| Tobacco brown spot,caused by Alternaria alternata,is a serious fungal disease to tobacco production.It often occurs at the later maturity stage of tobacco leaves,and significantly influence the yield and quality of tobacco production.Planting resistant varieties is the most economical and effective way to control this disease.However,the interaction between Alternaria alternata and tobacco has not been illuminated and none of the resistant genes has been cloned yet,which restrict the selection and utilization of resistance sources.In this project,we use resistant variety Jingyehuang（JYH）,susceptible variety NC82 and F2:3:3 family group to build the resistant parent pool（JYH）,susceptible parent pool（NC82）,resistance group pool（R-Pool）and susceptible group pool（S-Pool）.We carry out the proteomic analysis in different pools by the method of iTRAQ technology to analysis resistance mechanism,and further identify resistant proteins.1.A total of 3,992 differentially expressed proteins were obtained（FDR≤1%）by iTRAQ technology;605 differentially expressed proteins were identified by a standard of 2-fold（or more）difference relative to control.Among which,355 different proteins were identified in JYH vs NC82,and 225 different proteins were identified in R-pool vs S-pool.Both in JYH vs NC82 and R-pool vs S-pool,there are 12up-regulated proteins and 11 down-regulated.2.GO terms of differentially expressed proteins are mainly enriched in biological processes such as photosynthesis,redox and reactive oxygen metabolism,et al.,which are mainly located in cell components such as photosynthesis related components,cell wall structure and cytoplasm.They have molecular functions such as oxidoreductase activity,oxidative phosphorylation,and chitinase activity.The KEGG metabolic pathway of differentially expressed proteins are mainly involved in the metabolism of photosynthesis-related substances,and also includes the pentose phosphate pathway,the flavonoid biosynthesis pathway,and the peroxisome metabolic pathway,et al..It is indicated that the resistant variety（JYH）may improve its disease resistance by enhancing energy metabolism,regulating cell wall structure and increasing the expression of defense protein.3.MLP423（Major latex-like protein 423）protein was significantly up-regulated in both JYH and R-pool,and the ORF sequences of MLP423 encoding genes were cloned from nicotiana tabacum and Nicotiana benthamiana,named NtMLP423 and NbMLP423 respectively.Bioinformatics analysis showed that NtMLP423 and NbMLP423 protein sequences contain 152 amino acids,with Betv1domain and Gly-rich loop domain,no signal peptide and transmembrane domain,which were typical members of MLP protein family.Induced by Alternaria alternata,NtMLP423 and NbMLP423 could respond rapidly,and the expression of NtMLP423 in JYH was significantly higher than that in NC82.4.The expression vector of RFP-NbMLP423 was constructed,and NbMLP423 protein was mainly located in cytoplasm and nucleus.NbMLP423 was expressed in leaves,roots,flowers and fruits analyzed by qRT-PCR,with the highest expression in leaves.After abiotic treatment,the expression of NbMLP423 was up-regulated after NaCl,4℃and drought stress,indicating that the expression of NbMLP423 could induced by abiotic stress.5.The VIGS silencing system of NbMLP423 was successfully constructed using TRV vector,and the transient silencing of NbMLP423 did not significantly affect the resistance to Alternaria alternata.The over-expression system of NbMLP423 was constructed with the high expression efficiency.The over-expression of NbMLP423 significantly enhanced the resistance to Alternaria alternata,and the overexpression of NbMLP423 promotes up-regulation of pathogenesis-related protein PR10（NtPR10）,which was significantly higher than that in control tobacco plants.6.Exogenous spraying of plant hormones such as SA,MeJA and ET have significant induction effect on NbMLP423,and the induction effects of SA and MeJA were more significant with inoculation of Alternaria alternata.It further indicated that NbMLP423 was involved in regulating the resistance to Alternaria alternata through plant endogenous hormone signaling pathway. |
