MAIN CONCLUSION CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species. Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, is an effective technique for analysing the functions of genes in plants. However, no VIGS vectors have been available for Brassica oleracea until now. Here, tobacco rattle virus (TRV), pTYs and cabbage leaf curl virus (CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using the phytoene desaturase (PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression of PDS and observed photobleaching phenomena in cabbage in response to pTYs and CaLCuV, with the latter being more easy to operate and less expensive. The parameters potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including the targeting fragment strategy, inoculation method and incubation temperature, were then compared. The optimized CaLCuV-based VIGS system involves the following: an approximately 500 bp insert sequence, an Agrobacterium OD600 of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22 °C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected the Mg-chelatase H subunit (ChlH) gene in cabbage and knocked down its expression, and we observed yellow leaves, as expected. We successfully applied the CaLCuV-based VIGS system to two other representative Brassica crop species, B. rapa and B. nigra, and thus expanded the application scope of this system. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crop species.

中文翻译：

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使用基于卷心菜卷叶病毒 (CaLCuV) 的载体的芸苔属植物功能基因组学的有效病毒诱导基因沉默 (VIGS) 系统

主要结论 基于 CaLCuV 的 VIGS 在卷心菜中有效发挥作用，并有助于在芸苔属作物物种中进行有效的功能基因组学研究。病毒诱导基因沉默（VIGS）是一种转录后基因沉默方法，是分析植物基因功能的有效技术。然而，到目前为止，还没有可用于芸苔的 VIGS 载体。在这里，选择烟草脆裂病毒 (TRV)、pTYs 和卷心菜卷叶病毒 (CaLCuV) 基因沉默载体 (PCVA/PCVB) 来改进卷心菜中的 VIGS 系统，使用八氢番茄红素去饱和酶 (PDS) 基因作为有效的视觉指标维格斯。我们成功地沉默了 PDS 的表达并观察到卷心菜中响应 pTYs 和 CaLCuV 的光漂白现象，后者更易于操作且成本更低。然后比较了可能影响CaLCuV在卷心菜中沉默VIGS效率的参数，包括靶向片段策略、接种方法和孵育温度。优化的基于 CaLCuV 的 VIGS 系统包括以下内容：大约 500 bp 的插入序列、1.0 的农杆菌 OD600、使用在芽阶段应用的真空渗透方法以及 22°C 的孵化温度。使用这些参数，我们实现了 65% 的稳定消音效率。为了进一步测试该系统的有效性，我们选择了卷心菜中的镁螯合酶 H 亚基 (ChlH) 基因并敲低了它的表达，我们观察到了黄叶，正如预期的那样。我们成功地将基于 CaLCuV 的 VIGS 系统应用于另外两种具有代表性的芸苔属作物物种 B. rapa 和 B. nigra，从而扩大了该系统的应用范围。我们在此描述的 VIGS 系统将有助于对芸苔属作物物种进行有效的功能基因组学研究。