Analysis of the function of miRNA on the resisitance to white-rot disease in Vitis davidii based on microRNA sequencing

Abstract: 【Objective】Grape white-rot [Coniella diplodiwlla (speg.) Petrak & Sydow] is one of themost destructive diseases in grape. The traditional fungicide control not only could increases the produc-tion cost, but also would affect the environment. Therefor, it is necessary to use the resistance genes ofwild grape in China and improve the resistance of new varieties. An wild grape strain 0943 (Vitis davidii) was found to be resistant to the grape white rot in our previous study. It has been known that mi-croRNAs (MiRNAs) play important role in plant disease resistance. This study intended to explore themiRNA mechanism of expression regulation in V. davidii after infection by pathogenic bacteria.【Methods】For providing plant tissues for sRNA sequencing analysis, 2-year-old plant of Vitis davidii and Vitisvinifera'Manicure Finger'were grown in a greenhouse at 28 ℃ with a 16 h photoperiod. These plantswere inoculated with C. diplodiella by fixing four mycelium gelose discs (diameter of 2 mm) on eachleaf with small pins and covering the leaf. Leaf samples were collected at 0, 12 and 36 hours post inocu-lation (hpi) . 3 μg of total RNA for each sample was taken to create a small RNA library. Gene Ontolo-gy's study of the distribution of candidate target genes in Gene Ontology would elucidate how the sam-ple differences in experiments were reflected in gene function. KEGG analysis, and significant enrich-ment through pathway were used to identify the most important biochemical and signal transductionpathways for candidate target genes involved. KEGG enrichment analysis was made using KOBAS.【Results】We sequenced three small RNA libraries from Vitis davidii. In this study, we obtained 3.282 Gdata, each library data was over 0.5 G. 106 miRNA mature bodies were obtained through sequencing, ofwhich 91 were able to match the miRNA precursor, and the types of miRNA that could match were6 567. After statistics, the miRNA number of V. davidii was more thanthat of the'Manicure Finger'at0 hpi, but the induced expression of miRNA was lower than the'Manicure Finger'. The number ofmiRNA species increased at 12 hpi and 36 hpi points in both V. davidii and'Manicure Finger', and themiRNA species of V. davidii increased from 862 to 1 300 and 1 115, while'Manicure Finger'miRNAspecies increased from 828 to 1 384 and 1 078. In the differential expression analysis of miRNAs, themiRNAs with significantly different expressions at 12 hpi and 36 hpi were used as references for thethorny grape at the 0 hpi treatment point, and their target genes were analyzed by KEGG enrichment.The analysis showed that miRNAs played a role in 11 life pathways through 847 target genes, of whichthe target genes involved in the protein processing in endoplasmic reticulum pathway were the most, and the target genes involved in the Pantothenate and CoA biosynthesis pathway were the least, accounting for 23. Through the prediction of miRNA target genes, the basal metabolic pathways with sugar me-tabolism as the core were analyzed. The results showed that in spiny grapes, all basal metabolic path-ways including the Citrate cycle and the oxidative phosphorylation of the NADH energy synthesis path-way, were in a down regulation mode; CoA-mediated energy release Coumaroyl-CoA pathway was inan up regulation mode. In this study, the differences between V. davidii and'Manicure Finger'were an-alyzed. The different expression of 150 was obtained. And 44 miRNA was different between V. davidiiand 'Manicure Finger'. At the same time, 5 miRNA were specifically expressed in V. davidii, but werenot expressed in 'Manicure Finger'. Target gene prediction showed that the target gene includedWRKY, SPL, EFR and other transcription factors related to disease resistance, and also included LRRdisease-resistant genes. 【Conclusion】In this study, 5 specific expressions of candidate miRNA, mir172 a, miR172 b, miR845 a, novel＿81 and miR159 a in V. davidii were screened, which could be usedfor the study of resistance to grape white rot in V. davidii.