KR20050062477A - Transgenic rice line producing high level of flavonoids in the endosperm - Google Patents

Abstract

The present invention seeks to develop transgenic rice that produces high levels of flavonoids in whole endosperm. Rice varieties, Oryza sativa japonica cv. Hwa-Young is transformed with maize C1 and RS genes, which together activate most structural genes in the flavonoid biosynthetic pathway. Expression of the C1 and RS transgenes is endo-specific for promoter utilization of the 13-kD rice prolamin gene, which is specifically expressed throughout the endosperm. For the staining variation of the 27 individual C1 / RS transformation lines, the T1 grains of most lines are light brown and distinctly darker than wild type grains as well as transgenic rice plants with vectors alone. T2 and T3 grains of homozygous transformation lines are darker colored and have smaller T1 grain sizes.

Description

Transgenic rice line producing high level of flavonoids in the endosperm

The present invention relates to transgenic rice containing high concentrations of various flavonoids in rice grains. The present invention also relates to transgenic rice transformed with maize C1 and RS genes.

Research on crop plants has significantly improved productivity through breeding for high yields, disease / pest-resistance and anti-dwarfism. Recently, genetic engineering tools, along with conventional breeding methods, are readily used to develop new crop varieties with improved nutrition as well as enhanced nutritional quality.

One well-known example is the provitamin A-producing "Golden Rice" genetically designed with three genes whose endosperm is involved in the carotenoid biosynthetic pathway (Ye et al., 2000). Cahoon et al. (2003) report the development of transgenic maize that produces four to six times more vitamin E in the embryo by overexpressing barley homogenicic geranylgeranyl transferase. .

More than 2,000 flavonoids produced in plants are involved in the coloring of flowers and fruits, protection against UV and pathogens, signaling for symbiotic organisms and male fertility in some plant species (Shirley, 1996; Winkel-Shirley, Considered in 2001a, 2001b and 2002). All flavonoids are synthesized through successive condensation reactions of p-coumaroyl-CoA with three molecules of malonyl-CoA. In addition to the minor modifications of phenylbenzopyrone itself, flavonoids are diversified by the addition of various moieties such as hydroxyl, methyl and sugar groups to the basic structure.

Flavonoids have been extensively studied for their biological activity, which includes antioxidant, antiviral, anticancer, anti-aging, hepatoprotective effects, cancer prevention, strengthening immune system and consequently improving serum lipid quality which lowers the risk of cardiovascular disease (Dixon and Steele, 1999; Yousef et al., 2004).

While most of the healthy-beneficial roles are primarily associated with the antioxidant properties of flavonoids that function as reducing agents, hydrogen donors and free radical quenchers, many non-antioxidant activities have inhibitory effects on carcinogenesis (Jankun et al., 1997; Ren et. al., 2003) and the regulatory effects on receptors and intracellular signaling enzymes (William et al., 2004). Pinent et al. (2004) also reported that procyanidins, oligomeric flavonoids have antidiabetic properties, possibly through interactions with signaling components such as phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinases. . In a similar study, Enomoto et al. (2004) demonstrated the inhibitory effect of 3-methoxy quercetin on human aldose reductase, platelet aggregation and blood coagulation.

Among the major secondary metabolic pathways in plants, flavonoid biosynthesis is best characterized for the molecular genetics of the genes and enzyme mechanisms involved in that pathway (Holton and Cornish, 1995; Winkel-Shirley, 2001a and 2001b). Members of the C1 and R regulatory gene families, each encoding a Myb-form transcription factor and a basic helix-loop-helix-form transcription factor, individually or mutually activate different sets of structural genes within the flavonoid biosynthetic pathway (Quattrocchio et al., 1993; Quattrocchio et al., 1998).

Both gene family members have been isolated from many monocots as well as dicotyledons and are known to be highly conserved within the corresponding families. For this reason, some C1 and R family members are functionally interchangeable between different plant species (Schijlen et al., 2004). For example, the ectopic expression of both corn C1 and R was sufficient to produce anthocyanins in roots, drawing boards, and surgery, which are generally deficient in anthocyanins in Arabidopsis and tobacco (Lloyd et al., 1992). . Similarly, Bovy et al. (2002) reported that tomato plants transformed with both corn C1 and LC ( R gene family members) produced anthocyanins and additional flavonoids in leaves and pulp, respectively. In addition to regulatory genes, increased production of specific flavonoids in certain tissues of tomato fruit is evidenced by increased expression of biosynthetic genes for chalcone synthase, chalcone isomerase and flavonol synthase (Verhoeyen et al., 2002).

The technical problem to be achieved in the present invention is to provide a transformed rice containing a high concentration of various flavonoids in rice grains. The present invention also provides a transformed rice transformed with maize C1 and RS gene.

Plain white rice, Oryza sativa japonica cv. Hwa-Young was transformed with maize C1 and RS regulatory genes, respectively, under the control of an endosperm-specific promoter. The T2 and T3 kernels of the homologous transformation line, designated "Green Tea rice" (GT rice), are not colored in size and are slightly more pigmented than T1 and T2 semi-bonded grains, which are slightly darker than untransformed grains. Darker and smaller in size.

