Study On The Effect Of The Replication And Traffic Of Stem Loop Structure Variation Of Peach Latent Mosaic Viroid

The secondary structure of RNA is a key factor in the biological function of RNA.The stem-loop structure is the key structure in the secondary structure of viroid RNA.Different nucleosides can form different spatial structures,affect the binding of the loop region with different cytokines or proteins,and thus affect its biological function.In this study,a Peach latent mosaic viroid(PLMVd)isolate was identified,and the secondary structure variation of Peach latent mosaic viroid was studied.By preparing mutants that turn off different stem-loop structures,using in situ hybridization and real-time PCR to analyze the effects of different stem-loop structures on the replication and traffic of viroids.The main contents are as follows:(1)Using peach leaves as experimental material,total RNA was extracted and amplified by conventional RT-PCR to obtain a specific 339 bp band.After cloning and sequencing,the results showed that the isolate have 96-100%homology to those reported in GenBank.Phylogenetic tree analysis showed that this isolate was closely related to American isolate,but had a distant relationship with the Greek and Turkish isolates.In the phylogenetic tree,there were no obvious geographic correlations between the segregation in different regions.(2)By designing the mutant primers and using a site-directed mutagenesis kit,a plasmid containing the Peach latent mosaic viroid having different stem-loop structures was obtained.Using in vitro transcription technology,the closed stem-loop mutant plasmid was in vitro transcribed into viroid-like RNA,so as to obtain a potential mutant of viroid with different stem-loop structures that could have infectivity.(3)Using healthy tobacco leaves as experimental materials,light enzymolysis was avoided,and protoplast rupture was avoided.Via mesh filtration,W5 solution was washed by centrifugation and suspended in MMg solution.The mutants were then introduced into protoplasts using polyethylene glycol(PEG)method.RNA was extracted after culture and the replication of viroid was detected by real-time PCR.(4)Virus mutants were inoculated into healthy tobacco seedlings by rubbing inoculation method.After two weeks of culture,inoculated leaves and adjacent leaves,which were taken,were used as experimental materials.Paraffin sections were analyzed by in situ hybridization and whole tissue in situ hybridization.The specific distribution of viroid in the tissue was examined.Finally,combined with the results of real-time PCR and in situ hybridization,it showed that the tranffic and replication of mutant of loop 7 were not affected;the tranffic of mutants of loop 1,6,10,12,17,18,19,and 20 was damaged.Most of these loops are concentrated in the variable and right-end regions,as well as the junction between the disease-prone region and the central region.For mutants of loop 5,11,14,16 traffics were damaged.For mutants of loop 4,8,9,13,15 replication is destroyed.For mutants of loop 2,loop 3 replication ability weakened.