Study On CsTCTPs Mediate TOR Signaling Regulates Disease Resistance Of Cucumber

Cucumber(Cucumis sativus L.)is the largest facility vegetable in China,and its planting area and output rank first in the world.Cucumber powdery mildew [Sphaerotheca fuliginea(Schlecht)Poll.] is seriously harmful,which seriously affects the yield,quality and income of cucumber.Therefore,it is of great significance to carry out in-depth research on the molecular mechanism of cucumber in response to the infection of powdery mildew and to find new resistance gene resources.In the preliminary study of this laboratory,it was found that Cs TCTPs played a negative regulatory role in the response of cucumber to powdery mildew,but its mechanism of action is still unclear.TCTP is ubiquitous and highly conserved in eukaryotes.In animals,TCTP can regulate and control disease occurrence and immune response by activating the upstream regulator of the TOR signaling pathway,Rheb.Based on this,this study used Cucumber cotyledon transient expression technology to silence and overexpress Cs TCTPs,and RT-PCR was used to detect the changes of gene expression of TOR signaling pathway and downstream regulatory factors,and to confirm the role of Cs TCTPs in regulating TOR signaling pathway.Combined with TOR inhibitor,the effect of inhibiting TOR signal on cucumber powdery mildew resistance was analyzed,and the role of TOR signal in cucumber defense reaction was determined.At the same time,cucumber callus was genetically transformed by Agrobacterium-mediated method to obtain Cs TCTPs expression.Inhibition and overexpression of cucumber callus laid the foundation for further investigation of the molecular mechanism by which Cs TCTPs gene mediates TOR signaling to regulate cucumber disease resistance.The main findings are as follows:(1)Construction of Cs TCTP1/2 silencing and overexpression vector,transient transformation of cucumber cotyledons,inoculation of powdery mildew after 24 h of overexpression or 7 d of silencing,respectively,and detection of cucumber TOR signaling pathway element genes Cs TOR,Cs LST8 by real-time quantitative PCR The expression levels of Cs RAPTOR and TOR downstream effectors Cs S6 K,Cs E2 Fa and Cs E2 Fb were changed.The results showed that the disease resistance of cucumber cotyledons was enhanced when Cs TCTP1/2 was silenced,while the resistance of cucumber cotyledons was decreased when Cs TCTP1/2 was overexpressed.At the same time,the expression of TOR signaling pathway-related genes was not affected by silencing or overexpressing Cs TCTP1.Large and insignificant,Cs TCTP2 negatively regulates gene expression changes in TOR signaling element genes(Cs TOR and Cs LST8)and downstream regulatory genes(Cs S6 K and Cs E2Fa).It is speculated that Cs TCTP2 may regulate the defensive response of cucumber by directly regulating TOR signal;while Cs TCTP1 may regulate TOR signal by indirect influence.(2)The TOR inhibitors Rapamycin,AZD8055 and Torin1 were used to inhibit the TOR signal of cucumber seedlings,and the effects of TOR signal on the growth and disease resistance of cucumber seedlings were analyzed.The results showed that 10 μmol·L-1 Rapamycin,5 μmol·L-1 AZD8055 and 10 μmol·L-1 Torin1 inhibited the growth and development of cucumber seedlings to some extent,but the disease resistance was significantly improved.Combined with the identification of disease resistance and the analysis of resistance physiological indexes,AZD8055 with the optimal inhibitor of 5 μmol·L-1 was screened out.The cucumber hydroponic seedlings were pretreated with 5 μmol·L-1 AZD8055 for 7 days,then inoculated with powdery mildew,and the enzyme activities and lignin changes of cucumber seedling resistance-related enzymes were determined.The results showed that under the infection of powdery mildew,inhibition of TOR signal could promote the accumulation of lignin and increase the enzyme activity of lignin synthesis related enzyme PAL.At the same time,it enhanced the enzyme activities of active oxygen metabolism related enzymes SOD,POD and CAT,and slowed MDA.The extent of the increase in content.In summary,the TOR signaling pathway negatively regulates cucumber disease resistance.(3)The Cs TCTPs gene was overexpressed and silenced in cucumber callus by genetic transformation.The best medium for screening for callus induction was MS+6-BA 2.0 mg·L-1+NAA 0.1 mg·L-1+NH4NO3 2.0 g·L-1+ yeast extract 5.0 g·L-1.The optimal time for Bacillus infection was 20 min.Among them,Cs TCTP2 gene was successfully transferred into cucumber callus,which laid the experimental foundation for further exploring the molecular mechanism of Cs TCTP2-mediated TOR signaling pathway to regulate cucumber powdery mildew resistance.