangzhou University,Yangzhou 225009,China
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Abstract

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.

Key words

Raphanus sativus L./VIGS/CRISPR/Cas9/Agrobacterium rhizogenes/A.tumefaciens/RsPDS

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基金项目

Jiangsu Seed Industry Revitalization Project,China(JBGS2021071)

中央高校基本科研业务费专项(YDZX2023019)

国家自然科学基金(32172579)

earmarked fund for Jiangsu Agricultural Industry Technology System,China(JATS2023421)

Jiangsu Postgraduate Scientific Research Innovation Plan,China(KYCX21_0610-2021)

Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)()

出版年

2024

农业科学学报(英文)

中国农业科学院农业信息研究所

农业科学学报(英文)

CSTPCD

影响因子：0.576

ISSN：2095-3119

参考文献量56

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