Studies On Genetic Transformation Of2-Cys Prx In Tobacco Leaves And Its Photoprotective Functions Under Salt Stress

Plant cells produced reactive oxygen species (ROS) mostly mediated by photosynthesis under environmental stresses, but ROS in turn lead to the destruction of pant photosynthetic mechanism by attacking photosynthetic electron transport, photosynthetic membrane and PS II etc. Therefore, removal of ROS in chloroplast especially H2O2of the chloroplast membrane was to protect plant photosynthetic mechanism from hurting under stresses. Under the stress of adversity, ascorbate peroxidate (APX) in chloroplast was extremely sensitive to ROS, ROS could passivate and deactivate APX. When the APX had passivated, double cysteine thioredoxin peroxidase (2-Cys Prx) could clear ROS in chloroplast.2-Cys Prx was newly found in the yeast as a thioredoxin peroxidase (Prxs), it could clear ROS. Higher plant had2-Cys Prx in chloroplast, however, its photoprotective mechanism was unclear. In this study,2-Cys Prx gene was cloned from leaves of tobacco, and transformed it into tobacco. The function of clearing H2O2of2-Cys Prx in chloroplast was researched using2-Cys Prx overexpression transgenic tobacco, the photoprotective mechanism of2-Cys Prx was explored under stress, and theoretical basis and technical support for plant defense physiology were provided.The2-Cys Prx cDNA was cloned from tobacco leaves of longjiang911through reverse transcription RT-PCR. The DNA sequencing analysis showed that the size of the cloned2-Cys Prx was816bp, which contained the whole encoding sequence of the gene, and the similarity with the published gene sequence was above99%. ProkⅡ and2-Cys Prx were used to construct the overexpression vector, and constructed the inhibition expression vector through Gateway using pK7GWIWG2(Ⅱ) and2-Cys Prx, and then transformed them into Agrobacterium LBA4404. Leaf disc transformation was respectively peformed to tobacco, and transgenic plants were obtained after the detection of genomic DNA PCR and semi-quantitative RT-PCR.The photochemical characterization of PS II of2-Cys Prx overexpression transgenic tobacco was measured before and after photosynthetic dark reaction activation under salt stress to test the photoprotective function of2-Cys Prx with wild tobacco as control. The results showed that:under salt stress, the APX activity of wild tobacco was lower, the H2O2content was higher, but the H2O2content in leaves of2-Cys Prx overexpression transgenic tobacco was lower than that of wild tobacco, it showed that2-Cys Prx could clear H2O2when the APX activity was lower, reducing the oxidative damage under salt stress. It was also found that the reduction of PS II original light energy conversion efficiency (Fv/Fm) of2-Cys Prx overexpression transgenic tobacco was lower than wild tobacco under salt stress, as well as chlorophyll fluorescence intensity and K point (the characteristic loci of about300μs after illumination), it showed that2-Cys Prx could allevitate the damage degree of PS II donor side under salt stress. Futher analysis found that2-Cys Prx significantly increased the electron acceptor capacity (Sm) of PS Ⅱ receptor side in leaves, the probability of photosynthetic electron in the electron transport chain more than the electron acceptor of QA-increased, reduced the excessive light enegy reduction for Qa, and the reduction rate of QA(Mo) slowed down. At the same time, it was found that the percentage declines in non-QA-reducting reaction center and non-QB-reducting reaction center of2-Cys Prx overexpression transgenic tobacco was higher than wild tobacco under salt stress, it showed that2-Cys Prx increased the number of PS II activated reaction center, capyured light energy for photochemical reaction, formatted assimilatory power by electron transfer and coupling photophosphorylation to promote carbon assimilation reaction, and reduced the injury of photosynthetic mechanism under salt stress.2-Cys Prx overexpression and suppression expression transgenic tobacco were obtained by tobacco transformation, proved2-Cys Prx could clear ROS (H2O2) as antioxidase, and played a role in clearing ROS when APX had passivated under salt stress. Especially photosynthetic mechanism had excess light energy under salt stress,2-Cys Prx could change the distribution direction of photosynthetic electron flow, reduce the extent of water cracking system (OEC) and the repressed ahead of QA receptor side, increase the electron acceptor capacity of PS II receptor side, enhance the number of PS Ⅱ activated reaction center, reduce the damage of salt stress. It demonstrated that2-Cys Prx played an important role in the photoprotective mechanism under salt stress.