Cloning And Functional Analysis Of LetAPX In Tomato Under Environmental Stress

Abiotic stress is the major enviromental stress for agriculture production worldwide, severely affecting the growth, yield and quality of kinds of crops. Reactive oxygen species (ROS) tend to increase if plants were exposed to stressful conditions such as low or high temperature, drought, salinity, air pollution and herbicides. Oxygen is essential for the existence of aerobic life, but toxic ROS, including superoxide anion radical (O 2), hydrogen peroxide (H2O2) and hygroxyl radical(·OH), are generated in all aerobic cells during the processes of metabolism. Oxidative damage caused by these ROS is one of the major damaging factors in plants exprosed to enviromental stresses. Therefore, plants have developed a number of homeostatic antioxidant defense machanisme to protect themselves against ROS. These machanismes employ antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and catalases (CAT). APX is localized in chloroplasts including thylakoid-bound APX (tAPX) and stroma APX(sAPX), cytosol(cAPX), mitochondria and peroxisomes. APX is the key enzyme for scavenging H2O2 in chloroplasts. The expression of LetAPX gene was induced by stress treatments. It is very improtant to investigate the relationship between tAPX and stress tolerance in crops.The tAPX gene from tomato leaf (LetAPX ) was introduced into tobacco. Wild plants(WT), sense-transgenic lines T2-2(+), T2-3(+)and T2-6(+)of tobacco were used to measure tolerance against environmental stresses. Northern blot analysis indicated that the expression of LetAPX was induced by mutiple stresses. Stess tolerance of transgenic tobacco plants with over-expression of LetAPX increased much greater than that of WT. The main results are as follows:1. The degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves on the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The full length of the cDNA was isolated by 5'-RACE and 3'-RACE. The clone contains 1369 nucleotides, comprising 377 amino acid. The deduced amino acid sequence showed high identities with chloroplast tAPX from tobacco, Arabidopsis. This gene was designed as LetAPX.2. Northern blot analysis showed that expression level of LetAPX was higher in leaf of tomato than in young fruit, stem and calyx, and expression of LetAPX was lest in root and flower at normal temperature. The results indicated that the expression of LetAPX in tomato was induced by stress treatments including low temperature, high temperature, salnity, drought and H2O2, and the expression levels varied with the time of treatment. The expression levels of LetAPX in transgenic tobacco plants are higher than that of WT under 4℃for 12 h.3. APX activity, H2O2 content, MDA content and relative electric conductivity of leaves in both the transgenic plants and WT under different stress conditions were investigated. The results showed that APX activity under stress conditions was higher than that under normal conditions, and APX activity in the transgenic lines increased more evidently than that in WT. The level of H2O2, MDA content and relative electric conductivity of leaves increased notably of all the tested lines under stress treatment, but the measured value of transgenic lines increased much more slowly than WT. This suggests that higher APX activity and the slower increase in H2O2 content and MDA content in transgenic plants might correlated with the much stronger tolerance than WT against oxidative stresses.4. To test for the germination ability of seeds under stress conditions, seeds from WT and several homozygous transgenic lines were placed on MS medium under different stress conditions. The results showed that the seeds of WT was more sensitive to stress than transgenic lines, and transgenic plants appeared to remain more healthy. The fresh weight, dry weight of transgenic plants were higher than those of WT under stress treatments. The results indicate that the overexpression of LetAPX results in the increased tolerance of the transgenic tobacco plants to stress.5. The photosynthetic rate showed that photosythetic efficiency of the tested lines was reduced under stress treatments, while the reduction was higher in WT. The results showed that the maximal photochenmical efficiency of PSII (Fv/Fm) decreased with the stress treatment, but the Fv/Fm of the transgenic plants was higher than that of WT. The transgenic plants showed could maitain higher photochemical efficiency of PSII than that of WT after treatments.