| Melon(Cucumis melo L.)as cucurbitaceae annual trailing herbs,is one of the important horticultural crops.Mitogen-actived protein kinase(MAPK)is a serine/threonine protein kinase activated by extracellular stimulation through MAPKKK-MAPKK-MAPK cascades.MAPK cascade pathway is a widely existing and highly conserved signal transduction pathway in eukaryotes,which amplifies the upstream signal cascade to the downstream response molecules through protein phosphorylation.This pathway plays a key role in plant response to biological and abiotic stress and hormone signal transduction.Therefore,in this study,MAPK cascade pathway gene family members in melon were identified from the whole genome level,and their classification,conserved structural domain,gene structure and expression characteristics were analyzed by bioinformatics method.In order to explore the expression pattern of CmMPKs in adversity,non-biological stress,hormone signal induction,light signal induction and biological stress were conducted on melon seedlings in this study,and the relative expression was determined by qRT-PCR technology.Meanwhile,the exogenous injection of MAPK inhibitor PD98059 verified whether the MAPK cascade pathway was a necessary condition for activating the LOX pathway.The main research results are as follows:(1)Homologous comparison and HMMER search were used to find 84 genes from the melon genome website,including 14 genes belonging to MAPK family,6 genes belonging to MAPKK family,and 64 genes belonging to MAPKKK family.These genes were named and identified.The phylogenetic tree of melon and arabidopsis revealed that 14 Cm MPKs and 6 Cm MKKs identified in this study were divided into four subgroups: A,B,C and D,and 64 Cm MAPKKKs were divided into three subgroups: MEKK,RAF and ZIK,revealing the homology of melon and arabidopsis.Chromosomal localization analysis showed that 84 members were distributed on 13 different chromosomes,and these genes were involved in tandem replication as well as chromosome fragment replication events.Finally,protein conserved motifs and protein functional domain analysis showed that MAPK cascade pathway genes in melon were very conserved in structure,so we could predict the functions of MAPK cascade pathway related genes in melon based on the reported homologous genes in arabidopsis,cucumber and watermelon.(2)Through the qRT-PCR technology analyzes the Oriental melon seedling leaf in 14 MAPK genes expression patterns under adversity stress.The results show that under the drought stress,except Cm MPK19,Cm MPK20-1 and Cm MPK20-2 transcription levels drop,the rest are in stress after 6 h,12 h,24 h transcription levels significantly higher than control,and then return to the original level.Only Cm MPK3,Cm MPK7,Cm MPK20-1 and Cm MPK20-2 were significantly induced under salt stress.After salicylic acid(SA)induction,except the down-regulated expression of Cm MPK20-1 and Cm MPK20-2,the rest of the family members were induced to up-regulate the expression.At the late induction stage of methyl jasmonate(Me JA),all 14 Cm MPKs were strongly induced.After red light induction,the transcription levels of Cm MPK3,Cm MPK6-1 and Cm MPK7 were significantly increased compared with the control group,among which the transcription levels of Cm MPK3 were up to 5 times higher than that of the control group.The transcription levels of Cm MPK20-1 and Cm MPK20-2 were not significantly different from those of the control group,and the transcription levels of other gene family members were significantly down-regulated after red light induction compared with the control group.After inoculation of powdery mildew pathogen on melon leaves,except for Cm MPK6-1 and Cm MPK7,the rest of the Cm MPKs gene members were induced and significantly up-regulated on the third day after infection by powdery mildew,and the expression level gradually decreased except for Cm MPK3 and Cm MPK4-2 as the invasion time of powdery mildew increased.At the same time,after the induction of red light,all Cm MPKs were found to have significantly lower transcriptional levels than powder mildew inoculated alone,and were significantly upregulated at 7 and 9 days after inoculation.The expression level of Cm MPK3 and Cm MPK7 in a variety of stress was significantly higher than that in the control group,indicating that Cm MPK3 and Cm MPK7 may be involved in multiple different signal transduction pathways and play an important role in them.(3)After 12 h pretreatment with exogenous spraying inhibitor PD98059,the following treatments were performed,treatment 1: powdery mildew inoculation,treatment 2: powdery mildew inoculation after red light induction,control group: powdery mildew inoculation after 12 h pretreatment with exogenous spraying solvent 0.1% dimethyl sulfoxide(DMSO).The results showed that the disease index of treatment 1 and treatment 2 was significantly lower than that of the control,among which the disease index of treatment 2 was the lowest.According to the observation of leaf phenotype,the incidence of powdery mildew and the coverage area of powdery mildew bacteria in treatment 1 and 2 were significantly lower than those in control.DAB tissue staining results showed that the number of dead cells in leaves of treatment 1 and 2 was significantly lower than that of control,and the number of dead cells in leaves of treatment 2 was the lowest.The results of trypan blue tissue staining showed that the content of ROS in leaves of treatment 1 and 2 was significantly lower than that of control,and the content of ros in leaves of treatment 2 was the lowest.To sum up,we speculated that inhibition of MAPK kinase activity could improve the resistance of melon seedlings to powdery mildew,and the red light induction could further inhibit MAPK kinase activity.(4)In the absence of red light induction,the expression levels of CmLOX10 and Cm LOX12 were significantly increased by the exogenous injection inhibitor PD98059,but its LOX activity was not significantly different from that of the control group.In addition,red light induction significantly increased the expression level of four 13-LOXs and LOX activity.However,under the induction of red light,PD98059,an exogenous injection inhibitor,was found to down-regulate the expression levels of all four 13-LOXs compared with the treatment without inhibitor,in which Cm LOX12 was significantly down-regulated and LOX activity was significantly decreased.Further analysis showed that under the condition of PD98059,there was no significant difference between the expression level and LOX activity of the other three 13-LOXs and those not induced by red light after red light induction,except for Cm LOX10.Therefore,it is speculated that MAPK cascade pathway may be a necessary pathway for LOX activity activation in the red light induced defense response. |
PDF Full Text Request