College of Horticulture and Landscape, Yunnan Agricultural University, Kunming, Yunnan, 650201, People's Republic of China.
Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agriculture Sciences, Kunming, Yunnan, 650205, People's Republic of China.
BMC Genomics. 2022 May 5;23(Suppl 1):346. doi: 10.1186/s12864-022-08549-x.
The tomato (Solanum lycopersicum L.) is an economically valuable crop grown worldwide. Because the use of sterile males reduces the cost of F1 seed production, the innovation of male sterility is of great significance for tomato breeding. The ABORTED MICROSPORES gene (AMS), which encodes for a basic helix-loop-helix (bHLH) transcription factor, has been previously indicated as an essential gene for tapetum development in Arabidopsis and rice. To determine the function of the SlAMS gene (AMS gene from S. lycopersicum) and verify whether it is a potential candidate gene for generating the male sterility in tomato, we used virus-induced gene silencing (VIGS), CRISPR/Cas9-mediated genome editing and over-expression technology to transform tomato via Agrobacterium infection.
Here, the full-length SlAMS gene with 1806 bp from S. lycopersicum (Accession No. MK591950.1) was cloned from pollen cDNA. The results of pollen grains staining showed that, the non-viable pollen proportions of SlAMS-silenced (75%), -knockouted (89%) and -overexpressed plants (60%) were significantly higher than the wild type plants (less than 10%; P < 0.01). In three cases, the morphology of non-viable pollen grains appeared tetragonal, circular, atrophic, shriveled, or otherwise abnormally shaped, while those of wild type appeared oval and plump. Furthermore, the qRT-PCR analysis indicated that SlAMS in anthers of SlAMS-silenced and -knockouted plants had remarkably lower expression than in that of wild type (P < 0.01), and yet it had higher expression in SlAMS-overexpressed plants (P < 0.01).
In this paper, Our research suggested alternative approaches to generating male sterility in tomato, among which CRISPR/Cas9-mediated editing of SlAMS implied the best performance. We also demonstrated that the downregulation and upregulation of SlAMS both affected the pollen formation and notably led to reduction of pollen viability, suggesting SlAMS might be essential for regulating pollen development in tomato. These findings may facilitate studies on clarifying the SlAMS-associated molecular regulatory mechanism of pollen development in tomato.
番茄（Solanum lycopersicum L.）是一种具有经济价值的世界性作物。由于使用不育雄性可降低 F1 种子生产成本，因此雄性不育的创新具有重要意义。ABORTED MICROSPORES 基因（AMS），其编码一个碱性螺旋-环-螺旋（bHLH）转录因子，先前被表明是拟南芥和水稻绒毡层发育所必需的基因。为了确定 SlAMS 基因（来自 S. lycopersicum 的 AMS 基因）的功能，并验证其是否是番茄雄性不育的潜在候选基因，我们使用病毒诱导的基因沉默（VIGS）、CRISPR/Cas9 介导的基因组编辑和过表达技术通过农杆菌感染转化番茄。
从花粉 cDNA 中克隆出番茄全长 SlAMS 基因（1806 bp），其登录号为 MK591950.1。花粉粒染色结果表明，SlAMS 沉默（75%）、敲除（89%）和过表达植株（60%）的非活力花粉比例明显高于野生型（小于 10%；P<0.01）。在三种情况下，非活力花粉粒的形态呈四边形、圆形、萎缩、皱缩或其他异常形状，而野生型的花粉粒呈椭圆形和饱满状。此外，qRT-PCR 分析表明，SlAMS 在 SlAMS 沉默和敲除植株的花药中的表达明显低于野生型（P<0.01），而在 SlAMS 过表达植株中的表达更高（P<0.01）。
本文提出了在番茄中产生雄性不育的替代方法，其中 CRISPR/Cas9 介导的 SlAMS 编辑表现最佳。我们还表明，SlAMS 的下调和上调都影响花粉的形成，并显著导致花粉活力降低，这表明 SlAMS 可能对调控番茄花粉发育至关重要。这些发现可能有助于阐明番茄花粉发育中 SlAMS 相关分子调控机制的研究。
