Although male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

中文翻译：

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bHLH转录因子异常Tapetum 1的破坏导致西瓜雄性不育

尽管雄性不育已被确定为杂交活力利用和杂交种子生产的有用性状，但其在葫芦科物种中的潜在分子机制仍不清楚。据报道，一种自发的雄性不育西瓜突变体 Se18 的绒毡层发育异常，导致花粉粒完全流产。基于图谱的克隆证明了因果基因Citrullus lanatus 异常绒毡层 1(ClATM1) 编码具有 10 bp 缺失的基本螺旋-环-螺旋 (bHLH) 转录因子，并在 Se18 植物中产生没有 bHLH 相互作用和功能 (BIF) 结构域的截短蛋白。qRT-PCR 和 RNA 原位杂交表明ClATM1在花药发育的 6-8a 阶段，在绒毡层和小孢子母细胞中特异性表达。的遗传功能ClATM1通过 CRISPR/Cas9 介导的诱变验证了在调节花药发育中的作用。而且，ClATM1在 Se18 突变体中显着下调，在转录水平显示出明显的剂量效应。随后的双荧光素酶报告基因、β-葡萄糖醛酸酶 (GUS) 活性和酵母单杂交试验表明，ClATM1 可以通过启动子结合激活其自身的转录表达。集体，ClATM1是第一个从西瓜中克隆的雄性不育基因，其自我调节活性为植物花药发育的分子机制提供了新的见解。