| Isoflavones is important polyphenolic secondary metabolites in the soybean. Plant androgenbelonged isoflavones, which played positive role on decreasing the level of blood-lipid andrestraining fatty deposition. Daidzein may constitute30%~35%of total isoflavones, and it is oneof the main ingredient in isoflavones of soybean. chalcone reductase of the soybean was catalyzedsynthesis of the liquiritigenin that is precursor of daidzein biosynthetic pathways. chalconereductase (CHR) was specific existed in Biosynthetic pathway of daidzein where chalconereductase played important role, and there has effects on synthesis of the genistein.The results ofthe research as follows:1. Total RNA was extracted from leaves of Jinong17soybean. cDNA synthesis wasperformed according to the guidelines of RevertAid First Strand cDNA Synthesis Kit inFERMENTS company. The BLAST research on the NCBI website, the primers according to theconserved sequence of the novel chalcone reductase genes was designed for the RACE method.2. Chalcone reductase genes was linked to prokaryotic expression vector Pet28a, whichtransformed to Ecoli. BL21.. To verify the transformant, about969bp fragment ampliedby using colony PCR was sequenced, the result is consist with expectation.3. chr3was successfully expressed in Ecoli..BL21-pET28a-chr3was induced4hours byIPTG and ultrasonication, which was purified by Ni-NTA affinity chromatography. The proteinwas detected32.5KDa by SDS-PAGE.4. The expression product of chr3could be identified catalytic function by HPLC.Liquiritigenin was detected content changes of Before and after reaction, which Controlled forBL21-pET28a with only pET28a.The peak area of liquiritigenin was542256in the control,which was converted to1.597μmol/g. In the samples of recombinant bacteria, the peak area ofliquiritigenin increased clearly was1241766that is converted to3.673μmol/g. chr3protein wascatalyzed synthesis of liquiritigenin, that turns out biological function of chr3gene. |
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