An efficient virus-induced gene silencing vector for maize functional genomics research
An efficient virus-induced gene silencing vector for maize functional genomics research
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用于玉米功能基因组学研究的高效病毒诱导基因沉默载体
DOI：
10.1111/tpj.13142
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发表时间：
2016
期刊：
影响因子：
7.2
通讯作者：
Zhou Tao
中科院分区：
文献类型：
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作者：
Wang Rong;Yang Xinxin;Wang Nian;Liu Xuedong;Nelson Richard S.;Li Weimin;Fan Zaifeng;Zhou Tao
玉米是世界主要农作物，其丰富的遗传多样性为遗传研究提供了先进的资源。然而，一直缺乏可用于大多数玉米品种的玉米中快速瞬时基因功能分析的工具，导致依赖于耗时的稳定转化和突变研究来获得答案。我们基于天然感染玉米的黄瓜花叶病毒（CMV）株系ZMBJ-CMV开发了用于玉米的有效病毒诱导的基因沉默（VIGS）载体。构建了ZMBJ-CMV的侵染性克隆，并优化了农杆菌的维管束穿刺接种方法，以提高其在玉米CMV侵染中的应用。然后将ZMBJ-CMV修饰为用作VIGS载体。ZMBJ-CMV载体在许多玉米品系中诱导了轻度至中度症状，使其可用于极其重要的玉米栽培品种（例如测序的参考近交系B73）的基因功能研究。使用这种CMV VIGS系统，发现与非沉默组织相比，两种内源基因ZmPD和ZmIspH的表达分别降低了75%和78%。长度为100-300 bp的引物产生最完整的转录和视觉沉默表型。此外，自噬相关基因ZmATG 3和ZmATG 8a也被沉默，发现它们在叶片淀粉降解中起作用。这些结果表明，我们的ZMBJ-CMV VIGS载体为玉米中快速有效的基因功能研究提供了工具。
Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time‐consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus‐induced gene silencing (VIGS) vector for maize based on a naturally maize‐infecting cucumber mosaic virus (CMV) strain, ZMBJ‐CMV. An infectious clone of ZMBJ‐CMV was constructed, and a vascular puncture inoculation method utilizingAgrobacteriumwas optimized to improve its utility for CMV infection of maize. ZMBJ‐CMV was then modified to function as a VIGS vector. The ZMBJ‐CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes,ZmPDSandZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non‐silenced tissue. Inserts with lengths of 100–300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy,ZmATG3andZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ‐CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize.