| Germinated soybeans (Glycine max (L.),Which is rich in proteins, carbohydrates, vitamins and minerals, have been cultivated and used as human food for more than2000years in our country. Many previous studies have shown that series of biochemical and physical changes occur in the process of soybeans sprouting, in which residual enzymes of seeds are produced or activated, resulting in the decomposition of major components in sprouts such as starch and protein into usable smaller molecule. Actually, studies have shown that, as one of methods, the germination process can also be used to generate or improve the functional components of soybean like y-aminobutyric acid (GABA) and so on. The previous researchers has proved that GABA synthesis in plant is promoted via the pathways of GABA shunt and polyamine degradation; in the course of the synthesis, the glutamate decarboxylase (GAD, EC4.1.1.15) and the diamine oxidase (DAO, EC1.4.3.6) act as the most important rate-limiting enzymes respectively. Meanwhile, stressful environmental conditions, such as salt stress and hypoxia, could strongly promote activity of GAD and (or) DAO, resulting in the obvious accumulation of GABA in plant. In the research of this thesis, mobilization of stored reserves and the dynamic changes of GABA in soybean during germination were investigated; the accumulation and regulation mechanism of GABA in germinated soybean was carried out under hypoxia, salt stress and combined treatment; the different effects of endogenous ABA and Ca2+on expression level of GAD, DAO and CaM gene were explored and thus the accumulation and regulation mechanism of GABA in germinating soybean were revealed on the molecular biological levels, which were also helpful to improve synthesis capability and activity of GAD and DAO in germinating soybean under adversity stress conditions, so the effects of GABA accumulation will be improved. The main results were as follows:1. Soaked at30℃for4h, the soybean seeds had sufficient moisture content, satisfactory germination percentages and GABA accumulation. It is indicated from the result that, with the increase of culture temperature and time,the sprout length and respiration rate of the germinating soybean were enhanced,the contents of soluble sugar decreased, and the content of reducing sugar, soluble protein, free amino acids and GABA increased, but the dry matter content presented the downward trend along with culture time. Correlation analysis revealed, on the one hand, that the GABA accumulation had a significant positive correlation with sprout length, respiratory rate, soluble protein, free amino acid(r,0.878～0.943). By comprehensive consideration of quality and GABA accumulation in germinated soybean, the suitable germinating condition for high GABA germinated soybean was dark culture for5d at30℃;the activity of GAD and DAO in germinated soybean cotyledon and embryo first increased and then decreased; The GAD activity of cotyledon and embryo reached at maximum value when the germinated soybean was cultured3d and the DAO activity of cotyledon and embryo reached at maximum value when the germinated soybean was cultured5d and4d respectively.2. The Response Surface Methodology (RSM) were used to have a research into the effects of different NaCl content, culture time and culture temperature on the y-aminobutyric acid (GABA) accumulation during germination of soybean; results showed that the optimal condition with salt stress for GABA accumulation in germinated soybean was at a NaCl content of133.5mM, a culture time of5.5d and a culture temperature of33.3℃, and that the actual highest GABA yield was1197.36μg/g DW. Analysis of variance and confirmatory trials for the regression model suggested that the model can quite exactly predict GABA accumulation in germinated soybean under salt stress. The suitable stress opportunity was dark culture for2d with distilled water and then hypoxia stress in aerated culture medium for2d in a dark incubator at30℃.Under these conditions, the maximum GABA content was4.5times higher than that of the initial sample of aeration treatment (0h) and13.5times higher than that of the raw material.Correlation analysis also revealed that the GABA accumulation was significantly correlated to the corresponding physicochemical. Box-behnken experimental analysis showed that the optimal condition with aeration treatment for GABA accumulation in germinated soybean was at a temperature of30.5℃, a pH value of4.1and an air flow rate of0.9L/min; and that the actual highest GABA yield was2.65mg/g DW, which was16.6times of that in raw seeds.3. The length of sprout and GAD activity were not affected apparently by2.5mmol L-1AG treatment, however, the DAO activity of sprout was inhibited significantly. This result suggested that the hypoxia-induced accumulation of GABA in soybean embryo resulted in part from PA oxidation, whose contribution via DAO to GABA accumulation was about32%. There were significant differences on GABA accumulation of germinated soybean at different parts with different additives; namely, responses to different concentration of glutamate (Glu), pyridoxal phosphate (PLP), arginine (Arg), CuCl2, NaCl, CaCl1, significant differences which include accumulation of GABA, changes of GAD and DAO activity in germinated soybean under hypoxia, were occurred, and the maximum accumulation of GABA were4.07,3.02,3.50,3.26,4.00and3.30g kg-1DW respectively, which were significantly higher than those of germinated soybean under normal culture (CK) and hypoxia culture (CKo). The GAD and DAO had different distributions in the cotyledon and the embryo of germinated soybean, and the