Development and application of the TRV-induced gene-silencing system in different Rhododendron species

IF 2.3 3区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Plant Cell, Tissue and Organ Culture Pub Date : 2024-05-27 DOI:10.1007/s11240-024-02782-w

Yunyun Xu, Yumeng Cui, Hanyu Chen, Ya Pu, Chunying Zhang, He Huang

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Abstract

Rhododendron is one of the top ten traditional flowers in China, with high ornamental and medicinal values. However, molecular research on this species has been impeded by the lack of efficient molecular genetic techniques. Virus-induced gene silencing (VIGS) technology is an effective tool for analyzing gene functions, which has been successfully applied to many plant species. But there have been no reports of VIGS system for Rhododendron. In this study, tobacco rattle virus (TRV) was chosen to infect Rhododendron micranthum using phytoene desaturase (PDS) as the reporter gene. After the inoculation of pTRV2-RmPDS by leaf injection, photobleaching phenomena appeared in the newly developed leaves and the RT-qPCR assay demonstrated that RmPDS was successfully silenced. Then three parameters including the developmental stage, the Agrobacterium concentration and the inoculation temperature were examined to optimize the system. The silencing efficiency was increased from 2.4 to 11.4% and the optimized conditions were as follows: the developmental stage of the two true-leaf stage, the adjustment of the inoculation solution to a final OD₆₀₀ = 1.5 and the inoculation temperature of 18 ℃. To further validate the system, the most optimal combination was used to infect other six rhododendron genotypes. R. mucronulatum, R. ovatum, R. × pulchrum, R. simsii and R. yedoense displayed the silenced phenotype of PDS as expected. We successfully established TRV-mediated VIGS technology in Rhododendron which could evaluate and characterize the function of plant genes without the need for cumbersome tissue culture.

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在不同杜鹃花品种中开发和应用 TRV 诱导的基因沉默系统

杜鹃花是中国十大传统名花之一，具有很高的观赏价值和药用价值。然而，由于缺乏高效的分子遗传技术，该物种的分子研究一直受到阻碍。病毒诱导基因沉默（VIGS）技术是分析基因功能的有效工具，已成功应用于许多植物物种。但目前还没有关于杜鹃花 VIGS 系统的报道。本研究选择了烟草鼠疫病毒（TRV）感染杜鹃花小鳞茎，并使用植物烯去饱和酶（PDS）作为报告基因。通过叶片注射接种 pTRV2-RmPDS 后，新长出的叶片出现光漂白现象，RT-qPCR 检测证明 RmPDS 被成功沉默。随后，研究了发育阶段、农杆菌浓度和接种温度等三个参数，对系统进行了优化。沉默效率从 2.4% 提高到 11.4%，优化条件如下：发育阶段为两片真叶期，将接种液调整到最终 OD600 = 1.5，接种温度为 18 ℃。为了进一步验证该系统，我们使用了最理想的组合来感染其他六种杜鹃花基因型。R.mucronulatum、R.ovatum、R.×pulchrum、R.simsii和R.yedoense如期表现出PDS的沉默表型。我们成功地在杜鹃花中建立了 TRV 介导的 VIGS 技术，该技术无需繁琐的组织培养即可评估和鉴定植物基因的功能。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Plant Cell, Tissue and Organ Culture 生物-生物工程与应用微生物

CiteScore

5.40

自引率

13.30%

发文量

203

审稿时长

3.3 months

期刊介绍： This journal highlights the myriad breakthrough technologies and discoveries in plant biology and biotechnology. Plant Cell, Tissue and Organ Culture (PCTOC: Journal of Plant Biotechnology) details high-throughput analysis of gene function and expression, gene silencing and overexpression analyses, RNAi, siRNA, and miRNA studies, and much more. It examines the transcriptional and/or translational events involved in gene regulation as well as those molecular controls involved in morphogenesis of plant cells and tissues. The journal also covers practical and applied plant biotechnology, including regeneration, organogenesis and somatic embryogenesis, gene transfer, gene flow, secondary metabolites, metabolic engineering, and impact of transgene(s) dissemination into managed and unmanaged plant systems.