Optimization Of Extraction System For Soybean Isoflavone And Construction Of Silencing Expression Vector For Soybean Flavanone 3-Hydroxylase Gene (F3H)

In natural conditions, isoflavones can protect the soybean from the pathogenic microorganisms to grow well, and due to its bitter taste, the soybean is less grazed by herbivores.In application, it plays an important role in the body's health and disease treatment.In the study, we established a optimized system of extracting soybean isoflavone by ultrasonic wave for the first time,using the ultrasonic method to extract and the three-wave length UV spectrophotometry method determinated by Ju XY to determine.With selecting ethanol as solvent, single element effect test to extraction ratio of soybean was conducted under four factors, namely, concentration of ethanol, extraction time, ratio of material to solvent and ultrasonic power. Based on single element effect test, using L9(34) orthogonal table analyzed the influence of these factors to extraction content of soybean isoflavone. Finally, the optimal condition was that:concentration of ethanol 50%, extraction time 30min, ratio of material to solvent 1:20, ultrasonic power 100W; The relative standard deviation (RSD) was 1.7%, average recovery of recovery test was 95.0%, recovery rate between 95% and 105%, and the coefficient of variation was 4.00%. All above showed that the method was feasible.Soybean isoflavone content of 24 varieties were determined by the optimized system. We found that:soybean isoflavone content ranged from 0.11% to 0.31%, and changed significantly. In All species, the highest isoflavone content was in cultivar Jilin 32, lowest in Dongnong 42 and Dong 163;The results from the two methods showed that compared to high performance liquid chromatography (HPLC), the three wavelength method was more accurate. We could choose cultivar Jilin 32, the isoflavone content of which was the highest, as the experimental material in the next test;According to the flavanone 3-hydroxylase (F3H) cDNA sequence from NCBI database(GeneBank:AF 198451), we choosed 547bp fragment from 23-569bp of F3H gene to construct a sense plant expression vector pBI121-F3H-antisense-Q and pBI121-F3H-antisense-B and RNA interference plant expression vector pBI121-F3H-RNAi. The promoter of RNA interference plant expression vector pBI121-F3H-RNAi was CaMV35S promoter, CHS8 intron located between sense and antisense fragment, including NOS as a terminator.