Cloning Of Soybean (Glycine Max) Flavone Synthase Gene And RNA Interference Regulation In Isoflavone Production

Flavonoids are one kind of secondary metabolites that are widespread throughout thegrowth and development of plants, including isoflavone, flavone, chalcone, anthocyanidin.They play diverse roles in regulation of auxin transport, modulation of flower color,plant–microbe interactions, and have direct but complex effects on human health. Theresearch of flavonoids biosynthesis pathway plays important roles in plant defenseresponse and improving functional components of food.In all higher plants, flavonoids are produced through a branch of the phenylpropanoidpathway. At present, the research for phenylpropanoid pathway has been focus on the fieldof molecular biology, more and more coding genes in pathway has been cloned andidentified in soybean. On the other hand, the development of metabolic engineering,especially RNA interference technology, opens up new possibilities for the study ofregulation of plant secondary metabolite accumulation and their roles in plant defense.In this study, two FNS Ⅱ genes from soybean cultivar Hefeng47were clonedaccording to basic local alignment search tool (BLAST) contexts using flavone synthasesequences reported in other species. These were named GmFNSⅡ-1and GmFNSⅡ-2.Sequence alignments showed that the cDNA of GmFNSⅡ-1was identical to that ofCYP93B16, whereas GmFNSⅡ-2was clearly distinct. Both GmFNSⅡ genes wereanalysized by bioinformatics in their genome sequences, the nucleotide sequence, aminoacid sequences, physical and chemical properties of protein, and function.Functional assays in Escherichia coli and yeast (Schizosaccharomyces pombe)suggested that these two enzymes could convert (2S)-naringenin into apigenin. The twoGmFNSⅡ genes had similar tissue-specific expression patterns, and were significantlyincreased by osmotic stress like glucose and MeJA treatment. This demonstrates that the gene plays an important role in the response to defense signals in soybean.Here, a bivalent RNA interference (RNAi) plant-transformation vector wasconstructed to silence both the flavanone3-hydroxylase (F3H) gene and the flavonesynthase Ⅱ (GmFNSⅡ) gene. Moreover, two further unit RNAi vectors were constructedfor each of these two genes.RNAi-mediated suppression of those genes showed a significant reduction (to <10%)in gene expression in hairy roots that arose from soybean cotyledons transformed withAgrobacterium rhizogenes (ATCC15834). HPLC showed in hairy roots，RNAi-mediatedsuppression of these genes effectively regulated flavone and isoflavone production.Notably, the bivalent RNAi vector had a significantly greater effect for increasingisoflavone production compared with the two unit RNAi vectors.This present study highlights molecular methods that can be used to enhanceisoflavone production in soybean, and demonstrates the challenges associated with suchmetabolic engineering for the production of plant natural products.In this study, we tested two flavonoids, apigenin and genistein, in amended culturemedia against seven soybean pathogens including Colletotrichum truncatum,Macrophomina phaseolina, Phoma exigua, Phytophthora sojae, Pythium ultimum,Rhizoctonia solani, and Sclerotinia sclerotiorum. Both compounds restricted growth of allseven pathogens, although not all equally. For example, inhibition of growth was less (P>0.05) for C. truncatum than the other pathogens tested. There also was a dosage effect asincreased concentrations of either compound further restricted colony growth. The resultssuggest that through metabolic engineering, apigenin and genistein may be useful targetsfor overexpression in planta to enhance disease resistance in soybean.