关键词: 辣椒, 辣椒轻斑驳病毒, 原位RT-PCR, 地高辛, 组织定位, cDNA

Abstract: The aim of this study was to analyze the characterization of distribution of Pepper mild mottle virus（PMMoV）in tissue of peppers，and an in situ RT-PCR system was developed to locate PMMoV in tissue of peppers. Total RNA was extracted from pepper leaves with a modified CTAB method. The specific fragment was amplified by RT-PCR using total RNA as a temple，and it was testified that the fragment was PMMoV-specific one on the basis of cloning and sequencing. A specific digoxin-labeled cDNA probe of PMMoV was generated according to PCR labeling technique. A system for localization of PMMoV in tissue level was developed with indirect in situ RT-PCR，via optimization of in situ RT-PCR including section preparation（7 μm sections in the Superfrost plus positively charged slides），pre-treating（1 mg ? L-1 protease K digested for 5 min），RT-PCR（performing 2.5 h in 37 ℃ for reverse transcriptionand annealing in 58 ℃ for PCR），and detection of hybridization. The technique，which can analyze the distribution of PMMoV in tissue of peppers，was developed. The result of in situ RT-PCR showed that PMMoV-positive signals were the best in palisade tissues of pepper leaves，and it had lots of PMMoV-positive signals in palisade tissues. Epidermis had also few positive signals. In addition，few positive signals located mostly in epidermis could be found in petioles.

Key words: Capsicum, Pepper mild mottle virus, in situ RT-PCR, digoxin, location in tissue cDNA