| Melon(Cucumis melo L.)is one of the important economic crops in China which growth and development are often affected by various diseases,resulting in a decline in yield and quality.Powdery mildew is a vital disease in melon,which harms the whole plant and exists in the entire process of growth and development.When the plant is infected by pathogenic bacteria,there will be a series of physiological and biochemical changes in the plant,forming a complex signal transduction pathway,transmitting external signals into the cell,thereby causing the expression of resistance genes and resisting stress.The mitogen-activated protein kinase(MAPK)cascade pathway plays a necessary role in plant signal transduction.Previous research in our research group found that Cm MAPK3 and Cm MAPK7 were induced by red light(160 ?mol m-2s-1·6 h)and up-regulated after inoculated with powdery mildew in melon seedlings,while their expression level was lower inoculated with powdery mildew after irradiation of red light.Therefore,we constructed virus-induced gene silencing vectors,and made a preliminary study on the founction of Cm MAPK3 and Cm MAPK7 inoculated with powdery mildew.The main results are as follows:1.The In-Fusion method was used to construct Cm MAPK3 and Cm MAPK7 silencing vectors.When the cotyledons of melon seedlings were fully expanded,we infiltrate the bacterial solution(TRV:00,TRV:Cm MAPK3,TRV:Cm MAPK7)into the two cotyledons by the cotyledon injection method,and the silence was identified by q RT-PCR at 25 d after injection.The result showed that the expression levels of Cm MAPK3 and Cm MAPK7 in the silenced plants were 0.3~0.6 times as much as the control,respectively indicating that we obtained Cm MAPK3 and Cm MAPK7 transient silenced plants.2.Transient silenced seedlings of oriental melon at the three-leaf stage(TRV:00,TRV:Cm MAPK3,TRV:Cm MAPK7)were inoculated with powdery mildew and inoculated with powdery mildew after irradiation of red light respectively.It was found that the leaves had obvious symptom at 5 d after inoculation with powdery mildew.And the area of the lesions was constantly expanding with the extension of the infection time.However,Cm MAPK3 and Cm MAPK7 silenced plants(TRV:Cm MAPK3 and TRV:Cm MAPK7)had a smaller lesion area than control(TRV:00)of which the disease index was remarkably lower than that of control Moreover,the symptom of powdery mildew treatment after irradiation of red light were significantly reduced,and the disease index of plants inoculated with powdery mildew after irradiation of red light was lower than that treated with powdery mildew alone.Both DAB and trypan blue staining showed that reactive oxygen accumulation and the number of dead cells in silencing plants was reduced.The activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)at 5 d and 7 d after inoculation were higher than those of control.When plants were inoculated with powdery mildew after irradiation of red light,the activities of POD and CAT at 5 d and 7 d of silencing plants were higher than those treated with powdery mildew alone.The above results indicate that Cm MAPK3 and Cm MAPK7 silenced plants have enhanced disease resistance,and red light can reduce the occurrence of powdery mildew.3.By measuring the expression level of four 13-Cm LOXs genes in Cm MAPK3 and Cm MAPK7 silenced plants,it was found that Cm LOX10,Cm LOX12,and Cm LOX18 were all significantly up-regulated at 5 d after inoculation and gradually increased with the extension of infection time.Four 13-Cm LOXs genes were induced at 5 d when plants were inoculated with powdery mildew after irradiation of red light.The expression of Cm LOX10 and Cm LOX18 genes in Cm MAPK3 silenced plants was significantly higher than other treatments;the expression of Cm LOX12 in Cm MAPK7 silenced plants was remarkably higher than that of control.In addition,we also found that the LOX activity of silenced plants was significantly higher than that of unloaded plants at 7 d in the condition of inoculation with powdery mildew after irradiation of red light.It is showed that Cm MAPK3 and Cm MAPK7 may regulate 13-Cm LOXs in disease resistance response.4.After red light induction,Cm MAPKK2-1 and Cm MAPKK9 were significantly upregulated.After inoculation with powdery mildew alone,all Cm MAPKKs were induced to upregulate expression,which were higher than that of inoculation with powdery mildew after irradiation of red light.In addition,the yeast two-hybrid test results showed that Cm MAPKK2-1 interacted with Cm MAPK3 and Cm MAPK7,while Cm MAPKK9 interacted with Cm MAPK3.It is revealed that Cm MAPKK2-1 may participate in the regulation powdery mildew resistance through Cm MAPK3 and Cm MAPK7,and Cm MAPKK9 may participate in the regulation powdery mildew resistance through Cm MAPK3 in melon.In summary,Cm MAPK3 and Cm MAPK7 play a negative regulatory role in resistance to powdery mildew in melon.The upstream may involve Cm MAPKK2-1 and Cm MAPKK9,and the downstream may regulate 13-Cm LOXs. |
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