The Cloning Of Arabidopsis Glycosyltransferase Gene UGT76E12 And Its Role In Plant Adaptation To Stress

The responses of plants to abiotic stress are related to a complex series of physiological and biochemical processes, and they often involve interactions among multiple genes. Until now what we have found may simply reflect some aspects of the whole stress-adaptation mechanisms. Therefore, studying the plant adaptation to stress from a new angle could help us to better understand the plant stress response mechanisms in a comprehensive level. When plants are coping with adversities, the modification of small molecules can enable the plant to have greater flexibility under stress conditions, among them, the glycosylation modification of glycosyltransferase catalysis plays a very important role in regulating the solubility, stability and biological activity of small molecule.Glycosyltransferases are enzymes responsible for glycosylation of plant compounds, which catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, thus change their biological activity, water-solubility, transport characteristics, subcellular localization and binding properties with receptors. Recently, there are many researches on plant glycosyltransferases, which have proved that glycosyltransferase have many biological functions in plant. Recent studies have shown that plant glycosyltransferase is also involved in regulation of plant response to abiotic stress reaction process, however, the specific mechanism is unclear, so we should explore the molecular mechanism of glycosyltransferase involving in plant stress adaptation.In this paper, according to the microarray data, and combined with the real-time PCR analysis, we found that UGT73B4, UGT73B5, UGT73D1, UGT76E12 were induced by salt, mannitol, ABA and other stimuli. So, we constructed the plant overexpression vector of the four glycosyltransferase gene UGT73B4, UGT73B5, UGT73D1, UGT76E12 driven by CaMV 35S promoter. Then through molecular biology and reverse genetics methods, we mainly studied the function of UGT76E12 gene, trying to investigate the relationship between glycosyltransferase and the plant response to stresses through the model plant Arabidopsis thaliana.The main contents and results of this study are summarized as follows:1. The glycosyltransferase gene UGT76E12 was proved to play a significant role in plant response to stress.Based on our real-time PCR analysis, we proved that the expression levels of UGT76E12 gene was induced by salt, mannitol and ABA. Moreover, through analyzing the phenotype of UGT76E12 transgenic arabidopsis under stresses, we found that the germination rate and greening rate of 35S::UGT76E12 transgenic arabidopsis was lower than that of wild-type arabidopsis in conditions of salt, mannitol, ABA treatment. These discrepencies were only observed during seed germination and postgermination growth, demonstrating that UGT76E12 gene may affect the plants adaption to stresses during this specific stage.2. The glycosyltransferase gene UGT76E12 was found to affect the plant adaptaion to stresses via the ABA synthesis pathway.When the inhibitor of ABA synthesis pathway sodium tungstate was added in the culture medium, sensitive phenotype of 35S::UGT76E12 lines was restored. We infered that it may be associated with ABA. So we analyzed the expression level of several ABA synthesis pathway related genes in UGT76E12 transgenic arabidopsis thaliana. Results show that under mannitol treatment, the expression of some ABA synthesis related genes ABA3, AAO3, NCED in UGT76E12 transgenic arabidopsis thaliana were significantly higher than that in wild-type plants, demonstrating that UGT76E12 affects the plant response to stresses possibly via the ABA synthesis pathway3. The plant expression vector, GUS expression vector, prokaryotic expression vector of three additional Arabidopsis glycosyltransferases genes were also constrcted.Using RT-PCR method, we cloned the full-length cDNA of UGT73B4, UGT73B5 and UGT73D1 from Arabidopsis thaliana, and construct their plant expression vectors. Then through the Agrobacterium-mediated genetic transformation, kanamycin resistance screening and RT-PCR detection, we obtained the Arabidopsis transgenic plants of these three genes. We identified their T-DNA insertion mutants of these three genes by using three-primer PCR method. Salt and drought stress tolerance analyses using these materials were carried out. For the above material has carried on the preliminary analysis of the resistance to salt, drought tolerance experiment.In summary, through a series of experiments, we proved that the glycosyltransferase gene UGT76E12 is involved in the plant stress adaptation, providing a novel stress regulation mechanism in plants. At the same time, the other three glycosyltransferase genes were cloned and their transgenic plants were obtained. However, the mutant of UGT76E12 gene was not yet obtained, and its detailed molecular mechanism involved in plant stress adaptation needs further exploration.