In addition to expression of the C1 and RS transgene gene of the flavonoid biosynthetic pathway activated by the C1 and RS transgene was confirmed by RT-PCR using an RNA extracted sikimeuroseo developing grain of T3 homologous line. While the expression levels of phenylalanine ammonia lyase, chalcone synthase, flavonone 3-hydroxylase, flavonoid 3'-hydroxylase and dihydroflavonol 4-reductase in the activated genes were substantially increased in GT rice, However, these gene expressions were hardly detectable in the wild type. However, the anthocyanin synthase gene was not activated by the transgene, which explains the absence of anthocyanin production in GT rice grains.

Comparison of the HPLC flavonoid profiles showed that high concentrations of various flavonoids were produced in GT rice grains. It was not technically feasible to identify individual HPLC peaks by matching the retention time with the reference compound by numerous forms of flavonoids in the transformed kernel extract. Moreover, commercially available flavonoid standards are limited to HPLC analysis. To avoid these technical problems, many peaks in the HPLC profile were reduced by proper acid-hydrolysis and ethyl acetate partitioning. Then, was further analyzed using a major peak LC / MS / MS, and NMR, from which the dihydro quercetin (dihydroquercetin) (taksi morpholine (taxifolin)), sorbitan di-Hydro also netin (dihydroisorhamnetin) (3'- Ο - methyl taksi morpholine) and 3'- Ο - it was confirmed that methyl quercetin. Concentrations and total flavonoid content of Taxipoline, 3'- O -Methyl Taxoline and 3'- O -Methyl Quercetin, using certified taxolines as reference, were 150 μg, 330 μg, 55 μg and 12 per gram of dry grain, respectively. It was estimated in mg. Flavonoid fluorescence labeling with diphenylboric acid in T3 GT rice grains indicated that flavonoids were highly concentrated in the 4-5 layers of external endosperm. More importantly, moderate levels of flavonoids were also detected throughout the internal endosperm.

We also developed transgenic rice that produces flavonoids in endosperm by expressing the corn C1 and RS genes out of place on the following grounds: (1) Regular rice grains lack flavonoids, while "black rice" grains Includes a small class of flavonoids, anthocyanins, colored only within the rind that is completely removed by milling. (2) Dietary flavonoids have various health-promoting effects, such as antioxidant, antiviral, anticancer, anti-aging and hepatoprotective effects, as well as cancer prevention and immune system enhancement (Dixon and Steele, 1999; reviewed in Ren et al. , 2003). Moreover, Howitz et al. (2003) showed that flavonoids function to extend mammalian life based on the biological activity of activating genes involved in extending the life of yeast. (3) The C1 and RS genes, when introduced into the endosperm, are expected to activate most, but not all, structural enzyme genes within the flavonoid biosynthetic pathway.

Levels and forms of flavonoids vary significantly depending on plant species as well as tissue type. Grain endosperm, for example, lacks flavonoids, while green tea ( Camellia sinensis ) leaves contain high concentrations of flavonoids of 10-20% of their dry weight. For the "black rice" variant, some forms of anthocyanin, colored flavonoids are produced only in the skin. Therefore, we want to develop a transformation line in which flavonoids are produced over the entire endosperm of a grain. The strategy is to (1) transform normal japonica rice with maize C1 and RS (seed-specific members of the R gene family) to activate various structural genes within the flavonoid biosynthetic pathway, and (2) the nutritional tissue of the transgenic plant. Use of an endosperm-specific promoter that modulates expression of the transgene to prevent side effects in the stomach.

(Example)

C1 Of R-S  Drying of the transgene

Each p35SC1 and pBluescript sequence number within the SK of the EcoRI site: with a 1 DNA sequence maize C1 (gene bank accession number AF320614) and SEQ ID NO: Clone of the RS with the DNA sequence of Figure 2 (accession number X15806) is the US University of Missouri Karen Provided by the doctor. A clone of the prolamin NPR33 promoter (5'UTR between -652 and -13 from the ATG start site of the rice 13-kD prolamin gene) (SEQ ID NO: 3) in the pUC18 plasmid was identified by the Japan National Institute of Agricultural Science Fumio. Provided by Dr. Takaiwa. The promoter was subcloned into the HindIII / XbaI site of the modified pGEM7Zf (+) plasmid (Promega Co., Madison, Wis.) Containing Nos terminator (Tnos) at the SacI / EcoRI site and named pYMPP. C1 cDNA (822 bp) uses a p35SC1 plasmid containing C1 cDNA as a template, and an anterior primer with XbaI restriction site (5'-AT TCTAGA CGAGCTTGATCGACGAGAGAGCGAG-3 ') (SEQ ID NO: 4) and a reverse primer with SacI site It was amplified by PCR using (5′-C GAGCTC GACGTGTACTT GTTGTCTACGCAAG-3 ′) (SEQ ID NO: 5). The PCR product was digested with XbaI and SacI, ligated into the same restriction site of the pYMPP plasmid, again named pYMPPC1. At this stage, the RS cDNA (1839 bp) is a different primer set, 5'-GC TCTAGA CGTTCAG CAGGCGCGTGATG-3 '(SEQ ID NO: 6, corresponding to the RS sequence with the XbaI site at the front primer end and the SmaI site at the reverse primer end). ) And 5'-CC CCCGGG GGCTGCCCCTTCACCGCTTCCCT-3 '(SEQ ID NO: 7) in the same way subcloned into the pYMPP plasmid, again named pYMPPR-S. Reverse primers (5'-CG) for Tnos with BamHI site using pYMPPC1 plasmid as template and forward primer for NPR33 promoter with HindIII site (5'- AAGCTT GGTGTAGCAACAC GACTT-3 ') (SEQ ID NO: 8) GGATCC CGGATCTAGTAACATA GATGACAC-3 ') (SEQ. ID. NO: 9) using the PCR product was digested with HindIII and BamHI, T-DNA, as well as smGFP phosphino new tree (phosphinotricin) resistant bar gene under the CMV 35S promoter :: GUS inside the boundary The modified pCambia3301 binary vector was also incorporated into the same restriction site. Since, RS coding sequence with a promoter and NPR33 Tnos is used pYMPPR-S plasmid as a template, the forward primer for the NPR33 promoter with a BamHI site (5'- GGATCC GGTGTAGCAACACGAGTT-3 ') ( SEQ ID NO: 10) and EcoRI Amplified by PCR using reverse primers (5'-G GAATTC C GATCTAGTAACATAGATGACAC-3 ') (SEQ ID NO: 11) for Tnos with site. PCR products were digested with BamHI and EcoRI and ligated into the same restriction site of the modified pCambia3301 including C1 . A modified pCambia3301, each with a prolamin NPR33 promoter and a Nos terminator and coding regions of the C1 and RS genes fused at the general restriction site BamHI, was named pYMPPC1 / RS and was identified by Agrobacterium tumefaciens (freeze / thaw). Agrobacterium tumefaciens ) was transformed into LBA4404.

