Screening And Identification Of Grape LncRNA Related To Resistance To Downy Mildew

Grape downy mildew is a worldwide disease caused by Plasmopara viticola.It is widely distributed in all grape producing areas in China,especially in wet and humid areas,and mainly harms the leaves.The importance of long non-coding RNA(lncRNA)in plant disease resistance has been reported more and more in recent years,but there has been no specific report on lncRNAs in grape,especially on disease resistance.In the early stage of this study,transcriptome sequencing was performed on the leaves of Chinese wild Vitis piasezkii accession ’Liuba-8’ and European grape Vitis vinifera cultivar ’Pinot Noir’ at six time points of0 h,12h,24 h,48h,96 h and 120 h after inoculation with downy mildew.Based on transcriptome sequencing results,possible lncRNAs were screened out in this study and the characteristics of grape lncRNAs were analyzed.Then,the cis-and trans-regulatory target genes of lncRNAs were predicted,and the possible functions of lncRNAs were analyzed.Then the micro RNAs that lncRNAs might target were predicted and lncRNAs play a role as micro RNA endogenous target simulators.An lncRNA that might play a positive role in grape resistance to downy mildew was screened and preliminarily verified by transient overexpression.The main results are as follows:(1)4011 candidate long non-coding RNAs(lncRNAs)were screened and their characteristics were analyzed.Lnc RNAs were evenly distributed on each chromosome of grape.Similar to protein-coding genes,the gene density near telomeres was significantly higher than the heterochromatin region around centromeres.According to the relative position of the nearest protein-coding gene on the chromosome,lncRNAs can be divided into four types: righteous lncRNAs,antisense lncRNAs,intronic lncRNAs,and intergene lncRNAs.The number of four types of lncRNAs from Watson chain and Crick chain was similar.Grape lncRNAs are shorter and contain fewer exons than protein-coding genes.(2)Using bioinformatics software and some programming languages,3643 lncRNAs were predicted to cis-regulate 6622 protein-coding genes,Go function enrichment was performed on target genes to predict the possible functions of lncRNA,and significant enrichment was found in disease-resistance related modules such as ADP binding.and 91 lncRNAs acted on 31824 protein-coding genes through trans-regulation.Based on Weighted Gene Co-Expression Network Analysis(WGCNA),trans-target genes were divided into 18 modules to construct gene network map.117 grape micro RNAs were predicted to potentially target 184 lncRNAs,and 6 lncRNAs were predicted to be endogenous targeting mimics of 15 micro RNAs.The e TM binding site sequences of the same miRNA in grape(Vitis vinifera)and model plant Arabidopsis thaliana were compared,and the predicted miRNA binding sites were well conserved in the e TMs of the same miRNA in both species,while the nucleotides that formed the bulge regions in the e TMs were not conserved.(3)The differentially expressed genes were detected by real-time fluorescence quantitative PCR,and an lncRNA MSTRG.12742.1 was screened out,which may have a positive effect on resistance to grapes downy mildew.The target genes of MSTRG.12742.1were predicted according to Pearson coefficient of expression level,which were mainly enriched in photosynthetic membrane.The target genes with the highest positive correlation showed that it might regulate the level of stomatal opening in leaves.The grape leaves were transformed by vacuum and then inoculated by using "leaf disc method".Aniline blue staining was used to observe different periods after inoculation.It was found that the inoculated grape leaves had a certain inhibitory effect on downy mildew in the early stage.