| Reactive oxygen species (ROS) is the product of normal metabolism in plant cell.When plants suffer stress, ROS concentration in plants will increase. If an excess ofROS can not be cleared, the normal life activities of plants will be threatened.Therefore, it is very important for the normal growth and development of plants toremove ROS timely and effectively and keep the ROS concentration in a regular level.APX is one of the important enzymes to scavenge ROS in plants. APX participates inAsA-GSH cycle of plant, and most of its isozymes mainly exists in plant with the typeof cytoplasmic APX (cAPX). cAPX plays an irreplaceable role in resisting ROSdamage to plants.Populus tomentosa is a fine tree species for ecological afforestation andafforestation of fast-growing timber. In this study, using wild-type741populustomentosa as experimental materials, transgenic poplar plants of over-expressingcAPX genes successfully produced by the method of leaf disc transformation withcytoplasmic pBI121-cAPX recombinant plasmid. We studied the resistance of Populustomentosa to different stress (salinity, drought and oxidation) by overexpressing cAPXgenes, and the main results were shown as follows:1Using the method of leaf disc transformation with agrobacterium-mediated and20mg/L Kan to screen adventive shoots and root, we gained the regenerated poplarplants that had Kan resistance.2Using selective marker gene npt-Ⅱ primers and the poplar plants above toproceed PCR detection, the results showed that four kinds of positive populustomentosa strains were successfully obtained which overexpressed cAPX genes(APX-Ⅱ, APX-Ⅲ, APX-Ⅶ andAPX-Ⅸ).3In order to made a further test for the transgenic Populus tomentosa plants which were identified as positive by PCR, test-tube plantlets of Populus tomentosaplants were choosen for Gus histochemical staining. The resultes suggested thatexogenous gene was expressed in the leaf of transgenic plants.4We measured and compared the ratio of cAPX expression in differentcAPX-overexpressed Populus tomentosa plants by real-time quantitative PCR, theresults showed that the expression of cAPX genes in transgenic plants were higherthan that of the wild-type plants.5Under salt stress, the APX activity in vivo, AsA content, total chlorophyllcontent, leaf K+content and K+/Na+in transgenic plants were higher or significantlyhigher than that of the wild-type poplar. However, the H2O2and MDA contents in thebody, relative conductivity and the Na+content in the leaf of transgenic plants weresignificantly lower than those of the wild-type. This suggests that overexpressingcAPX increased the resistance to salt stress in transgenic plants.6After the drought treatment, compared to WT plants, the RWC, APX activity,AsA content and total chlorophyll content of transgenic Populus tomentosa werehigher. Comparing with the transgenic Populus tomentosa, the phenomenon of leavesinjury in the wild-type plants appearred relatively earlier and the cell membranepermeability increased obviously, the content of H2O2and MDA were higher. Ourresults showed that overexpressing cAPX enhanced plant drought resistance.7Under the oxidative stress by the methyl viologen, the transgenic Populustomentosa enzymes (APX and GR) activity, AsA content and total chlorophyll contentwere obviously higher than that of wild-type Populus tomentosa. Whereas wild-typepoplar H2O2content, MDA content and membrane permeability increasedsignificantly. The resultes suggested that overexpressing cAPX enhanced the cAPXpoplar resistance to oxidative stress.We can find from the above results that, compared with the wild-type poplar,overexpressing cAPX can increase the tolerance of transgenic poplars to abioticstresses (salt, drought and oxidation). This thesis laid the foundation for screeninghigh resistance of poplar varieties. |
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