Rice transformation

Callus comprises N6 base salts and vitamins, 1 g / L cassino acid, 30 g / L sucrose and 2 mg / L 2,4-D, and callus solidified by 2 g / L gellite -Rice Oryza sativa japonica cv. On induction medium (N6CI, pH 5.8). It was produced by germinating Hwa-Young's belly. After 1 or 2 weeks of secondary culture on fresh medium, the callus was infected with Agrobacterium tumefaciens LBA4404 incubated with the binary vector pYMprolaminProC1 / RS and subjected to Agrobacterium for 3 days at 25 ° C. on N6CI medium supplemented with 100 M acetosyringone. Co-cultured. Transformed callus cells were basal salts and vitamins, 1 g / L cassanoic acid, 30 g / L sucrose, 2 mg / L 2,4-D, 500 mg / L carbenicillin and 6 mg / It was selected by incubating for about 3 weeks on a selection medium consisting of L of phosphenothricin. The callus was then divided into N6 base salts and vitamins, 2 g / L casamino acid, 30 g / L sucrose, 30 g / L sorbitol, 1 mg / L 2,4-D, 0.5 mg / L 6 -Transferred to pre-regeneration medium containing benzyladenin, 0.25 g / L of Sephataxim and 6 mg / L of phosphinothricin. After 10 days of culture on pre-regeneration medium, callus was recovered from regeneration medium (MS base salt and vitamin, 2 g / L cassino acid, 30 g / L sucrose, 20 g / L sorbitol, 1 mg / L naphthalene Acetic acid, 5 mg / L of kinetin, 0.125 mg / L of Cytaxim and 6 mg / L of phosphinothricin). The greening of the caller and the regenerated seedlings from the callus were apparent after 4 weeks on the regeneration medium. Regenerated seedlings were transferred to growth medium (MS base salts and vitamins, 30 g / L sucrose, 3 g / L gellite, pH 5.8) and grown to about 10 cm-size before being transferred to soil.

GUS Analysis

Three to four leaf discs were punched from each plant after the T0 plants had grown for about one month and 2 mM 5-bromo-4-chloro-3-indole-BD glucu in 500 mM sodium phosphate buffer pH 7.0 Incubated for 18 hours in a GUS assay solution consisting of ronide, 1% dimethylformamide, 0.1 mM potassium ferricyanide, 0.1 mM potassium ferrocyanide and 1 mM EDTA. The leaf discs were then washed for 1 hour with 70% ethanol and 48 hours with 100% ethanol to completely remove chlorophyll.

Genomic DNA Isolation and Genomic DNA PCR to Validate Transgene Introduction

About 2 g of leaf tissue was ground to a fine powder in liquid nitrogen and 1% cetyltrimethylammonium bromide (w / v), 1% β-mercaptoethanol (v / v) in 100 mM Tris-HCl buffer (pH 7.5). ), Homogenized in 9 mL of extraction solution consisting of 700 mM NaCl and 10 mM EDTA. Homogenates were incubated at 65 ° C. for 1-1.5 hours with shaking every 15 minutes. After cooling 5 mL of chloroform / isoamyl alcohol (24: 1) was added and shaken slowly for 5 minutes. The mixture was centrifuged at 3,000 rpm for 10 minutes and the supernatant was transferred to a new 15 ml Falcone test tube. The extract was treated with RNase at a final concentration of 40-50 μg / mL at 35 ° C. for 30 minutes. Genomic DNA was precipitated by adding 6 mL of cooled isopropanol and centrifuged at 4,000 rpm for 5 minutes. DNA pellets were transferred to a 1.5 mL microfuge test tube and washed twice with 0.5 mL of 70% ethanol. The pellet uses about 10 ng of genomic DNA as a template and uses a primer set of bar gene, 5'-TACATCGAGACAAGCACGGTCAACTT-3 '(SEQ ID NO: 12) and 5'-TGCCAGAAACCCACGTCATGCCAG TT-3' (SEQ ID NO: 13). By amplification.

