| Isoflavones are phenolic secondary metabolites found mostly in legumes.Thesecompounds play key roles in many plant-microbe interactions and are associated withthe health benefits of soy consumption. Because of their biological activities,metabolic engineering of isoflavonoid biosynthesis in legume and nonlegume cropshave significant agronomic and nutritional impact by enhancing palnt diseaseresistance and providing dietary isoflavones for the improvement of human health.Daidzein, genistein and glycitein compounds comprise the most common groupof soybean isoflavonoids. These soybean secondary metabolites contribute to soybeanagainst the attack of pathogenic microorganisms. Related researches show daidzeinnot only involved in protecting the plants from diseases and gnawing by herbivores,but also owes other functions such as resisting the radiation of strong ultraviolet light,promoting the sedimentation of anthocyanidin and pollen germination. In soybean, theattack of pathogenic bacteria induces the synthesis of isoflavonoids, especiallydaidzein compound. Chalcone reductase is a key enzyme among daidzein biologicalsynthesis, which can catalyze4,2’,4’–trihydroxychalcone co-act with chalconesynthase(CHS). Isoliquiritingenin (4,2’,4’-trihydroxychalcone) is the precusorsubstances necessary for the biological synthesis of daidzein. Recently, the researchfocus on abilities of chalcone reductase (CHR) which is one of key enzyme during thedaizein pathway is still at the early-stage. Contrast to geinstein that is another normalsoybean isoflavone, the synthesis and regulation mechanism into daizein are notclarification enough to application. The diversity of function and structure ofisoflavonoids and the complicated regulation of various genes of its biologicalsynthesis pathway provide an ideal research model to research the interaction of geneexpression in the secondary metabolic pathways of plants.1. In this research, we scanned soybean EST library to obtain two gene segments ofchalcone reductase and used RACE technology to amplify the gene cDNAcomplete sequence, totally1417bp (Genbank code: KF758395), and contain969bp reading frame, the other (Genbank code: KF938604) is1410bp with codingregion of966bp. 2. We analyzed the conservative domains in the gene sequence and the copy numberof reductase gene in the soybean genome, and3. We also analyzed that the chr genes at the root had the severest variation ofexpression quantity under the stimulated induction of phytophthora root rot ofsoybean by real-time fluorescent quantitative PCR technology (Real-timePCR).Through high-performance liquid chromatography (HPLC), we analyzed thecatalytic product of the reductase4,2’,4’–trihydroxychalcone, namelyisoliquiritingenin, the content of which increased to3.972μmol/g.4. Meanwhile, compared with the known chalcone reductase gene of soybean, werevealed the difference of natures among different genes, especially in expressedlevel of the interested genes. The cloned new genes were transformed into thetobacco individually, and the catalytic function of the new gene expressionproduct was verified through real-time PCR and HPLC respectively. The resultshowed the interested genes were able to express significantly in defferent tissuesof tobacco, and the average accumulation of isoliquiritingenin are38.52μmol/gand9.63μmol/g for Gmchr2and Gmchr4gene individually.5. By molacular biotechnology, to co-express the gene of chalcone redectase andchalcone synthetase inside transformed Lactobacillus, analyze the product of thefermentation to comprehend interaction of two major enzyme in the pathway,clarify specific function and efficiency among the different typical chalconereductases and the regulation mechanism in the synthesis way.6. The results of combined fermentation suggested the key factors for combinedfermentation were temperature and pH value of the reaction. And under theoptimal condition, the isoliquiritingenin content was improved to10.3times thanthe origin. |
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