Molecular Mechanism Of Magnesium Regulating Tobacco Resistance To PVY~N

Potato virus Y necrosis strain（PVY^N）can cause necrosis of tobacco veins,significantly reducing the quality of tobacco leaves or even losing the value of baking.In recent years,the study of the interaction between mineral elements and plants to resist plant virus diseases has attracted more and more attention.Previous studies in this laboratory have found that magnesium can promote tobacco resistance to PVY^N,but the mechanism of action is not clear.In this paper,the molecular mechanism of Mg element regulating tobacco anti-PVY^Nwas studied by using transcriptome sequencing technology and virus-mediated gene silencing technology,and the main findings were as follows:1.Mg element can reduce the symptoms of PVY^Ninfection with tobacco,and can effectively reduce the accumulation of PVY^NRNA and CP in tobacco leaves.In this experiment,taking ordinary tobacco K326 as the test material,using Hogeland culture solution differential culture,five Mg²⁺concentration gradients were set up,namely 60mg/L,120mg/L,240mg/L,360mg/L,and 480mg/L,and the differences in the incidence of disease between different treatments after inoculation with PVY^Nwere observed.The results showed that the symptoms appeared at the base of the tobacco stem treated with Mg²⁺concentration of 480 mg/L,which was delayed by 2 days compared with other concentrations,and the symptoms were also mild;the accumulation of PVY^NRNA in the leaves was detected by real-time quantitative PCR（RT-q PCR）and the accumulation of PVY^NCP in the leaves was detected by western blot（Western blot）and found that mg element could reduce the accumulation of PVY^NRNA and CP in tobacco leaves,and480mg/The treatment of L was the most different from the treatment of 60 mg/L,and the accumulation of viral RNA was reduced by 14.53%.2.RNA-seq technology was used to analyze the differentially expressed genes after Mg regulated tobacco infection with PVY^Nin combination with bioinformatics methods.In the course of the analysis,we focused on the main aspects of the high degree of enrichment of physiological pathway differential expression genes,such as glutathione metabolism,MAPK signaling pathway-plant,phenylalanine metabolism,andα-linolenic metabolic pathway acid metabolism,correspondingly enriched in glutathione S-transferase（GST）,catalase isoenzyme（CAT）synthesis,phenylalanine ammonia-lyase（PAL）,ribose-1,5-diphosphate carboxylase/oxygenase（Ru BP）;The genes that regulate the synthesis of the above substances were further analyzed and screened out,and the genes with large differential expressions in each treatment were screened,namely Nb GST,Nb CAT,Nb PAL,and Nb Ru BP.3.Using TRV-mediated VIGS technology,the function of the above key genes is verified.In this experiment,key gene fragments were amplified and connected to the TRV vector to construct a VIGS system,and transgenic plants were obtained by infiltrating the lower leaves of Bunsen tobacco.After ten days of silencing PDS gene plants,the silencing efficiency of each treatment gene was about 70%,and PVY^Nwas inoculated.After 10 days of treatment,the accumulation of viral RNA and CP in the leaves on the 10th day after PVY^Ninoculation was detected by RT-q PCR and Western blot,and the related enzyme activities were detected at three time points:1 day before silencing the key gene,10 days after silencing the gene,and 10 days after PVYN inoculation.The results showed that the viral accumulation increased significantly after silencing the key genes,and the activities of enzymes related to the regulation of each gene decreased significantly,which verified that mg element can regulate the ability of the above four genes to induce tobacco to produce anti-PVY^N.