RNA isolation and RT-PCR to measure expression of transgenes and genes involved in the flavonoid biosynthetic pathway

Total RNA was minimally modified from the grains of the T3 homozygous line after 10-15 days, based on the protocol of "Purification of RNA from cells and tissues by acid phenol-guanidinium thiocyanate-chloroform extraction" in Sambrook and Russell (2001). Separated. The first strand cDNA of purified total RNA was synthesized for 60 minutes at 42 ° C. in a total volume of 25 μL containing 3 μg total RNA, 1.5 μg oligo dT and 200 units of M-MLV reverse transcriptase. RNA relative concentrations in other samples were assessed by a second PCR using a primer set of rice actin, 5'-AACTGGGATGATATGGAGAA-3 '(SEQ ID NO: 14) and 5'-CCTCCAATCCAGACACTGTA-3' (SEQ ID NO: 15). Relative expression levels of the C1 and RS transgenes as well as most genes in the flavonoid biosynthetic pathway were measured by second PCR using the same batch of the first strand cDNA.

Flavonoid Extraction and HPLC Analysis from Rice Grains

Flavonoids are extracted from 1 g of fine powder of T3 GT rice grains in 10 mL of 70% methanol by incubating with vigorous boxing for 2 minutes, sonication for 30 minutes and shaking for 3 hours at room temperature or overnight at 4 ° C. It became. The mixture was centrifuged at 3,000 rpm for 10 minutes, the supernatant was taken and filtered through a 0.45 μm syringe filter. After partitioning into 20 mL of n-hexane to remove lipids, an extract of 0.5 mL aliquot was vacuum-dried at room temperature for 2 hours. The dried aliquots were resuspended in 100 μL of 10% methanol and 20 μL of WatersTM with a Waters ^™ Symmetry C18 guard column, a Waters ^™ XTerra Phenyl column (250 mm × 4.6 mm id) and a Waters ^™ 996 photodiode array detector. Injected into 600 series HPLC system (Milford, MA, USA). The binary mobile phase consisted of 6% acetic acid (final pH 2.5, v / v, solvent A) in 2 mM sodium acetate and 25% solvent A (v / v, solvent B) in acetonitrile. The HPLC flow rate was 1 mL / min with a 10 minute 100% solvent A, 40 minutes 0-50% solvent B and 5 minutes 50-100% solvent B sensitization continuous program. Data was obtained and processed using Waters ^™ Millennium 32 software.

For proper acid-hydrolysis and ethyl acetate partition, each 0.5 mL extract of dried aliquat was dissolved in 100 μL of 1 N HCl with 0.1% ascorbic acid (w / v) and treated at 90 ° C. for 10 minutes. Three volumes of ethyl acetate were added to the acid-hydrolyzed mixture to purify aglycones of flavonoids and were simply vortexed. Ethyl acetate phase (top phase) was taken and vacuum-dried. 2 aliquots were dissolved into 100 μL of 10% methanol for injection into the HPLC system.

Liquid Chromatography Combined with Electrospray Ionization / Mass Spectrometry

Finnigan LCQ Advantage MAX ion trap mass spectrometer with Finnigan Surveyor Modular HPLC System (Thermo Electron Co., USA) with Waters XTerra MS C18 (5 μm, 2.1 mm × 150 mm) and Finnigan electronic spray source. , USA) were used for individual flavonoid separation and electrospray ionization mass spectrometry, respectively. The system was run under software Xcalibur (version 1.3 SP2, Thermo Electron). The HPLC mobile phase consisted of water (solvent A) and acetonitrile (solvent B) containing 0.1% formic acid. HPLC was performed at a flow rate of 0.2 mL / min with the following sensitization program: 0-3 minutes, 5% solvent B; 3 to 50 minutes, 5 to 50% solvent B; 50 to 55 minutes, 50 to 100% solvent B; And 55-75 minutes, 100% solvent B. Mass spectrometry was performed with an electronspray ionization source and the following conditions: spray needle voltage of 5 kV, ion transfer capillary temperature of 200 ° C., 60 arbitrary units of nitrogen sheath gas and 5 Auxiliary gas flow rate for any unit. Full-scan mass spectra were obtained in the 100-1000 m / z range with 3 microscans and a maximum ion implantation time of 200 ms. Data was obtained using software provided on LCQ mass spectrometers and systems in positive and negative modes.

C1 Of R-S  NMR Measurement of Flavonoids Isolated from T3 Grains of Transgenic Rice

1-3 mg of individual flavonoids isolated from HPLC were dissolved in 0.15 mL of CD ₃ OD. Each specimen was transferred to a 3 mm-diameter NMR tube and its ¹ H and ¹³ C NMR spectra were measured using a Varian Unity Plus 500 (Walnut Creek, CA, USA) at 500 MHz and 125 MHz, respectively. NMR gHSQC, gCOSY and gHMBC pulse sequences were used to determine the chemical structure of the isolated flavonoids.

