The Function Of Ubiquitin Gene In Salt Tolerance Of Tobacco And Its Physiological Mechanisms

Organisms must produce new functional proteins in the cell cycle, at the same time, it is necessary to degrade those invalid proteins which had finished their functions already. Protein degradation is as important as it's synthesize and post-translation, which plays a regulation role in physiological metabolism, growth and development of the organisms. Strictly selective degradation of proteins is respond to spatio-temporal regulation accurately in cells.Ubiquitin/26S proteasome pathway (UPP) is the most important selective proteolytic pathway. In this pathway, ubiquitin (Ub) is attached to proteins destined for degradation, the conjugated protein are then recognized and degraded by the 26S proteasome. Under stress condition, cell would accumulate a large number of abnormal and damaged proteins. It is very important that removing these damaged proteins in time to maintain cellular function.It is reported that the expression of ubiquitin is increased under stress condition, the degradation of short-lived regulatory proteins and abnormal polypeptides is accelerated. But till now, little work has been done in the effects of ubiquitin on regulating plant physiology by genetic engineering of over-expressing or inhibititing Ub expression. In our previous work, a monoubiquitin gene Ta-Ub2 from wheat (Triticumaestivum) was cloned, a sense vector with Ta-Ub2 was constructed and transformed into tobacco. Our results showed that overexpression of sense Ta-Ub2 gene enhanced the tolerance of transgenic tobacoos to drought and salt stress. Then, we cloned another Ub gene Nt-Ub1 from tobacco, constructed a antisense vector with Nt-Ub1 and transformed it to tobacco, and obtained some antisense transgenic lines in which Ub gene was repressed.In this study, the sense Ta-Ub2 transgenic plants and antisense Nt-Ub1 transgenic plants were used to study the responses of transgenic plants to salt stress. We investigated the seeds germination, seedling growth status, membrane damage, accumulation and removing of reactive oxygen species (ROS), ions homeostasis and photosynthesis, etc. The results showed that overexpressing Ub promoted plant growth and enhanced salt tolerance in the transgenic seedlings. In contrary, repressing Ub expression reduced the growth and salt tolerance of the tobacco plants. The main results are as follows:1. Immumology analysis of Ub protein in sense and antisense transgenic plants indicated that neither overexpressing Ta-Ub2 nor antisense repressing the expression of Nt-Ub1 could significantly alter the level of free ubiquitin pool in transgenic tobaccos. However, the accumulation of conjugated ubiquitin were increased in sense Ta-Ub2 transgenic plants, which were enhanced by salt stress; while repressing the expression of Nt-Ub1 reduced the accumulation of conjugated ubiqtuin.2. Under salt stress, overexpressing Ta-Ub2 enhanced germination and seedlings growth of sense transgenic plants, released the growth inhibition resulted from salt stress, and enhanced the salt tolerance. Antisense repressing the expression of Nt-Ub1 deferred the seeds germination and inhibited the growth of transgenic plants.3. Overexpressing Ta-Ub2 gene effectively protected the membrane integrity and stability under salt stress. Salt stress destroyed the membrane integrity and stability and induced Reactive Oxygen Species (ROS) production, and ultimately resulted in evident increase in malondialdehyde (MDA) content and electrolyte leakage. Several main antioxidant enzymes such as SOD, CAT, POD and APX in sense Ta-Ub2 transgenic plants maintained higher activity than WT under salt stress, which then inhibited the increase of ROS production, MDA content and electrolyte leakage caused by salt stress. And finally, overexpressing Ta-Ub2 effectively decreased the peroxidant level and protected the membrane integrity and stability of transgenic lines from salt stress; reversely, repressing the expression of Ub gene aggravated the membrane destruction results from salt stress in antisense transgenic plants.4. Overexpressing Ta-Ub2 gene maintained water status and ion homeostasis in transgenic plants effectively, mitigated the osmotic stress and the toxic effect of ions, and ultimately ameliorated the salt tolerance of sense Ta-Ub2 transgenic plants; antisense repression of Nt-Ub1gene expression enlarged the effection of osmotic stress and ions toxic under salt stress, reduced the salt tolerance of antisense transgenic plants.5. Compared with controls (WT or/and T-GUS, a transgenic plant without Ta-Ub2), many photosynthesis indexes of sense Ta-Ub2 transgenic plants were ameliorated. The decrease of CO2 assimilation and photochemical efficiency of PSII and degradation of pigments were alleviated. Repressing the expression of Nt-Ub1 gene reduced the photosynthesis in transgenic plants.6. ROS accumulation level showed that antisense transgenic tobaccos accumulated more ROS than control seedling, while overexpressing Ub reduced this accumulation. This suggests that ROS accumulation might be involved in the acclimation of sense and antisense transgenic plants to stress. Ub/26S proteasome pathway was involved in removing ROS.7. Overexpressing Ta-Ub2 gene enhanced the tolerance of transgenic plants to salt stress, high temperature and high light stresses combination, furthermore, alteration of Ub expressing affected plant flowering time. Antisense repression of Nt-Ub1 gene expression led to premature leafsenescence and necrotic lesions, which decreased the tolerance to salt stress. From all the results above, overexpressing the Ta-Ub2 gene enhanced the tolerance to salt stress and stress combination in transgenic plants. These results proposed a new stratagem for crop breeding and enhancing the tolerance of plants to diverse adverse conditions as well as stress combination.