EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, Tours, France.
Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Toulouse INP, Toulouse, France.
Methods Mol Biol. 2022;2505:1-32. doi: 10.1007/978-1-0716-2349-7_1.
As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus. RNA in situ hybridization has proven a key technological approach to unravel this complex spatial organization. Pioneer work in 1999 discovered the involvement of epidermis and laticifer/idioblasts in the intermediate and late steps of the pathway, respectively. The localization of the early steps of the pathway to the internal phloem-associated parenchyma later came to complete the three-tissular block organization of the pathway. Since then, RNA in situ hybridization was routinely used to map the gene expression profile of most of the nearly 30 genes involved in this pathway. We introduce here a comparison of advantages and drawbacks of in situ hybridization and more popular promoter: GUS strategies. Two main advantages of in situ hybridization are the suitability to any plant species and the direct localization of transcripts rather than the localization of a promoter activity. We provide a step-by-step protocol describing every details allowing to reach a medium throughput including riboprobe synthesis, paraffin-embedded plant tissue array preparation, prehybridization, in situ hybridization, stringent washing and immunodetection of hybridized probes, and imaging steps. This should be helpful for new comers willing to domesticate the technique. This protocol has no species limitation and is particularly adapted to the increasingly studied model, nonmodel species, nonamenable to promoter::GUS transformation, such as C. roseus.
作为应对其对宿主植物潜在细胞毒性的手段，次生代谢物通常被隔离在特定的细胞类型中。这种空间组织可能达到复杂的顺序多细胞区室化。到目前为止，特征最复杂的例子是长春花中抗癌单萜吲哚生物碱的顺序多细胞生物合成。RNA 原位杂交已被证明是揭示这种复杂空间组织的关键技术方法。1999 年的开创性工作发现，表皮和乳管/异形细胞分别参与途径的中间和晚期步骤。途径早期步骤定位于内部韧皮部相关的薄壁组织，后来完成了途径的三组织块组织。从那时起，RNA 原位杂交被常规用于绘制该途径中涉及的近 30 个基因的基因表达谱。我们在这里介绍了原位杂交和更流行的启动子：GUS 策略的优缺点比较。原位杂交的两个主要优点是适用于任何植物物种和直接定位转录本，而不是定位启动子活性。我们提供了一个逐步的方案描述，包括每个细节，允许达到中等通量，包括核糖探针合成、石蜡包埋植物组织阵列制备、预杂交、原位杂交、严格洗涤和杂交探针的免疫检测以及成像步骤。这对于希望驯化该技术的新手应该有所帮助。该方案没有物种限制，特别适用于越来越多的研究模型，即非模式物种，如长春花，不适合启动子：GUS 转化。