Fluorescent Labeling of Flavonoids in Rice Grains

After 25 days, the grains of wild-type and T3 Cl / RS transgenic rice were fixed in 3.7% paraformaldehyde and 0.2% picric acid in 50 mM potassium / 5 mM EGTA buffer, pH 6.8. After washing the fixative in the same buffer, the samples were dehydrated and infiltrated with paraffin. Paraffin-fixed tissue blocks were sectioned to a thickness of 30 μm. The thin section was dewaxed, rehydrated and labeled with saturated (0.25%, w / v) diphenylboric acid (DPBA) for 5 minutes. DPBA-labeled sections were examined with a fluorescence microscope (Olympus BX 51). Fluorescence images were obtained with a FITC filter set by using an Olympus digital camera mounted on a microscope.

Color Response Analysis of GT Rice Grain Extract with Vanillin and DMACA

A working solution of 1% vanillin was prepared by dissolving 0.1 g vanillin in 10 mL of 5 N HCl, while 5% dimethylaminocinnamaldehyde (DMACA) was prepared in methanol containing 5% H ₂ SO ₄ . Lyophilized extract against 100 mg of dry kernel was dissolved in 50 mL of vanillin or DMACA working solution. Taxifolin and catechin at concentrations of 100 ppm each were used as reference compounds for the color reaction. Vanillin is catechin-specific and produces a bright red color, so no color develops when reacted with taxoxyline. DMACA, which reacts with catechins, produces dark cyan, while reacting with taxoxyline produces purple.

result

corn C1  And R-S  Generation of Transgenic Rice Lines Expressing Transgenes

In two separate transformation events, Oryza sativa japonica cv. Callus induced by germinating Hwa-Young's embryos was infected with Agrobacterium tumefaciens LBA4404 incubating the C1 / RS transgene (FIG. 1). Selection and regeneration of infected callus resulted in a total of 27 individual primary transgenic plants (T0) and grown to adulthood in the greenhouse (FIG. 2). The presence of the transgene in the regenerated plants was conveniently measured by GUS analysis or genomic PCR for the bar gene (FIG. 3A). Germination of T1 kernels on agar plates containing 4 mg / L of phosphinothricin showed mostly lines with a single copy of the transgene except for some lines by two linked copies. T2 grains harvested from herbicide-resistant T1 plants showed no difference in germination and plant growth compared to wild type. However, the development of T2 kernels, particularly dehydration, was markedly delayed, resulting in fresh T2 kernels on the inflorescence and more than twice as soft as wild-type kernels. The growth of T2 plants in greenhouses during the winter rather than in summer resulted in the production of thin and light grains, indicating that grain filling was more adversely affected than the wild type by poor growth conditions in the transformation line. Some homozygous transformation lines where all T2 grains were herbicide-resistant were selected and their T3 grains were further analyzed.

Various flavonoids are produced in GT rice grains

The T2 and T3 kernels of the homozygous transform line were colored darker and smaller in size than the T1 kernels, which were slightly darker than the untransformed kernels (FIG. 4). Expression of the transgene was estimated by RT-PCR using RNA isolated from the developing kernel of the homozygous line and primer sets specific for the maize C1 and RS genes. Representative images are shown in FIG. 3B although RT-PCR was performed for a greater number of transformation lines. The amount of RT-PCR product as a relative level of expression ranges from little detectable as lines 4-1 to high levels as lines 2-1 and 9-2. However, the C1 and RS transgenes in each line were expressed at very similar levels. Flavonoids were extracted from the grains of Hwa-Young wild type, black rice cultivar Milyang 188 and homozygous GT rice in the same manner. HPLC profiles of extracts without additional treatment clearly demonstrated that the grains of GT rice contained various levels of flavonoid strains (FIG. 5). In contrast, no flavonoids were detected in the wild type grain extract, whereas only some flavonoids, perhaps anthocyanins, were observed in the black rice. It was technically not feasible to identify individual HPLC peaks based on retention times matched with reference compounds by the numerous forms of flavonoids produced in GT rice. Moreover, commercially available standards are limited to HPLC analysis. To prevent this technical problem, the extract was hydrolyzed by acidic conditions and then partitioned into ethyl acetate. The number of peaks in the HPLC profile was thus reduced, while at the same time the levels of some major peaks were significantly increased (FIG. 5D). The m / z values of the major peaks measured by LC / MS / MS were used for NMR analysis. Due to the limited separation ability of the HPLC column, peaks 5, 6 and 8 in the HPLC profile of FIG. 5D corresponding to peaks I, II and III of FIG. 6A accumulated in sufficient amounts for NMR analysis up to 12 times the injection of the extract. LC / MS / MS and NMR analysis on the basis of peak Ⅰ, Ⅱ and Ⅲ respectively dihydro quercetin (dihydroquercetin) (taksi morpholin) (taxifolin), sorbitan di-Hydro also netin (dihydroisorhamnetin), dihydro (3'- O - Methyl taxoxyline) and 3'- 0 -methyl quercetin. The amount of major compounds was estimated as shown in Table 1 using certified Taxiline and Waters ^™ Millennium 32 software as reference compounds.

