Isolation And Functional Identification Of Pumpkin Powdery Mildew Related Genes

Pumpkin is an important vegetable cultivation crop with high economic value.Powdery mildew,a major fungal disease of pumpkins,brings serious economic losses.At present,most of the main cultivars of pumpkin do not possess resistance to powdery mildew.The control of chemical agents is liable to produce drug resistance and cause environmental pollution.In this study,the high-resistant powdery mildew inbred line“112-2”and the highly susceptible powdery mildew inbred line“Jiujiangjiaoding”of C.moschata Duchesn were taken as the object of study.Analysis of transcriptome data of“112-2”after 24h and 48h of inoculation with powdery mildew,it was found that the expression levels of 5 differentially expressed genes?DEGs?of“112-2”were significantly different with“Jiujiangjiaoding”.Homologous cloning method was used to isolate resistant candidate genes,Real-time quantitative techniques are used to analyze the expression of different treatments.Agrobacterium-mediated leaf disc transformation method was used to transform tobacco,the resistance of transgenic and non-transgenic tobacco was identified,the expression patterns of signal transduction related genes were analyzed.the results of the study are as follows:?1?The ORF of CmSGT1 is 1080 bp,encoding 360 aa.H₂O₂ and SA treatment induced the expression of CmSGT1 in“112-2”and inhibited the expression of CmSGT1 in“Jiujiangjiaoding”.Overexpression of CmSGT1 in tobacco promoted the accumulation of H₂O₂ and accelerated cell necrosis.Overexpression of CmSGT1in tobacco induced the up-regulation of PAL and PR1a,indicating that CmSGT1 may participate in the reaction against powdery mildew through the SA signal transduction pathway.Overexpression of CmSGT1 reduced tobacco resistance to bacterial wilt and scab.?2?The ORF of CmPR1 is 597 bp,encoding 198 aa.CmPR1 was induced by pumpkin powdery mildew,Eth,and MeJA.CmPR1 overexpression promoted H₂O₂accumulation and accelerated cell necrosis;CmPR1 overexpression in tobacco induced the up-regulation of PAL,NPR1,PR5 and PR1a,and induced the down-regulated of PDF1.2 expression,suggesting that CmPR1 may participate in resistance to powdery mildew through SA and JA signal transduction pathway.Overexpression of CmPR1 increases tobacco resistance to bacterial wilt and scab.?3?The ORF of CmMYB1 is 858 bp,encoding 285 aa.CmMYB1 was induced by pumpkin powdery mildew.MeJA and Eth inhibited the expression of CmMYB1 in“112-2”and induced CmMYB1 expression in“Jiujiangjiaoding”.Overexpression of CmMYB1 promoted the accumulation of H₂O₂,induced the up-regulation of PR5,and inhibited the expression of PDF1.2.It was suggested that CmMYB1 may be involved in resistance to powdery mildew mainly through SA and JA signal transduction pathway.?4?The ORF of CmERF is 681bp,encoding 226 aa.CmERF was induced by powdery mildew,MeJA and Eth.The expression of CmERF in“112-2”was induced and the expression of CmERF in“Jiujiangjiaoding”was suppressed.The overexpression of CmERF accelerated cell necrosis and induced the up-regulated expression of PAL,NPR1,and PR1a,and inhibited the expression of PDF1.2.This indicated that the CmERF gene may increase resistance to powdery mildew mainly through participating in SA and JA signal transduction pathway.Overexpression of CmERF in tobacco reduces resistance to bacterial wilt and scab.?5?The ORF of CmbHLH87 is 1068 bp and encodes 355 aa.Treatment with pumpkin powdery mildew and SA inhibited the expression of CmbHLH87 in“112-2”.Overexpression of CmbHLH87 promotes H₂O₂ accumulation and accelerates cell death;induces upregulation of PAL,PR1a,and PR5 and inhibits expression of PDF1.2,indicating that CmbHLH87 may increase tobacco resistance to powdery mildew mainly by participating in SA and JA signal transduction pathway.Overexpression of CmbHLH87 increases resistance to tobacco bacterial wilt and scab.