Zhang Ruimin, Chang Jingjing, Li Jiayue, Lan Guangpu, Xuan Changqing, Li Hao, Ma Jianxiang, Zhang Yong, Yang Jianqiang, Tian Shujuan, Yuan Li, Zhang Xian, Wei Chunhua
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
State Key Laboratory of Vegetable Germplasm Innovation, Tianjin Kernel Vegetable Research Institute, Tianjin, 300384, China.
Hortic Res. 2021 Dec 1;8(1):258. doi: 10.1038/s41438-021-00695-9.
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.
尽管雄性不育已被确定为利用杂种优势和生产杂交种子的有用性状，但其在葫芦科物种中的潜在分子机制仍不清楚。在此，报道了一个自发的雄性不育西瓜突变体Se18，其绒毡层发育异常，导致花粉粒完全败育。基于图谱的克隆表明，致病基因西瓜异常绒毡层1（ClATM1）编码一个具有10bp缺失的基本螺旋-环-螺旋（bHLH）转录因子，并在Se18植株中产生一种没有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。通过CRISPR/Cas9介导的诱变验证了ClATM1在调节花药发育中的遗传功能。此外，ClATM1在Se18突变体中显著下调，在转录水平上呈现明显的剂量效应。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可以通过与启动子结合来激活其自身的转录表达。总的来说，ClATM1是从西瓜中克隆的第一个雄性不育基因，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。