 Estimated concentration of major peaks in HPLC profiles. Peak #, retention time (RT) and absorption unit (AUs) corresponded to the HPLC profile of FIG. 5 (D). Peaks # 5, 6 and 8 corresponded to peaks I, II and III of FIG. 6 (A) identified by LC / MS / MS and NMR analysis, respectively. The concentration μg / g is μg per gram of dry T3 GT rice grains. peak# RT (minutes) AUs Μg / g One 6.33 3.20 268.9 2 9.85 0.35 29.4 3 11.20 0.42 35.3 4 20.97 2.10 176.5 5 26.91 1.85 155.5 6 35.66 3.93 330.3 7 36.86 1.19 100.0 8 47.67 0.66 55.5 9 49.22 0.64 53.8

3'- Ο of having a broad distribution of the concentration of compound 9 in the peak 6-methyl taksi morpholine (330.3 ㎍ / g) was reached at the highest level. Wild-type, was estimated by A ₂₈₀ value of flavonoid extract for existing rice, rice T3 GT and the morpholine concentration taksi total flavonoid content is known in the tea leaves ₂₈₀ A value for (Table 2).

Total flavonoid concentrations estimated by using taxoxyline as reference at 280 nm wavelength. SD, standard deviation calculated from 5 measurements per sample. a, low levels of flavonoids in wild-type kernels may be due to their tummy. specimen mg / g dry weight SD Hwa-Young WT 0.414 ^a 0.0144 Black rice 1.900 0.0036 C1 / R-S T3 12.880 0.0467 green tea 151.660 0.0856

Based on this schematic estimate, the total flavonoid content in GT rice was at least 30 and 6 times higher than wild type and existing black rice, respectively. However, GT rice contained about 1/10 of the total flavonoid content of the compared green tea leaves. About 15% of the total flavonoid content of the dried green tea leaves is generally within the range of 10-20% reported by the other groups, indicating that the quantification system is discreet. The total of the major compounds in Table 1 was about 1.2 mg / g, calculated as about 1/10 of the estimated total flavonoid content in GT rice grains. Catechin-specific dyes and vanillin were used for color reactions to test whether flavonoid extracts of GT rice contained catechins and catechin derivatives. The catechin reacted at a concentration of 100 ppm to give a bright red color, but the GT rice extract did not show any reaction with vanillin, indicating no catechin-type flavonoids. Conversely, the presence of taxifolin-form flavonoids in GT rice was confirmed by DMACA color analysis (FIG. 7).

C1 Of R-S  Flavonoid Biosynthetic Pathway Gene Activated by Transgene

Genes activated by the C1 and RS transgenes were identified by RT-PCR using primer sets for various genes involved in flavonoid biosynthetic pathways and RNA extracted from developing grains. Genes investigated were phenylalanine ammonia lyase ( pal ), chalcone synthase ( chs ), chalcone isomerase ( chi ), flavone synthase ( fns ), 2-hydroxyisoflavone synthase ( ifs ), flavonone 3-hydroxy Silase ( f3h ), flavonoid 3'-hydroxylase ( f3'h ), flavonol synthase ( fls ), dihydroflavonol 4-reductase ( dfr ), leucoanthocyanidin reductase ( lar ) , Anthocyanidin synthase ( ans ) and glutathione-S transferase ( gst ). The expression of pal, chs, f3h, f3'h and drf among these genes increased substantially in both transformation lines, while these gene expressions were almost undetectable or very low in the wild type (FIG. 8). By the absence of activation of lar and ans by the transgene, the pathway appears to have progressed to the biosynthesis stage of dihydroflavonols such as dihydrocaempferol and dihydroquecetin (FIG. 9). The results of RT-PCR were consistent with the biochemical analysis of flavonoids extracted from GT rice.

Flavonoids are produced by whole draining of GT rice

In the general labeling of flavonoids, thin sections of GT rice and wild type grains were incubated with saturated diphenylboric acid (DPBA). Specimen using an epifluorescence microscope with a FITC filter set showed that the DPBA-induced fluorescence levels were much higher in the GT rice section than the wild type (FIG. 10). Since DPBA fluorescence appears only upon binding to flavonoids, high fluorescence signals in GT rice grains indicate the presence of high levels of flavonoids. The concentrated fluorescence signal of the cell layer of outer endosperm in the labeled section of GT rice grains appeared as a thick green fluorescence band (FIG. 10A). Higher fluorescence bands at higher magnifications were determined to consist of 4-5 cell layers, including a heterologous layer (FIG. 10D). The fluorescence signal is suddenly diluted inside the starchy endosperm of the highly fluorescent band by starch particles that limit the free cytoplasm and at the same time block the fluorescence signal of DPBA-labeled flavonoids.

Grains such as corn, wheat, barley and rice lack flavonoids in the endosperm, but some grain varieties with colored grains produce some form of anthocyanin in the rind. The production of flavonoids is also limited to the peels of berries such as apples and tomatoes.

Since genes involved in flavonoid synthesis are not expressed in the endosperm, the introduction of regulatory genes to activate them is necessary.

The rice genome has three members of the R gene family with about 75% identity to the amino acid sequences of maize R-S and LC transcription factors (Hu et al., 2000). High homology with maize R (Lc) gene. Expression of the rice Rb2 gene along with the corn C gene in corn suspension cells induced the production of anthocyanins (Hu et al., 2000).

