A Study of the Distribution of Apple stem pitting virus in Tissues of Pear Tree Using In Situ Hybridization and In Situ RT-PCR

To understand the distribution of Apple stem pitting virus (ASPV) in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL−1and 0.4 mmol L−1 for RNasin and dNTPs respectively, 0.1–1.3 U μL−1 SuperScript II, 0.6–0.8 μmol L−1 primer concentration, and above 0.5 U 100 μL−1LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60°C with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.