| Flagllin Sensitive 2（FLS2）is the first gene with disease resistance discovered in the Leucine-Rich Repeats RLK（LRR-RLK）gene family,which is widely exist in plant tissues and organs.FLS2 is involved in plant growth,development and plant hormone transduction.FLS2 recognizes flagellin of pathogens through the extracellular LRR domain and mediates the immune response of plants.Flagllin sensitive 2-like gene（Fl S2-like）exists in Physcomitrella patens,which has a domain similar to FLS2 and is named Physcomitrella patens Flagllin sensitive 2-like（PpFLS2L）in this thesis.At present,relevant studies on the mechanism of action of PpFLS2 L in plant immune response of P.patens have not been reported.In this study,the function and the possible mechanism of PpFLS2 L involved in immune response were investigated by using P.patens as experimental material,and the following conclusions were drawn:1.The PpFLS2 L knockout plants were successfully obtained in this study.A PpFLS2L-PTN182 knockout vector was constructed,and then PpFLS2L-PTN182 was transferred into the protoplast of P.patens by PEGmediated genetic transformation using homologous recombination technique.We used two different concentrations of G418 antibiotics for screening and performed molecular level verification,indicating that PpFLS2 L knockout plants were successfully obtained.2.The transcriptional expression of the chitin receptor gene CERK1 was effected by PpFLS2 L.Chitin is the inducer of plant disease resistance,and CERK1 is the cell membrane receptor of chitin.The expression of CERK1 family in wild-type（WT）plants and PpFLS2 L knockout plants was analyzed by RT-PCR,and the results showed that: the main participant in chitin recognition of WT plants may be CERK1-3.After PpFLS2 L knockout,the expression of the three CERK1 genes was increased,especially the effect was significant at 0.5 h and 4 h of chitin treatment.These results indicated that PpFLS2 L effected the expression of CERK1 and PpFLS2 L may be negatively correlated with CERK1.3.PpFLS2 L effects the transcriptional expression of genes related to the downstream CERK1 signal pathway.It has been reported that CERK1 of P.patens induces relevant defense responses by activating the MPK pathway.This study analyzed MPK activity and the results showed that:Activity of the MPK increased after chitin treatment in WT plants,and chitin also induced an increase of activity of the MPK after PpFLS2 L knockout and the increase was higher than WT plants,indicating that PpFLS2 L knockout effected the activity of the MPK.At the same time,the expression of MPK gene family members in WT plants and the PpFLS2 L knockout plants was also analyzed,and the results showed that MPK gene family members were up-regulated to varying degrees in chitin-treated WT plants;MPK gene family members also showed up-regulated expression and the expression of MPK gene family members were significantly higher than WT plants at 0.5h and 4h after PpFLS2 L knockout.Therefore,under chitin induction,PpFLS2 L may effect the MPK signal transduction pathway by effecting the expression of CERK1.4.The CERK1-mediated expression of defense-related genes was effected by the PpFLS2 L.It has been reported that CERK1 of P.patens can mediate the expression of resistance genes such as DOX,ERF2,CHS,LOX7,and PAL.The results of this study showed that after treatment with chitin,the expression of ERF2,CHS,LOX7,and PAL increased in the PpFLS2 L knockout plants compared with WT plants,except for the decreased expression of DOX.5.PpFLS2 L may be negatively correlated with chitin-induced programmed cell death.Programmed cell death is an anti-disease response that is often accompanied by an increase in reactive oxygen species.First,this study analyzed the cell death of WT plants and the PpFLS2 L knockout plants treated with chitin respectively,and the results showed that both genotypes had cell death after chitin treatment,but the number of cell death in the PpFLS2 L knockout plants was significantly higher than in WT plants.In addition,this study also analyzed the expression of reactive oxygen species and related genes after chitin treatment in WT plants and the PpFLS2 L knockout plants,and the result showed that,after chitin treatment in the PpFLS2 L knockout plants,the expression of RBOHD gene related to the production of reactive oxygen species and the amount of reactive oxygen species were significantly increased compared with WT plants.After chitin treatment,the activities of SOD,CAT,AOX,and POD in WT plants and the PpFLS2 L knockout plants were all increased,while the expression of the three genes was basically higher in the PpFLS2 L knockout plants than in the WT plants,except for the activity of POD.In conclusion,PpFLS2 L may negatively mediate downstream related signal pathways and related defense responses by effecting CERK1 gene expression. |
