Identification And Functional Analysis Of The Propamcarb-related Gene CsDIR16 In Cucumber

Cucumber(Cucumis sativus L.)is a very popular and widely cultivated vegetable.However,the high moisture levels and temperatures in greenhouses used to grow the plant allow insect pests and plant diseases to become established,especially downy mildew,which is one of the most damaging diseases.Propamocarb(propyl-3-(dimethylamino)propylcarbamate,PM)is a low toxicity fungicide with systemic action after absorption through leaves,stems,and roots,and by transportation throughout the treated plant via the vascular system.PM is an effective control for downy mildew;however,residues can remain in cucumber plants after the disease has been controlled.There is increasing interest in identifying biotransformation mechanisms measures that can be used to lower pesticide residues.In a previous study,we analyzed the transcriptome changes in cucumber fruit of cultivar ‘D0351' in response to PM treatment.The transcriptome data indicated that CsDIR16 with |log2Ratio|?1,P-Value?0.01,FDR?0.001,showed the highest differential expression between plants treated with PM versus water.Although many plant genes are induced by biological and abiotic stresses,such as insects,fungi,drought,and high-salinity,and their gene products function not only in stress responses but also in stress tolerance we found that only CsDIR16 showed significantly differential expression patterns between ‘D0351' and ‘D9320',and speculated that it may play important roles in response to PM.In order to explore the fuctional of CsDIR16 in reducing PM residues,bioinformatics analysis and molecular biology technique are mainly used in this study.The main results are as follows:(1)The full-length cDNA sequence of CsDIR16 was cloned from cucumber fruit of ‘D0351' by RT-PCR.CsDIR16 contained an open-reading frame(ORF)of 588 bp that encoded 195 amino acids with molecular formula was C977H1515N259O277S7 and a calculated molecular weight of 21.55 k Da.There is an N-terminal signal peptide cleavage site between the amino acids 28 and 29,a transmembrane helix between residues 10 and 27.CsDIR16 shares 99 % similarity with the Cucumis melo gene CmDIR7.The predicted three-dimensional structure showed that CsDIR16 has a typical ?-barrel structure with a hydrophobic chamber in the center.Transcriptome analysis showed that only four of the 23 CsDIR genes in cucumber responded to PM treatment.Of these four genes,CsDIR16 had the highest level of expression.(2)The expression patterns of CsDIR16 in fruit of cultivar ‘D0351' and ‘D9320' were determined in control and PM-treated plants.In ‘D0351',CsDIR16 expression was up-regulated and reached its maximum at 6 h after PM treatment.We observed a gradual decrease in CsDIR16 expression with time.In contrast,CsDIR16 expression followed a different pattern in ‘D9320'.A peak of expression was seen at 3 h,and there were no statistically significant differences to controls except at 9 h.(3)To compare CsDIR16 expression patterns in response to PM in different tissues,we analyzed leaves,stems,and roots at 6 h in ‘D0351' and ‘D9320' plants.CsDIR16 was expressed in all three tissues,but the patterns of expression differed between the two cucumber cultivars.CsDIR16 was upregulated by PM treatment in leaves and stems of ‘D0351'.In contrast,CsDIR16 was expressed at extremely low levels in leaves and stems of ‘D9320'.There were no statistically significant differences between roots of ‘D0351' and ‘D9320'.These results indicate that CsDIR16 expression was significantly induced by PM treatment in the fruit,leaves and stems of ‘D0351',suggesting that these are the major organs for metabolism of the fungicide.(4)The subcellular localization of the CsDIR16 protein was investigated using a CsDIR16-eGFP fusion gene driven by a 35 S promoter;transient expression in Arabidopsis protoplast cells showed that the CsDIR16-eGFP fusion protein was enriched in the nucleus.The results clearly indicated that CsDIR16 is a nuclear-localized protein.(5)We generated transgenic cucumbers overexpressing CsDIR16 under the control of the strong constitutive CaMV35 S promoter.Transgenic cucumbers T0 and T1 were identified by PCR,and the expression of CsDIR16 was measured by qRT-PCR.Overall,we obtained 13 CsDIR16(+)and 15 CsDIR16(-)T0 transgenic cucumbers.The level of residual PM was lower in CsDIR16(+)transgenic plants than wild-type ‘D9320' and CsDIR16(-)plants after PM treatment.The levels of PM did not differ significantly between wild-type ‘D9320' and CsDIR16(-)transgenic plants.The results indicate that CsDIR16 overexpression is likely to reduce PM residue levels by accelerating the degradation of the fungicide.(6)There were interaction between CsDIR16(+)and soluble sugar and POD,but there were no obvious relationship with soluble protein,SOD,GST and GR.MDA content can effectively confirm that CsDIR16(+)gene had the ability to repair the membrane lipid and leaded to reduce the PM residues.(7)Based on these results,we speculated PM activated phenylpropanoid pathway,activities of POD enzyme increased,the phenoxy radical obtained by POD enzyme oxidized in the fruit,leaves and stems produce pinoresinol by Cs DIR16 protein,which rapidly reduced the damage caused by PM,so as to effectively reduce the PM residual in ‘D0351'.