enzyme activity mainly located in the embryo of germinated soybean. Treated with optimal additive concentration of Glu and NaCl in culture medium, the GABA content in germinated soybean reached the maximum value than the others which reached4.07and4.00mg/g DW.4. Under hypoxia-salty stress, the GAD activities of cotyledon and the embryo of germinated soybean were1.64and1.76times of that under hypoxia stress respectively. Based on it, the GAD activities were promoted further after adding CaCl2which were1.80and2.26times of that under hypoxia, but Glu contents were decreased by44.50%and44.37%. The result indicated that Ca2+had an obvious activation on GAD activity under hypoxia-salty stress, Ca2+could form a chelated structure by adding EGTA in germinated soybean, which blocked combination of Ca+and CaM so that GAD activities were decreased. In turn, the decrease of GAD activities leads to Glu accumulation. Glu contents of cotyledon and embryo were thus increased by18.32%and35.49%compared with that under hypoxia-salty stress.5. The DAO activities of cotyledon and embryo of germinated soybean under hypoxia-salty stress, increased by36.52%and28.43respectively than that under hypoxia stress; however, the fPut contents decreased by7.15%and37.98%respectively. The results showed that hypoxia-salty stress promoted the transition from fPut to GABA by polyamines degradation. Under hypoxia-salty stress, exogenous Ca2+can increase fPut content and GAD activity of germinated soybean. It can be speculated that Ca2+promoted GAD activity. Hence GABA accumulation was strengthened by GABA branch. As a regulator, polyamine degradation pathway was inhibited indirectly so that the fPut was accumulated in germinated soybean.6. Under hypoxia-salty stress, the GABA chunt and polyamine degradation were simultaneously strengthened. The GABA contents in cotyledon and embryo were1.47and2.00times of that under hypoxia stress. The results indicate that the accumulative effects exist in hypoxia stress and salty stress. Under hypoxia-salty stress, the gene expression of GAD and DAO of cotyledon and embryo were increased by53.76%and173%respectively when AG was added. Meanwhile, expression of CaMs was changed correspondingly. It can be concluded that formation of GABA by polyamine degradation was blocked when AG inhibited DAO activity, which induced strong genes expression of GAD, so that the decrease of GABA contents due to inhibition of DAO was supplemented. Under hypoxia-salty stress, five expressions of CaMs genes in soybean were higher than the ones under hypoxia stress. The results showed joint stress promoted the expressions of CaM genes, which in turn promoted GAD activity as well. Accordingly, the promoted expressions of CaM genes as well as the promoted GAD activity lead to GABA accumulation in germinated soybean. Under hypoxia-salty stress, the gene expression levels of GAD of cotyledon and embryo increased by0.54and2.98times when Ca2+was added, in which the combination of Ca2+and CaM activated strongly the gene expression of GAD; gene expression of GAD in turn, contributed to GABA accumulation content through Ca2+increase.7. Under hypoxia-salty stress, the GAD activity of germinated soybean gradually increased from Coty to RadⅡ. GAD activity of RadⅡ is3.69times of Coty. Treated with exogenous ABA, endogenous ABA and Water-soluble Ca2+content increased, and gene expression of SCaM1and SCaM2were strengthened too, which increased GAD activity but decreased Glu content. After adding Flu, the endogenous ABA, Water-soluble Ca2+content and GAD activity decreased significantly, and thus induced accumulation of Glu. It showed that Ca2+content in germinated soybean had rapidly increased stimulated by exogenous ABA, and correspondingly, gene expression of CaM had been strengthened simultaneously, so GAD activity was activated, which sped up transformation of Glu. Compared with the control treatment, GAD activity of germinated soybean increased when it was treated with ABA, but expression of GAD gene showed no significant difference; whereas, GAD activity was decreased, but gene expression of GAD was strengthened when it was treated with Flu. It showed that GAD mRNA expression is not the only factor to increase GABA of germinated under hypoxia-salty stress; the activation of GAD protein synthesized in germinated soybean may also be involved in as a main factor.8. The DAO activity in germinated soybean is highest in the Rad I. GAD activity of Rad Ⅱ was decreased but was higher than that in the cotyledon. Compared with the control treatment, under hypoxia-salty stress, fPut and fSpd content of germinated soybean from Coty to Rad Ⅱ were higher when adding ABA, but both decreased when Flu was added, which showed that accumulation of fPut and fSpd can be improved by ABA, and that the gene expression of DAO in germinated soybean was significantly higher than that of Coty. The increase of DAO activity and the accumulation of polyamines work together to accelerate the transformation from fPut to GABA, resulting in the increase of GABA content in germinated soybean. Under hypoxia-salty stress, the gene expression of DAO of Rad Ⅱ in germinated soybean decreased when ABA was added to, but gene expression of DAO showed not significant difference in other parts of the soybean. Whereas, when treated with Flu, DAO activity decreased, but gene expression of DAO was strengthened. As a result, the activities of GAD and DAO, as well as the GABA content in embryo were higher than those in cotyledon, which showed that the embryo of germinated soybean was the vital part in GABA formation. |
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