Analysis Of Molecular Mechanism Of Rapamycin Target Protein Regulating Powdery Mildew Resistance Of Cucumber Based On Transcriptome

Cucumber(Cucumis sativus L.)is a major vegetable crop widely cultivated in China.Powdery mildew(PM)is one of the main diseases endangering cucumber production,which seriously affects the yield and quality of cucumber.Previous studies in our laboratory showed that the ability of cucumber to resist powdery mildew was significantly improved,and the growth and development of seedlings were inhibited to a certain extent using AZD8055 to restrain the activity of target of rapamycin(TOR)kinase.TOR is an evolutionarily conserved serine/threonine protein kinase in eukaryotes.Plant TOR kinase can integrate nutrition,energy,hormones and environmental signals,and coordinate and participate in various life stages such as embryogenesis,meristem activation,vegetative organ growth,flowering and aging.Recent studies have also found that TOR plays an important regulatory role in plant response to biological stress,but the mechanism is unclear.In this study,highthroughput sequencing RNA-Seq and q RT-PCR were used to analyze the changes of signaling pathways related to cucumber powdery mildew resistance when the activity of TOR kinase was inhibited at the transcriptional level,in order to identify cucumber differentially expressed genes in response to powdery mildew infection,and analyzed the mechanism of TOR signaling regulating cucumber powdery mildew resistance.The role of differential gene Cs MYB62 in regulating cucumber powdery mildew resistance was explored by using cucumber gene transient expression technology,which laid a foundation for further exploring the mechanism of TOR regulating cucumber growth and development and defense response.The main results were as follows:1.Combined with the pretreatment of TOR inhibitor azd8055,the expression changes of genes related to cucumber powdery mildew resistance under the inhibition of TOR kinase activity were studied by high-throughput RNA-Seq technique.(1)When the TOR was inhibited(DMSO vs AZD),the differential genes were mainly enriched in the components of the membrane in cucumber seedlings.The enriched KEGG pathways mainly included phenylpropane biosynthesis pathway,linoleic acid metabolism pathway,phenylalanine metabolism pathway,plant pathogen interaction pathway,starch and sucrose metabolism pathway,glutathione metabolism pathway,MAPK signal pathway,diterpene biosynthesis pathway,ubiquinone and other terpene quinone biosynthesis pathway,and plant hormone signaling transduction pathway.(2)When cucumber seedlings were infected with powdery mildew(DMSO vs DMSOP),the differential genes were mainly enriched in photosynthesis.The enriched KEGG pathways included photosynthesis signaling pathway,phenylpropane biosynthesis pathway,flavonoids biosynthesis pathway,pentose and glucuronic acid mutual transformation pathway,plant hormone signaling transduction pathway,and MAPK signaling pathway.(3)When TOR was inhibited,the differential genes caused by inoculation with powdery mildew fungus(AZD vs AZDP)were mainly enriched in photosynthesis,redox reaction and other functions.The enriched KEGG pathways included photosynthesis pathway,linoleic acid metabolism pathway,carotenoid synthesis pathway,niacin and nicotinamide metabolism pathway,selenium compound metabolism pathway,and porphyrin and chlorophyll metabolism pathway.(4)Under the infection of powdery mildew,the differential genes caused by AZD pretreatment(DMSOP vs AZDP)were mainly concentrated in photosynthesis and chloroplast.The enriched KEGG pathways mainly included photosynthesis pathway,carotenoid biosynthesis pathway,porphyrin and chlorophyll metabolism pathway,phenylalanine metabolism pathway,phenylpropane biosynthesis pathway,flavonoid biosynthesis pathway,starch and sucrose metabolism pathway,and glyoxylic acid and dicarboxylic acid metabolism pathway.(5)The differential expression patterns of some candidate genes were analyzed by q RT-PCR.The results showed that transcription factors could be strongly induced by powdery mildew of cucumber,such as MYB and photosynthesis related genes.2.The transient silencing vector and transient overexpression vector of Cs MYB62 gene were constructed by In-Fusion homologous recombination method.The transient silencing and overexpression lines of Cs MYB62 gene were successfully obtained by Agrobacterium transformation technology,and the disease resistance was identified after inoculation with powdery mildew.The results showed that Cs MYB62 transient overexpression could reduce the resistance of cucumber to powdery mildew,while Cs MYB62 transient silencing could improve the resistance of cucumber to powdery mildew.It was preliminarily clear that Cs MYB62 gene played a negative regulatory role in the process of cucumber powdery mildew resistance.