Overexpression Of MsAPX2Gene Improves Stress Tolerance In Plants

To be one of importance member of scavenging enzymes in reactive oxygen species (ROS) system, ascorbate peroxidase (APX) plays major roles in plants to the stress responses. In this study, a new APX gene, MsAPX2, was cloned and then introduced it into the genome of tobacco to improve tolerance to stresses. The functional experiments demonstrated that over-expression of MsAPX2in transformed tobacco plants could elevate the tolerance to multiple stresses. The results are listed as following.1. The MsAPX2gene with an ORF sequence of753bp, encoding251amino acids, was successfully achieved. The putative amino acid sequence contains typical heme-binding site, substrate-binding site and K+-binding site, and MsAPX2therefore is logically regarded as a typical APX gene.2. A transformation vector, pCBC-MsAPX2, was constructed and applied in genetic transformation in tobacco. The T1transgenic plants were tested by PCRs, RT-PCRs and bar gene test strips. The results elucidated that MSAPX2gene has integrated into the tobacco genome with stable inheritance to next generation. Meanwhile, the bar gene could also transfer to the progenies and express protein properly.3. Experiments were conducted for T1plants to confirm salt tolerance of T1transgenic plants. The results revealed that the transgenic plants could survived under150mM of NaCl whereas the wild-type plants could not even under100mM. Leaf discs from transgenic plants kept green after3days’treatment with2M NaCl but the leaf discs from wild-type exhibited chlorosis. Watered with100mM NaCl for3days, the amounts of AsA, praline and APX activity as well in transgenic plants increase remarkably than that in the wild-type. The MDA levels, in contrast, are rather lower in transgenic plants. These results clearly indicated that over-expression of MsAPX2can promote drought tolerance in transgenic tobacco.4. Evaluation of drought tolerance for T1plants was performed. The leaf discs from wild-type plants showed water deficiency after3days’ treatment with15%PEG, the transgenic leaf discs, however, did not change obviously. After6days’treatment without watering, wild-type plants displayed symptom of dehydration while performance of the transgenic ones was almost normal. The figures of several physiological indices including amounts of AsA, praline and MDA as well as APX activity demonstrated that over-expression of MsAPX2can enhance drought tolerance in transgenic tobacco.5. For cold tolerance test in T1transgenic plants, after4℃treatment for6days, the figures of amounts of AsA, proline and MDA, together with APX activity, proved that over-expression of MsAPX2can improve cold tolerance of transgenic tobacco.6. Experiments for anti-oxidation were carried out with H2O2treatments. When the leaf discs were dipped in30%H2O2for3days, the discs from wild-type turned yellow whereas the transgenic showed little damages. When treated with36%H2O2for36hours, the data for amounts of AsA, proline and MDA as well as APX activity made it clearly that over-expression of MsAPX2enable to boost the resistant level of anti-oxidation in transgenic tobacco plants.