The Analysis Of Key Genes In Magnaporthe Oryzae In Response To Citral

Citral has good antibacterial activity against M.oryzae in vitro and in vivo.Previous studies have shown that citral can cause cell wall dissolution of M.oryzae,and interfering with its physiological and biochemical metabolism.However,the key genes responsible for citral response in cell wall of M.oryzae have not been identified yet.Therefore this study under the action of citral is determined by method of enzyme activity of M.oryzae’s chitinase activity,and the differential genes of M.oryzae in response to citral stress were determined by RNA-seq and RT-qPCR,and the mechanism of action of citral on the cell wall of M.oryzae was established.The results provided a reference for exploring the action mechanism of citral on M.oryzae.The main contents are as follows:1.The key period of action of citral on chitinase from M.oryzae was determined.The chitinase activity of M.oryzae treated with citral was determined by enzyme activity method,The chitinase activity of M.oryzae increased significantly at 9,12 and 24h after treatment with citral at the same concentration for different time.The chitinase activity of M.oryzae at 12 h under citral stress at 50 μg/m L was0.0824(U/mg prot),1.77 times higher than that of the control group.The chitinase activity of M.oryzae under citral stress at 100 μg/m L and 200 μg/m L was0.0928(U/mg prot)and 0.0742(U/mg prot)at 9 h,which was 3.23 times and 2.59 times higher than that of the control group.At the same time,the chitinase activity of M.oryzae treated with citral at different concentrations for 9,12 and 24 h changed significantly,and reached the maximum value at 100 μg/m L,and the enzyme activity was 0.0928,0.0837,0.0826(U/mg prot),respectively.2.56,2.31,2.28 times higher than 3 h treatment,respectively.In conclusion,the key time points of citral on M.oryzae were 9,12,24 h,and the key concentration was 50,100 μg/m L2.The differential genes in response to citral stress in M.oryzae are associated with cell wall integrity.The changes of gene transcription level in response to citral stress in M.oryzae were analyzed by RNA-seq technique.Volcano map and Venn analysis showed that the number of differentially expressed genes and specific differentially expressed genes were 2301 and 649,respectively,after treated with citral at 100 μg/m L for 24 h,The GO enrichment analysis showed that the differential genes mainly enriched the transmembrane transport of developmental conidia in mycelium,etc.the cell component mainly concentrates the integral component of the membrane,the integral component of the plasma membrane,the cell plasma membrane,etc.the molecular function mainly concentrates the structural composition of oxidoreductase activity ribosome ATP binding and so on.KEGG enrichment analysis showed that the differentiated genes of M.oryzae were mainly enriched in glycolysis/gluconeogenic,amino sugar and nucleotide sugar metabolism,pathways of glyoxalic acid and dicarboxylic acid metabolism.Moreover,the metabolic pathways of amino and nucleotide sugars include chitin synthesis pathway and UDP synthesis pathway,indicating that citral has a significant effect on the differential expression of cell wall genes of M.oryzae.3.The mechanism of action of citral on the cell wall of M.oryzae was established.Using Pathway analysised the differential genes in response to citral stress of M.oryzae,we found that nine enzymes were differentially expressed in amino and nucleotide sugar metabolism pathways of M.oryzae after treatment with citral at 100 μg/m L for 24 h.among which five enzymes were different in the chitin synthesis pathway,and two enzymes were different in the UDP glycosynthesis pathway.And then,the 22 key genes were screened out in differentially expressed genes in amino sugar and nucleotide sugar metabolism pathways of M.oryzae were analyzed,among them,14 genes were involved in the chitin synthesis pathway and 8genes were involved in the UDP glycosynthesis pathway.Then,a model diagram of the action mechanism of citral on the cell wall of M.oryzae was constructed with Photoshop.The model diagram showed chitin synthase gene expression was down-regulated and chitin synthase gene expression was up-regulated in the chitin synthesis pathway of M.oryzae,which resulted in reduced chitin content in the cell wall of M.oryzae and damaged the cell wall of M.oryzae.The expression levels of22 key genes were determined by RT-qPCR,and the results verified the accuracy of the schematic diagram of the mechanism of action.