Whole-mount RNA in situ hybridization technique in Torenia ovules
Whole-mount RNA in situ hybridization technique in Torenia ovules
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DOI：
10.1007/s00497-022-00455-2
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发表时间：
2022-12
影响因子：
3.4
通讯作者：
Shihao Su;Xuan Zhou;T. Higashiyama
中科院分区：
文献类型：
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作者：
Shihao Su;Xuan Zhou;T. Higashiyama
感兴趣基因在细胞水平上的表达模式为其功能提供了强有力的证据。RNA原位杂交技术已被证明是检测基因在不同生物体中时空表达模式的有力工具。然而，经典的RNA原位杂交（ISH）技术是耗时的，需要复杂的切片技术。因此，我们建立了一种以植物繁殖研究的模式种蓝猪耳胚珠为材料的全胚原位杂交（WISH）方法。Fournieri的胚珠上有突出的胚囊，通过简单的操作，很容易观察和成像。为了确定经典ISH和我们新建立的WISH的效果，我们使用这两种方法检测了D类基因TfSTK3的表达。TfSTK3在经典ISH和WISH中的表达模式相似，证实了WISH方法的可靠性。与WISH相比，经典ISH常导致胚囊变形，难以区分雌配子体内部的信号。为了了解我们的WISH方案是否也能很好地检测胚囊内表达的基因，我们进一步检查了增效物富集的候选物TfPMEI1的表达，并清楚地观察到两个增效细胞内的特异性信号。总之，我们的WISH技术允许可视化的基因表达模式在胚珠T。Fournieri在一周内，并将有利于未来的植物繁殖领域。
The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial–temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules ofTorenia fournieri, which is a model species in the study of plant reproduction.T. fournieripossesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene,TfSTK3, using both methods. The expression patterns ofTfSTK3are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate,TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules ofT. fournieriwithin one week and will benefit the field of plant reproduction in the future.