Induction Of Isoflavonoid Secondary Metabolites In Soybean Cotyledons And Primary Study Of The Related Microsomal Proteins

Soybean [Glycine max (L.) Merr.] not only can supply us plentiful high quality proteins and oil, but also is an important source of isoflavonoids with a variety of bioactivities. Prenylated flavonoids are being noted for their increasingly outstanding bioactivities and limited spreading. As the main product of hypersensitive response in soybean, glyceollins is just such kind of prenylated isoflavonoid.In the present study, we used multiple modern technologies such as analytical HPLC, preparative HPLC, isotope-labeled, thin layer chromatography, Q-TOF/MS/MS and 2D electrophoresis to assess theaccumulation of glyceollins and their precursor-glycinol induced by silver nitrate and jasmonic acidin Zhonghuang No. 13 and "Cultivar 1". The dynamic isoflavonoids profiles during the induction were also discussed. At the same time, the prenyltransferase catalyzing the formation of glyceollins from glycinol in the soybean was also assayed. Furthermore, a primary study on the microsomal proteins of the induced soybean cotyledons was also conducted. Main conclusions are listed as follows:1. Zhonghuang No.13 and "Cultivar 1" perform differently to the extraneous stimulates. ZhonghuangNo. 13 appears more sensitive than "Cultivar 1".2. In the case using silver nitrate as the stimulate, the ratio of glyceollin isomers doesn't change duringthe incubation. The levels of daidzein and genistein increase and those of glycosides decrease. In contrast, the ratio of glyceollin I and HI decease and the amount of glyceollin II increase in the jasmonic acid treated samples. At the same time, the content of free aglycons falls off and that of their conjugates goes up. It suggests that silver nitrate and jasmonic acid maybe act to the soybean via different mechanisms, although both stimulates can induce the accumulation of glycinol and glyceollins. Silver nitrate of 0.01M and jasnomic acid of 10μM were found to be most effective to induce the formation of glyceollins.3. The compounds associated with the formation of glyceollins in the phenypropanoid pathway can notonly transform to each other, but also transfer spatially during the accumulation of glycinol and glyceollins. Phytoalexins accumulate only in the point of treatment. The level of the isofavonoids related to the production of phytoalexins in them is higher than that of the neighboring health cells. There is a tendency that these isoflavonoids are liable to being transferred from the surrounding health cells to the point of treatment. The associated compounds in the vertical orientation can move to the treated sites much easier than those in the horizontal orientation.4. High quality glycinol was purified from soybean cotyledons induced by 0.01M AgNO₃ using preparative HPLC. It provides the rare substrate for the prenyltransferase assay in soybean. At the same time, it supplies an effective method for the isolation and purification of glycinol.5. Prenyltransferase assay was determined using [³H] DMAPP and glycinol as substrates in the presentstudy. The products of enzymatic reaction were detected and separated using TLC and identified as glyceollidins by Q-TOF/MS/MS.6. The conditions for the separation of microsomal proteins by 2D electrophoresis were optimized. Inthe optimized conditions, 3-(4-hepty) phenyl 3-hydroxy propyl)(dimethylammonio propane sulfonate) was used to solubilizing of the membrane proteins and tributyl phosphine was used as the reducing agent.7. The results of 2D electrophoresis indicated that the control contained more proteins than that of theinduced sample. The isoelectric points (pi) of most microsomal proteins in soybean cotyledons were found to be pH 4⁶. Fourteen novel spots with pis of 3⁶ (4 spots) and 5-8 (10 spots) were observed in the sample induced with AgNO3. There was no such induced spot in pH 7-10.8. The results of Q-TOF/MS/MS analysis and amino acid sequence analysis suggested that AgNO3 canelicit the production of soybean Kunitz trypsin inhibitor and its 5kDa subunit and "SAM22_SOYBN".