Virus-induced gene silencing (VIGS) is a powerful tool for high-throughput analysis of gene function. Here, we developed the VIGS vector pCF93, from which expression of the cucumber fruit mottle mosaic virus genome is driven by the cauliflower mosaic virus 35S promoter to produce viral transcripts in inoculated plants. To test the utility of the pCF93 vector, we identified candidate genes related to male sterility (MS) in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. Specifically, we exploited previously reported reference-based and de novo transcriptome data to define 38 differentially expressed genes between a male-sterile line and its fertile near-isogenic line in the watermelon cultivar DAH. We amplified 200- to 300-bp fragments of these genes, cloned them into pCF93, and inoculated DAH with the resulting VIGS clones. The small watermelon cultivar DAH enabled high-throughput screening using a small cultivation area. We simultaneously characterized the phenotypes associated with each of the 38 candidate genes in plants grown in a greenhouse. Silencing of 8 of the 38 candidate genes produced male-sterile flowers with abnormal stamens and no pollen. We confirmed the extent of gene silencing in inoculated flowers using reverse transcription-qPCR. Histological analysis of stamens from male-fertile and male-sterile floral buds and mature flowers revealed developmental defects and shrunken pollen sacs. Based on these findings, we propose that the pCF93 vector and our VIGS system will facilitate high-throughput analysis for the study of gene function in watermelons.

中文翻译：

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病毒诱导的基因沉默，用于葫芦科基因功能的植物验证。

病毒诱导的基因沉默（VIGS）是基因功能高通量分析的强大工具。在这里，我们开发了 VIGS 载体 pCF93，黄瓜果实斑驳花叶病毒基因组的表达由花椰菜花叶病毒 35S 启动子驱动，在接种的植物中产生病毒转录本。为了测试 pCF93 载体的实用性，我们鉴定了与西瓜 (Citrullus lanatus) 雄性不育 (MS) 相关的候选基因，该基因难以遗传转化。具体来说，我们利用先前报道的基于参考的转录组数据来定义西瓜品种 DAH 中雄性不育系与其可育近等基因系之间的 38 个差异表达基因。我们扩增了这些基因的 200 至 300 bp 片段，将它们克隆到 pCF93 中，并用所得的 VIGS 克隆接种 DAH。小西瓜品种 DAH 能够利用小种植面积进行高通量筛选。我们同时表征了温室植物中与 38 个候选基因相关的表型。38 个候选基因中的 8 个被沉默，产生了具有异常雄蕊且没有花粉的雄性不育花。我们使用逆转录 qPCR 确认了接种花中基因沉默的程度。对雄性可育和雄性不育花蕾和成熟花的雄蕊进行组织学分析，发现发育缺陷和花粉囊萎缩。基于这些发现，我们建议 pCF93 载体和我们的 VIGS 系统将促进西瓜基因功能研究的高通量分析。