High levels of flavonoids are produced throughout the endosperm of GT rice and the grain size is significantly reduced. The first report is that grain endosperm produces flavonoids, even for cereal varieties.

Although there was no difference in growth behavior between GT rice and wild type, ripening of the grains was significantly delayed in GT rice. This can be explained by the role of flavonoids, generally auxin transport. Inhibition of auxin transport in the endosperm can delay or reduce ethylene production, which is directly related to grain ripening such as dehydration and induction of aging.

The choice of promoter for the transgene appears to be important for the generation of transgenic crops with predicted phenotypes. Expression of the transgene was specifically directed in the endosperm. Prolamin NPR33 flavonoids are endosperm-specific but occasionally leak into the trophic tissue.

Reduced grain size results from inhibition of grain filling or problems of carbohydrate metabolism. Penetration of the rind occurs into the internal drainage and was found in the grain section. Somewhat transparent starch embryos indicate abnormal embryonic development. Not all genes are activated by transgenes.

LC / MS / MS results and color reaction data of the major peaks indicate that the flavonoid biosynthetic pathway is activated up to the stage of dihydroflavonol, in which various derivatives are produced by various modifications. Similar results were reported by the group that generated the C1 / LC transgenic tomato lines. His case was somewhat different from us.

Tissue-specificity of flavonoid biosynthesis: Out-of-site expression of chalcone synthase results in huge flavonoid increases in the dermal tissue rather than the flesh (Verhoeyen, 2002).

The total flavonoid content in the estimated GT rice seems discernible because the sum of the major peaks is somewhat less than the estimated total content. Moreover, the total flavonoid content in green tea leaves is in the range reported in other studies, which are 10-20% dry weight.

Delayed grain ripening of transformed rice is associated with altered metabolism or transport of plant hormones such as auxin, cytokinin, ABA, GA and ethylene. Buer and Muday (2004) reported that flavonoids negatively influence the transport of auxin in apocytic roots, thus promoting the bending of roots in response to gravity.

The effect of the present invention is to provide a transformed rice containing a high concentration of various flavonoids in rice grains. The present invention also provides a transformed rice transformed with maize C1 and RS gene.

references

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1 shows the C1 / RS transgene constructs used for transformation. The construct backbone was derived from the binary vector pCAMBIA3301. RB, T-DNA right border; Tnos, nopalin synthase gene terminator; smGFP :: GUS , a soluble modified green fluorescent protein gene fused with a β-glucuronidase (GUS) coding sequence; 35S P , CaMV 35 S promoter; ProP , rice 13-kD prolamine NPR33 promoter; C1 , maize MYB-form transcription factor (accession number AF320614); RS , maize bHLH-form transcription factor (accession number X15806); bar , bialaphos-resistant gene; Tcmv, CaMV 35S terminator; LB, T-DNA left border.

2 shows the production of transgenic rice transformed with maize C1 and RS transgenes.

(A) Oryza sativa japonica cv. Callus Induced by Germinating Hwa-Young's Belly

(B) Callus infected with Agrobacterium tumefaciens LBA4404 bearing a transgene on a bassta-containing selection medium

(C) Callus to regenerate plantlets

(D) Regenerated seedlings transferred to magenta boxes containing MS medium

(E) Transgenic Plants Moved from Greenhouse to Soil

(F) Adult T0 transgenic plant with panicle

FIG. 3 is an image of ethidium bromide-stained product of genomic PCR and RT-PCR to confirm the presence of transgenes in the genome and to measure the relative expression levels of C1 and RS transgenes in developing transgenic kernels, respectively. Indicates. Arrows indicate 1 kb bands of DNA size ladder.

(A) PCR products were amplified against genomic DNA isolated from transformation lines 1, 2, 3, 4, 6, 9 and 19 using the bar gene primer. Plasmid DNA (PL) containing the transgene was included as a PCR positive control, while wild type genomic DNAs (WT) were included as a negative control.

(B) RT-PCR products show relative expression levels of C1 and RS transgenes in the developing kernels of wild type (WT), 2-1, 4-1 and 9-2 T2 transformation lines.

4 shows a comparison of untransformed T1, T2 and T3 rice grains.

T2 (C) and T3 (D) homozygous transformed grains are darker in color and smaller in size than untransformed (A) and T0 semi-conjugated transformed (B) rice grains.

FIG. 5 shows HPLC profiles of flavonoids extracted from Hwa-Young wild type (A), existing black rice Mil-Yang 188 Ho (B) and T3 GT rice (C) kernels without acid hydrolysis and ethyl acetate partitioning. (D) HPLC profiles of acid hydrolysis and ethylacetate split flavonoids extracted from T3 GT rice grains. Minutes, retention time; AU, absorption unit.

6 shows the chemical formulas identified by LC / MS / MS analysis and NMR analysis of flavonoid extracts of T3 GT rice grains.

(A) LC chromatogram of acid-hydrolyzed ethyl acetate partitioned extract.

(B-D) MS / MS spectra for compounds I (B), II (C) and III (D) in (A). The formula identified by NMR analysis is shown below the MS / MS spectrum of each compound.

7 shows a color reaction assay of flavonoids. The dry grain of T3 GT rice, 100 mg of vanillin- or DMACA-stained extract color, was compared to 100 ppm taxoxyline and 100 ppm catechin. Vanillin, 1% vanillin in 5N HCl; DMACA, 0.5% dimethylaminocinnamaldehyde (DMACA) and 5% H ₂ SO _{4 in} MeOH.

