Function Validation Of Dihydroflavonol 4-reductase (DFR) Gene From Populus Tomentosa With Expression In Transgenic Tobacco

Flavonoids are plant-specific secondary metabolites derived from the phenylopropanoid pathway. They are involved in various biological processes during plant growth and development, male fertility, cell cycle regulation and auxin transport. They also play key roles in symbiotic interactions between plants and microbes. A lot of researches have indicated that flavonoids have multi-biologically active in our body, including anti-oxidation characteristic, clearing free radicals, resisting virus, resisting inflamemation, vein expanding and so on.Through an elaborate network of pathways that share common precursors to flavonoids biosynthetic pathway, until recently, functional dissection of these networks has depended largely on molecular information stored in the genome of Arabidopsis, an annual herb. Now that the Populus genome sequence is available, the potential for understanding and exploiting secondary metabolism in tree species comes closer to realization.In the present overview, genomic information pointing to greatly expanded gene complexity and function of the phenylpropanoid pathway in Populus is summarized. Phenylpropanoid-derived flavonoid and salicylate phenolics occur in numerous functionally distinct forms, and can account for 50%of leaf biomass in Populus and other fast-growing tree taxa. Their potential effects on tree growth and their documented impacts on ecosystem diversity and productivity justify molecular dissection of secondary metabolism in Populus.Dihydroflavonol 4-reductase (DFR; EC 1.1.1.219) is a later key enzyme controlling flux into biosynthetic pathway branches leading to anthocyanins and condensed tannins (CTs). DFR preparations from several plants catalyze the reduction of the three dihydroflavonols dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM) into leucoanthocyanidins, which are common precursors for anthocyanins and CTs (proanthocyanidins) synthesis.Two DFR cDNA clones (PtDFR1 and PtDFR2) were isolated from Populus tomentosa. Construct the plant expressional vector of sense and antisense DFR gene under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. PBK-DFR1-S and pORE4-DFR2-S carried the sense DFR gene, PBK-DFR-As carried the antisense DFR gene, and then transferred into Agrobacterium tumefaciens (LBA4404), respectively. DFR gene was introducted into Nicotiana tabacum by leaf dish transformation. Transgenic plants were obtained under the selection pressure of kanamycin concentration (50 mg/L).To identify transformed plants carrying integrated DNA, PCR amplify the target gene (NPTâ…¡and DFR) from genomic DNA of transgenic tobacco plants. Wild-type tobacco is negative control, while the plasmid that carry target gene is positive control. The results have shown that the target gene was inserted into the genome of transgenic plants successfully.Total RNA was isolated from leaves of both transgenic lines and wild-type plants, and then reverse transcription synthesize cDNA. To 18s rRNA gene as an internal, adjust the concentration of the same cDNA template. PtDFR transcript levels, as indicated by reverse transcription polymerase chain reaction (RT-PCR), were increased in the sense transgenic lines contrasting to untransformed tobacco, but were significantly inhibited in the antisense transgenic lines contrasting to sense ones.The flowers of the PtDFR expressing plants had distinct pigment differences from wild-type plants, which produce corolla pink flowers under standard greenhouse conditions. There were visible increases in the color of transgenic flowers, whereas overexpression of PtDFR2 did not. The DFRlexp sense flowers were dark pink and even pistils were in some lines. The DFRlexp antisense flowers were white, only had the visible light pink on the edge of the corolla.Anthocyanins were eatraced from petals of wild-type and transgenic tobacco plants. HPLC analysis of the extract, cyanidin-3-arabinoside is the primary anthocyanin in the tobacco. Compared with wild-type tobacco, the peak area of DFRlexp sense plants was larger, whereas DFR2exp sense plants did not. The peak area of DFRlexp antisense plants was smaller.Total flavonoids were determined by colorimetric method with rutin. Compared with the wild nature of tobacco, the total flavonoids in transgenic plants is not a significant difference in wear.In this study, the function of DFR gene from Populus tomentos on anthocyanin synthesis was verified by expression in transgenic tobacco. This contributed to study the flavonoid pathway on the perennial. Advances in phytochemical regulation should elevate the potential for improved biomass quality and production through genetic selection or metabolic engineering in Populus.