Localization Of Apple Chlorotic Leaf Spot Virus In Apple Tissue

Apple virus disease has seriously affected the yield and quality of fruit trees. Apple chlorotic leaf spot virus (ACLSV) is one of the latent viruses which can turn the apple leaf yellow, lead to fruit malformation, poor quality, and even cause large areas of fruit to die. So the establishment of rapid detection technology and the distribution characteristics of analysis of the virus place an important role in controlling the harm of virus and understanding the virus pathogenic mechanism. In this experiment we mainly detect the virus rapidly by molecular hybridization and position the virus which is in the tissue of apple leaf by in situ reverse transcription PCR (IS-RT-PCR) technology.Firstly, this research aims to explore and establish a system for detecting ACLSV by molecular hybridization technology. The total RNA which was extracted from the leaves of infected apple by CTAB method was used as template to amplify the expected fragment with the ACLSV specific primers, and the specific fragment of ACLSV was verified by cloning and sequencing. Plasmid with the specific fragment was used as template to prepare the digoxigenin-labeled cDNA probe by PCR. The system of detecting ACLSV with cDNA probe was established. The plasmid was used as template which can effectively prepare a number of non-radioactive digoxigenin-labeled probes which are relatively safe, highly sensitive and have important application value. Probes detected as effective can be used for localization of tissue by in situ PCR.Firstly, sections of apple leaf tissue biopsies were fixed on superfrost plus for carrying out the reaction of in situ RT-PCR. Secondly, the effectively digoxigenin-labeled probes prepared in advance were used in the reaction of in situ hybridization. Finally, the section of apple leaf tissue biopsies was observed under the inverted microscope. The result showed that the palisade tissue which in the mesophyll cells of infected apple leaf was found a lot of purple spots, spongy tissue also presented blue-violet in individual position, and the negative control did not appear chromogenic reaction. So it can be speculated that the spongy tissue has low levels of virus, and the virus mainly focus on palisade tissue. A large number of chloroplasts were existed in the palisade tissue, the virus may interfere with the formation of the chloroplast through affecting the replication of chloroplast. And it may further hinder the photosynthesis of leaves and inhibit the growth of plant leading to plant dysplasia in the end.In the situ PCR reaction, the Poly-L-Lysine coated slides was compared to the superfrost plus one, which showed that the Poly-L-Lysine coated slides in preventing the slice falling off from glass slides was slightly inferior to superfrost plus one, and combining the method of packaging the superfrost with hybrid bag can successfully solve the problem of slice off in situ PCR reaction.