利用VIGS和CRISPR/Cas9技术验证萝卜RsPDS基因功能

doi:10.1016/j.jia.2024.03.059

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (5): 1557-1567.DOI: 10.1016/j.jia.2024.03.059

利用VIGS和CRISPR/Cas9技术验证萝卜RsPDS基因功能

收稿日期:2023-05-08 接受日期:2024-01-06 出版日期:2024-05-20 发布日期:2024-04-23

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

Jiali Ying¹, Yan Wang¹, Liang Xu¹, Tiaojiao Qin¹, Kai Xia¹, Peng Zhang¹, Yinbo Ma², Keyun Zhang³, Lun Wang², Junhui Dong¹, Lianxue Fan¹, Yuelin Zhu¹, Liwang Liu^{1, 2#}

¹National 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
²College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
³College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China

Received:2023-05-08 Accepted:2024-01-06 Online:2024-05-20 Published:2024-04-23
About author:Jiali Ying, E-mail: 2019204031@njau.edu.cn; #Correspondence Liwang Liu, E-mail: nauliulw@njau.edu.cn
Supported by:
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) and the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).

摘要/Abstract

摘要： 病毒诱导的基因沉默（VIGS）和成簇规律间隔的短回文重复序列/CRISPR相关蛋白（CRISPR/Cas）系统是现代植物生物技术中快速、精准进行基因功能验证的有效手段。然而，关于萝卜基因沉默和编辑的研究仍然有限。在本研究中，利用烟草脆裂病毒（TRV）和芜菁黄化花叶病毒（TYMV）介导的基因沉默技术敲减萝卜中八氢番茄红素脱氢酶基因（RsPDS），获得具有白化表型的株系；且TYMV介导的基因沉默效率高于TRV。萝卜基因型‘NAU-067’叶片中RsPDS的表达水平显著下降。构建带有2个RsPDS靶序列的2300GN-Ubi-RsPDS-Cas9载体，采用发根农杆菌介导的遗传转化对‘NAU-067’无根苗进行侵染。结果发现毛状根中9条不定根经GUS染色呈现蓝色，Sanger测序进一步发现其中4条不定根在RsPDS基因的靶序列1上发生了编辑。此外，通过根癌农杆菌介导的萝卜子叶转化获得了白化株系。株系1在靶序列2处发生了5个碱基替换和3个碱基缺失，株系2在靶序列1处发生了3个碱基插入和3个碱基替换。利用VIGS和CRISPR/Cas9技术可以精准验证萝卜基因的生物学功能，因此，它将促进萝卜育种计划中重要园艺性状的遗传改良。

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 program

Key words: Raphanus sativus L. , VIGS , CRISPR/Cas9 , Agrobacterium rhizogenes , A. tumefaciens , RsPDS

. 利用VIGS和CRISPR/Cas9技术验证萝卜RsPDS基因功能[J]. Journal of Integrative Agriculture, 2024, 23(5): 1557-1567.

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.

Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish [J]. Journal of Integrative Agriculture, 2024, 23(5): 1557-1567.

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利用VIGS和CRISPR/Cas9技术验证萝卜RsPDS基因功能

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

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