Salt Tolerance Analysis On Transgenic Rice Overexpressing SAC Gene

Rice (Oryza sativa L) is the main source of food, which supplying 60% of the people’s life on Earth, salt stress is the most common abiotic stress in rice. Its early seedling stage classified as salt sensitive crop, high salt concentration (300mM-0.5mM NaCl) inhibit most enzyme activity by disturbing hydrophobic the electrostatic force, which maintain balance between protein structure. Many metabolic reactions and membrane function will be strongly influenced in plants under these salt concentrations. In order to improve the yield under salt stress conditions, it is necessary to understand the basic molecular mechanisms of the body when suffer stress. In the past few years, many salt,drought and alkaline stress-related genes have been isolated and characterized, sulfur is the most important key elements to help plants cope with this stress. However,salt, drought and alkaline resistance is controlled and expressed by the complicated of multiple genes to participation in various biological processes, In general, the products of these genes can reduce the damage caused by stress, or directly enhance plant tolerance to stress.SAC protein is a bifunctional enzyme with ATP sulfurylase (ATPS) activity and adenosine-5’-phosphoryl sulfuric acid kinase (APSK) activity, is the key enzyme of activation SO42-, and is currently found only with the APS channel effect of inorganic sulfate. ATPS catalytic inorganic sulfate and ATP form APS (Adenosine-5’- phos phosulfate) and PPi, APS was oxidation to form 3’-Adenosine phosphate-5’- phosphoryl (PAPS) by APSK, or reduced to form sulfite by APSR. PAPS increase accumulation of cysteine in plants, and cysteine, glutamic acid and glycine synthesized glutathione. GSH is a low molecular weight antioxidant that play a vital role in plant metabolism and ROS stress tolerance. The mechanisms of antioxidant enzymes and synthesis of GSH can be adjusted and influenced through plant growth under salt stress. Many studies have shown, when plants affected by external stress, jasmonate can directly regulate the process of plant cell death that caused by the active oxygen through the signal nets, and exogenous JA induce the protective mechanisms of plants, this attracted the attention of scientists. Some other evidence suggest that there is a certain link between the redox metabolism of S elements and JA response. Exogenous JA can upregulate the expression of cysteine, methionine, and glutathione (GSH) synthesis, as well as gene expression in some S reduction process, recent studies have further elaborated how to influence the reaction of jasmonic acid with S oxidative metabolism.In this experimental study, we identify transgenic plants through the GUS and Western Blotting techniques and obtain two high expression of transgenic plants SHC-7 and SHC-10. We analyzed the expression levels between SHC-7 and SHC-10, and selected SHC-10 and WT for further study. at physiological levels, content of MDA is decreased in transgenic plants SHC-10, however, GSH, POD and chlorophyll has significantly improved when SHC-10 and WT are under salt stress condition. The reasons for this result, we believe that the SAC protein activity may affect certain hormone levels in rice.In order to verify the above assumptions, we have found response gene OsJAZ8 that can reflect the level of jasmonic acid in rice, the relative transcript levels of WT and SHC-10 were measured by using semi-quantitative PCR and RT-PCR and the result show that content of endogenous jasmonic acid in WT is 0.54 times than SHC-10 when treated with OmM NaCl. However, the difference between the two kinds of rice is very obvious, SHC-10 is 19 times than WT at 60mM, SHC-10 is 1 times the WT at 100mM. Expression of OsJAZ9 indicate that SHC-10 higher than WT, SHC-10 is 140 times than WT at OmM, it have biggest difference under this concentration.we found SAC protein is modified in rice compare with control, but SAC protein activity is measured that APSK Vmax= 344.5(±28.611)μM/min, Km =0.23(±0.103)μM,ATPS Vmax=236.5(±3.19)μM/min, Km=0.04(±0.005)mM.In summary, this study shows that APSK and ATPS can improve salt tolerance in rice. Expression of SAC gene promote cysteine level, which is one of raw material for GSH in plants. Thus, the elevated levels of GSH helps eliminate an oxidizing environment in rice. Jasmonic acid plays an important role in plant when under abiotic stress conditions, the content of GSH also affects Os,JAZ8 gene expression response to jasmonic acid in rice. analysis of OsJAZ9 expression show that SHC-10 is better than WT at salt tolerance. The results indicate that there have a definite link in antioxidant metabolism between sulfur metabolism-related genes and jasmonic acid,GSH that can provides us with new insights.