Phytopathology. 2003 Mar;93(3):278-85. doi: 10.1094/PHYTO.2003.93.3.278.
ABSTRACT In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.
摘要 原位反转录聚合酶链反应（RT-PCR）用于杏仁（Prunus dulcis，核果）的幼叶（来自树木和体外芽）和花蕾中，以定位李痘病毒（PDV，属于 Ilarvirus 属）。从固定在甲醛中并包埋在石蜡中的样品中获得的切片在甲醛中重新固定，以增加 RT-PCR 步骤中组织的保存。PDV 的外壳蛋白基因被用作目标，以产生 cDNA 拷贝，该拷贝通过 PCR 扩增，并使用带有 digoxigenin 标记核苷酸的直接检测方法可视化。优化了蛋白消化、PCR 和检测策略，以提高组织保存和信号强度。在幼叶和花蕾的维管束组织以及发育中的花器官的叶肉以及花粉粒的生殖细胞和营养细胞中均发现 PDV 感染样品。PDV 信号在环绕细胞核的环中观察到，并在细胞质中扩散。根据技术优化和 PDV 在组织中的分布以及通过花粉传播的情况讨论了结果。优化的原位 RT-PCR 方案是一种揭示低丰度 RNA 种类的强大技术。因此，它适合研究木本植物中 RNA 病毒的细胞和亚细胞分布。
