Analysis Of MiRNAs And Construction Of Infectious Clones In Virus/viroid Infected Peach

Peach is one of the most important cultivated fruit trees in China,peach trees mainly propagate asexually by grafting,which can easily lead to the accumulation and transmission of viruses.In recent years,with the increase of peach planting areas,the infection and spread of virus/viroid has gradually become an important factor affecting the healthy development of peach industry.In this study,miRNAs and their target genes from nectarine leaf and fruit tissues infected with different virus/viroid combinations were identified and characterized.We amplified the full-length genome sequences of NSPaV and PLMVd,constructed the full-length infectious c DNA clone of NSPAV and infectious clone of dimeric PLMVd and the inoculation of the above infectious clone were preliminarily completed.Our study provided a reference for exploring the regulatory mechanism of miRNA in peach in response to virus/viroid infection,and for studying the interaction between different viruses and viroid in peach.1.In this study,four high-quality s RNA libraries of peach infected by NSPaV and PLMVd were constructed,miRNAs and their targets in peach leaves and fruit tissues were comprehensively analyzed by high-throughput sequencing technology.(1)A total of 116 known peach miRNAs belonging to 49 miRNA families were identified,including some highly conserved miRNAs,such as miR156,miR166,miR167,miR390,miR395,and miR482.303 novel miRNAs were predicted,most of which were 24-nt in length,and most of the new miRNAs were from the miR5271 and miR7717 families.The target gene prediction of miRNAs differentially expressed between the groups showed that these miRNAs targeted 2785 genes,which involved transcriptional regulation,morphogenesis,signal transduction,pathogen response and other aspects.(2)Comparative profiling revealed that 45 miRNAs belonging to 27 known miRNA families exhibited significant differential expression between leaf and fruit tissue.Moreover,the expression levels of these miRNAs were significantly affected in the two tissues despite the fact that the trees were infected by different virus/viroid combinations;for example,miR167 a,miR172a-3p,miR319 a,miR395a-5p,and miR398a-3p were down-regulated in fruit tissue,while miR1511-5p,miR156 a,miR159,miR162,miR164 a,and miR171d-5p were up-regulated in fruit tissue,the expression of those miRNAs appears to be tissue-specific.(3)There were many more miRNAs differentially expressed in leaf tissues than in fruits under combined infection of different viruses/viroid,and a few miRNAs were identified that play important roles in the response to virus/viroid infection(miR482d-5p,miR6271,pp06-33439 and so on).Their predicted target genes encode disease-related genes such as calmodulin binding protein,ubiquitin protein ligase,zinc finger protein and RPM1 interaction protein.2.We amplified the full-length genomes of NSPaV and PLMVd by RT-PCR and RACE.We constructed the full-length infectious c DNA clone of NSPaV and infectious clone of dimeric PLMVd with skeleton of p CB301 and p GEM-T vector respectively.The above infectious clones were inoculated into the leaves of common herb indicator plants with injection of agrobacterium and friction inoculation respectively.NSPaV and PLMVd were detected in tomato/cucumber in the early stage,which need to be confirmed later.We inoculated the infectious clone into peach seedlings,suspected positive spots appeared in the Dot blot one month after inoculation,but no positive plants were detected in the subsequent 2-4 months.The possible reason might be that peach genetic transformation system is very complex,the infection of virus/viroid could be latent.We have re-inoculated the plants and will test them continuously.