Molecular Mechanism Of Antioxidant Of Grafted Cucumber Leaves Under Low Temperature And NaCl Stress

Cucumber(Cucumis sativus L.)is one of the main crops planted in facilities in North of China. However the yield and quality of cucumber was severely affected by the soil salinity accumulating resulted from planting year after year. Cucumber is cold-sensitive, and could be damaged when the temperature belows 10℃. In North of China, it often drops to about 5℃in greenhouse in winter and spring. The low temperature could make cucumber hurt, result in reduction of cucumber or even no yield. It is urgent to resolve salt injury and chilling damage in current greenhouse cucumber production.It is an effective way that using rootstock for grafting cucumber cultivation to overcome the soil salinization and improve the cold resistance of the plant. The cold resistance of grafted cucumber's leaves can be enhanced by 3℃. Meanwhile, the salt tolerance of cucumber significantly enhanced. The activities of SOD, POD, CAT and APX of the grafted cucumber leaves are higher than that of own-rooted cucumber. The increase of activities of antioxidant enzymes and the content of AsA is the main reason that the grafted cucumber increase its resistance.In order to identify the molecular mechanism of salt-tolerance and cold resistance of grafted cucumber, firstly the middle region of Cu / Zn-SOD, Mn-SOD, CAT, GalLDH, APX, DHAR and GR gene in cucumber leaves was cloned in this experiment. Then taking grafted and own-root cucumber (Junlv No.3) as trial material, the changes of expressions of genes, the activities of antioxidant enzyme, and the content AsA, GSH, H202 and MDA of grafted cucumber and own-root under NaCl stress and the low-temperature stress were analyzed.The results showed that:1. Cu/Zn-SOD,Mn-SOD,CAT,GalLDH,APX,DHAR and GR genes in cucumber leaves was cloned respectively. And the middle region of Mn-SOD,GalLDH,DHAR and GR genes were firstly cloned from cucumber leaves. Through analyzing the homology of the cloned gene, it was registered in gene bank, the number are EF121763, EF203086, EF468517, EF468516, EF468515, EF426538 and EF530128 respectively.2. The changes of activities of Cu/Zn-SOD and Mn-SOD correspond to the changes of their expression under low temperature stress, but the changes of activities of CAT were not the same as that of mRNA. SOD activity of grafted cucumber is higher than that of own-root cucumber under low-temperature stress. The key factor is that Cu/Zn-SOD and Mn-SOD mRNA in grafted cucumber leaves increases. And no significant change of CAT activities was observed between grafted and own-root cucumbers under low temperature stress. Thus CAT is not the main factor that enhanced the cold resistance of grafted cucumber.3. Under low temperature stress, the relative expression of DHAR, GR mRNA were all higher in leaves of grafted cucumber than that of own-root cucumber, while no significant change of the relative expression of GalLDH, APX mRNA was observed between them. The content of AsA, GSH and the ratio of AsA/DHA, GSH/GSSG were all higher in grafted cucumber than that of own-root cucumber, while the content of DHA, GSSG and H2O2 were all lower. Which demonstrates the higher content of AsA,GSH in the grafted cucumber plants enhance their chilling tolerances. And the higher relative expression of DHAR, GR mRNA resulted in the higher activities of DHAR, GR in grafted cucumber leaves, which resulted in the higher content of ASA and GSH in grafted cucumber leaves.4. Under NaCl stress,the relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA were all higher in leaves of grafted cucumber than that of own-root cucumber, and the activities of SOD, Cu/Zn-SOD, Mn-SOD and CAT in leaves of grafted cucumber were higher too. It could be concluded that the higher relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA resulted in the higher activities of SOD, Cu/Zn-SOD, Mn-SOD and CAT in grafted cucumber leaves. With NaCl stress time prolonging, the relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA both increased in grafted and own-root cucumber leaves, but the changes of the activities of Cu/Zn-SOD, Mn-SOD and CAT were not the same as that in mRNA, indicting that the activities of SOD and CAT were also regulated by other factors. The MDA content and electrolytic leakage in grafted cucumber plants was lower than that of in own-root cucumber plants, which demonstrates the grafted cucumber plants have higher scavenging metabolism that can decrease the oxidative damage and increase their salt tolerances.5. Under NaCl stress, the activities of GalLDH, DHAR, APX, GR were all higher in leaves of grafted cucumber than those of own-root cucumber. The relative expression of GalLDH, DHAR and APX mRNA were all higher in leaves of grafted cucumber than those of own-root cucumber, while no significant change of the relative expression of GR mRNA was observed between grafted and own-root cucumbers. The content of AsA, GSH and the ratio of AsA/DHA, GSH/GSSG in leaves of grafted were all higher than that of own-root cucumber, while the content of DHA, GSSG and H2O2 were lower. This demonstrates the higher content of AsA, GSH in the grafted cucumber plants enhance their salt tolerances. And the higher content of AsA and GSH in the grafted cucumber plants was resulted from the higher activities of GalLDH, APX, DHAR and GR. The higher activities of GalLDH, APX and DHAR was regulated by the higher relative expression of GalLDH, APX and DHAR mRNA. Under NaCl stress, the activities of GR were also regulated by other factors.