| Oriental melon is an important fruit-type vegetable widely grown in China and East Asian countries.It has shallow root system,large and thin leaves,and demands large amount of water,insufficient irrigation or delayed irrigation often reduce the yield and quality of melon.Powdery mildew is one of the diseases that often occur in the production of melon,causing a large amount of pesticide used,which pollutes the environment and reduces the safety of the products.Therefore,improving the drought resistance and powdery mildew resistance of oriental melon is of great significance for the healthy development of the melon industry.Light signals are the basis of plant growth and development,appropriate light intensity,especially red light(RL),can improve the defense response of plants,but the relationship between red light and defense response of oriental melon has not been reported.Lipoxygenases(LOXs,EC 1.13.11.12)are widely involved in plant growth and development,fruit maturation and aging,and stress,but the mechanism of the lipoxygenase family genes in stress resistance in melon is still unclear.Our research team previously identified 18 LOX family genes(Cm LOX01-18)from the melon genome website.In this thesis,oriental melon ’YMR’ was used as the research material,Cm LOX10 and Cm LOX13 with different expression patterns under drought stress were selected by RT-q PCR analysis for the study of the mechanism under drought stress.The suitable red light intensity and time were selected to induce defense response to powdery mildew.And two transcription factors bound to the Cm LOX10 promoter were screened by yeast one-hybrid(Y1H)method,and bioinformatics analysis was performed.The main results are as follows:1.Simulated drought stress treatment with PEG6000 for 0,3,6,12 and 24 h.The results of RT-q PCR analysis showed that the expressions of Cm LOX01,Cm LOX02,Cm LOX04,Cm LOX05 and Cm LOX06 did not change significantly within 24 h after treatment;Cm LOX03,Cm LOX07,Cm LOX08,Cm LOX09,Cm LOX14,Cm LOX15,Cm LOX16,and Cm LOX17 slightly up-regulated expression;the other five genes were strongly induced by PEG6000,of which Cm LOX10 and Cm LOX11 had the same expression pattern,immediately up-regulated expression after treatment and the expression level reached a peak at 6h after treatment,Cm LOX12 and Cm LOX13 had the same expression pattern,and the expression was significantly up-regulated at 6 h and reached the peak at 12 h after treatment.Similarly,Cm LOX18 was significantly upregulated at 6 h after treatment,however,the expression was highest at 24 h.The above results indicate that there are 5 LOX family genes in oriental melon respond to drought stress.Cm LOX10 and Cm LOX11 may function in the early stages of drought,Cm LOX12 and Cm LOX13 function in the middle stage of drought stress,and Cm LOX18 functions in the later stage of drought stress.2.We used virus-induced gene silencing(VIGS)technology to obtain Cm LOX10 silenced oriental melon plants(TRV-10),and heterologously expressed Cm LOX10 in Arabidopsis to obtain Cm LOX10 overexpressing plants(Cm LOX10-OX).TRV-10 and Cm LOX10-OX Arabidopsis plants were treated with drought stress.Compared to control plants,Cm LOX10-OX has higher survival rate after drought stress,electrolyte permeability(EL),malondialdehyde(MDA),hydrogen peroxide(H2O2)levels,stomatal aperture and transpiration water loss rates were significantly reduced,but jasmonate(JA)levels and expression of synthetic genes were increased.In contrast,Cm LOX10 silenced plants reduced resistance to drought stress,and the levels of EL,MDA,H2O2,as well as stomatal opening and transpiration loss were significantly higher than those of the control,while JA content was significantly lower than the control.There was no significant change in abscisic acid(ABA)content in the silenced plants and over-expressed plants comparing to their control plants.Exogenous JA treatment can alleviate the damage caused by drought stress in melon seedlings and reduce stomatal opening.Analysis of the Cm LOX10 promoter revealed that it contained methyl jasmonate(Me JA)response elements and that the expression of Cm LOX10 was induced by JA.In addition,JA signal pathway core transcription factor Cm MYC2 can also be induced by JA and PEG6000.The promoter region of Cm LOX10 contains multiple MYC binding elements.YIH results indicate that Cm MYC2 directly binds to the Cm LOX10 promoter.These results indicate that Cm LOX10 enhances plant drought tolerance by promoting JA accumulation and stomatal closure,and is also regulated by feedback from JA signals.3.The expression pattern of Cm LOX13 under PEG6000 was different from that of Cm LOX10.The expression level of Cm LOX13 began to increase significantly at 6 h after treatment and reached a peak at 12 h.Phenotypic and physiological analysis showed that heterologous overexpression of Cm LOX13(Cm LOX13-OX)improved drought tolerance in Arabidopsis.Endogenous ABA contents significantly increased in the Cm LOX13-OX lines after drought treatment.RNA-seq and q RT-PCR results showed that the transcription of NCED3 and NCED5,key genes in ABA biosynthesis process changed little,while the expression of four key genes CYP707A1 to CYP707A4 in ABA catabolism pathway were all significantly downregulated in transgenic plants.However,JA contents in Cm LOX13-OX lines were lower than that in WT under control and drought conditions accompanied by the higher expression of the JA catabolism genes.Exogenous ABA and its inhibitor can alter drought stress resistance both in WT and Cm LOX13-OX.In addition,physiological and transcriptome analysis indicated that stomatal closure was mediated by ABA signal transduction pathway.Our results demonstrated that Cm LOX13 functioned as a positive regulator by modulating ABA level through decreasing catabolism related genes expression and ABA-induced stomatal closure in response to drought stress.4.The suitable red light intensity and time for inducing the defense response of powdery mildew powdery mildew were screened at 160 μmol m-2 s-1 and 6 h.Pretreatment with 160 μmol m-2 s-1 red light for 6 hours can significantly reduce the disease index of powdery mildew powdery mildew.Treatment with LOX activity inhibitor NDGA found that red light-induced defense against powdery mildew depends on LOX activity.RT-q PCR analysis showed that the expression of Cm LOX10 and Cm LOX13 after each treatment was consistent with the change trend of LOX activity.Compared with the control,the silent plants of Cm LOX10 and Cm LOX13 had larger lesions and a higher disease index than the control,suggesting that Cm LOX10 and Cm LOX13 played a positive role in the resistance to powdery mildew.Cm LOX10 and Cm LOX13 silenced plants reduced the induction of red light on powdery mildew resistance.Using the Cm LOX10 promoter to screen the YIH library,two transcription factors,MELO3C016281 and MELO3C019925,belong to the WRKY family and the b ZIP family,and were named Cm WRKY41 and Cm ABL5-2.Protein subcellular localization prediction revealed that these two transcription factors are localized in the nucleus,and analysis of Plant CARE revealed that there are multiple light-responsive elements and defense-responsive elements in the promoter region of these two transcription factors.These results suggest that Cm LOX10 and Cm LOX13 play a positive role in the response of red light to defense against powdery mildew powdery mildew,and may be regulated by Cm WRKY41 and Cm ABL5-2. |
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