An efficient virus‐induced gene silencing (VIGS) system for gene functional studies in Miscanthus
An efficient virus‐induced gene silencing (VIGS) system for gene functional studies in Miscanthus
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DOI：
10.1111/gcbb.13051
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
Guo He;Xuhong Zhao;Yan Xu;Yu Wang;Zhihai Zhang;Liang Xiao;M. Hudson;Ruibo Hu;Shengjun Li
中科院分区：
文献类型：
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作者：
Guo He;Xuhong Zhao;Yan Xu;Yu Wang;Zhihai Zhang;Liang Xiao;M. Hudson;Ruibo Hu;Shengjun Li
病毒诱导的基因沉默（VIGS）是植物中瞬时基因功能分析的有力工具，特别是对于单子叶植物物种（例如，草类）对转化反应迟钝。尽管已经在不同的植物物种中开发了各种VIGS系统，但以前没有一种可用于生物能源作物芒草。本文报道了烟草摇铃病毒（TRV）介导的芒属植物VIGS系统的建立。我们首先研究了可能影响基因沉默效率的各种因素的影响，使用芒草八氢番茄红素脱氢酶（MsPDS）基因作为光漂白的视觉指标。然后，我们使用正交实验设计优化了TRV诱导的VIGS程序，其中四个因素（芽大小，农杆菌浓度，真空渗透时间和共孵育时间）各为三个水平。以下导致最高的沉默效率（~76%）：接种发芽幼苗（1.0-2.0 mm），根癌农杆菌培养物生长至在600 nm处的光密度（OD 600）为0.4，真空渗透90 min，并且共孵育5 h。所建立的VIGS系统对两种M. sinensis和M. lutarioriparius，具有相当的基因沉默效率。我们通过MYB转录因子MsMYB 112在盐胁迫耐受性中的作用的功能表征来验证VIGS系统的功效。MsMYB 112的表达使用VIGS系统成功地敲低，这导致沉默的芒属植物的耐盐性受损。因此，建立的基于TRV的VIGS系统可以大大促进芒属植物的功能基因组研究。
Virus‐induced gene silencing (VIGS) is a powerful tool for transient gene functional analysis in plants, especially for monocot species (e.g., grasses) that are recalcitrant to transformation. Despite various VIGS systems that have been developed in different plant species, none was previously available for the bioenergy crop Miscanthus. Here, we report the establishment of an efficient and robust VIGS system mediated by Tobacco Rattle Virus (TRV) in Miscanthus. We first investigated the impact of various factors that may affect gene silencing efficiency using the Miscanthus sinensis Phytoene Desaturase (MsPDS) gene as a visual indicator of photobleaching. Then, we optimized the TRV‐elicited VIGS procedure using an orthogonal experimental design with four factors (sprout size, Agrobacterium concentration, vacuum infiltration time, and co‐incubation time) each at three levels. The following led to the highest silencing efficiency (~76%): inoculation of germinating seedlings (1.0–2.0 mm), Agrobacterium tumefaciens culture grown to optical density at 600 nm (OD600) of 0.4, vacuum infiltration for 90 min, and co‐incubation for 5 h. The VIGS system established was applicable for both M. sinensis and M. lutarioriparius, with comparable gene silencing efficiency. We verified the efficacy of the VIGS system via the functional characterization of the role of a MYB transcription factor, MsMYB112, in salt stress tolerance. Expression of MsMYB112 was successfully knocked down using the VIGS system, and this led to compromised salt tolerance in the silenced Miscanthus plants. The TRV‐based VIGS system established may, therefore, substantially facilitate functional genomic studies in Miscanthus.