FIG. 8 shows RT-PCR for flavonoid biosynthetic pathway using RNA extracted from developing grains of wild type (WT) and GT rice homozygous lines (2-1 and 9-2). Actin, used as reference to the amount of PCR template; pal , phenylalanine ammonia lyase; chs , chalcone synthase, f3f , flavonone 3-hydroxylase; f3'h , flavonoid 3'-hydroxylase; dfr , dihydroflavonol 4-reductase; gst , glutathione-S transferase.

9 shows the structural genes activated by the C1 and RS transgenes, shown in flavonoid biosynthetic pathways and in bold capital letters. PAL, phenylalanine ammonia lyase; c4h, cinnamate-4-hydroxylase; 4cl, 4 coumarate CoA ligase; CHS, chalcone synthase; chi, chalcone isomerase; fns, flavone synthase; ifs, 2-hydroxyisoflavone synthase; F3H, flavonone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; fls, flavonol synthase; DFR, dihydroflavonol 4-reductase; lar, leucoanthocyanidin reductase; ans, anthocyanidin synthase; gst, glutathione-S transferase.

10 shows sections of T3 GT rice (A, D) and wild type (C, E) grains, labeled with diphenylboric acid (DPBA). Labeled flavonoids were indicated by green fluorescent signals. (B) Sections of DPBA unlabeled T3 GT rice grains as labeled negative controls. Intraaxial signal of wild-type kernel (C) acts as a label positive control. (D) and (E) are more enlarged insets of (A) and (C), respectively. Magnification of (A), (B) and (C), 0.5 mm; (D) and (E), 0.2 mm.

<110> KOREA KUMHO PETROCHEMICAL CO., LTD. <120> Transgenic Rice Line Producing High Level of Flavonoids in the Endosperm <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 855 <212> DNA <213> Maize C1 cDNA <400> 1 gagcttgatc gacgagagag cgagcgcgat ggggaggagg gcgtgttgcg cgaaggaagg 60 cgttaagaga ggggcgtgga cgagcaagga ggacgatgcc ttggccgcct acgtcaaggc 120 ccatggcgaa ggcaaatgga gggaagtgcc ccagaaagcc ggtttgcgtc ggtgcggcaa 180 gagctgccgg ctgcggtggc tgaactacac atcaggcgcg gcaacatctc ctacgacgag 240 gaggatctca tcatccgcct ccacaggctc ctcggcaaca ggtggtcgct gattgcaggc 300 aggctgcctg gccgaacaga caatgaaatc aagaactact ggaacagcac gctgggccgg 360 agggcaggcg ccggcgccgg cgccggcggc agctgggtcg tcgtcgcgcc ggacaccggc 420 tcgcacgcca ccccggccgc gacgtcgggc gcctgcgaga ccggccagaa tagcgccgct 480 catcgcgcgg accccgactc agccgggacg acgacgacct cggcggcggc ggtgtgggcg 540 cccaaggccg tgcggtgcac gggcggactc ttcttcttcc accgggacac gacgccggcg 600 cacgcgggcg agacggcgac gccaatggcc ggtggaggtg gaggaggagg aggagaagca 660 gggtcgtcgg acgactgcag ctcggcggcg tcggtatcgc ttcgcgtcgg aagccacgac 720 gagccgtgct tctccggcga cggtgacggc gactggatgg acgacgtgag ggccctggcg 780 tcgtttctcg agtccgacga ggactggctc cgctgtcaga cggccgggca gcttgcgtag 840 acaacaagta cacgt 855 <210> 2 <211> 1863 <212> DNA <213> Maize R-S cDNA <400> 2 ttcagcaggc gcgtgatggc cgtttcagct tcccgagttc agcaggcgga agaactgctg 60 caacgacctg ctgagaggca gctgatgagg agccagcttg ctgcagccgc caggagcatc 120 aactggagct acgccctctt ctggtccatt tcagacactc aaccaggggt gctgacgtgg 180 acggacgggt tctacaacgg cgaggtgaag acgcggaaga tctccaactc cgtggagctg 240 acatccgacc agctcgtcat gcagaggagc gaccagctcc gggagctcta cgaggccctc 300 ctgtcgggcg agggcgaccg ccgcgctgcg cctgcgcggc cggccggctc tctgtcgccg 360 gaggacctcg gcgacaccga gtggtactac gtggtctcca tgacctacgc cttccggcca 420 ggccaagggt tgcccggcag gagtttcgcg agcgacgagc atgtctggct gtgcaacgcg 480 cacctcgccg gcagcaaagc cttcccccgc gcgctcctgg ccaagagcgc gtccattcag 540 tcaatcctct gcatcccggt tatgggcggc gtgcttgagc ttggtacaac tgacacggtg 600 ccggaggccc cggacttggt cagccgagca accgcagctt tctgggagcc gcagtgcccg 660 acgtactcgg aagagccgag ctccagcccg tcaggacgag caaacgagac cggcgaggcc 720 gcagcagacg acggcacgtt tgcgttcgag gaactcgacc acaataatgg catggacata 780 gaggcgatga ccgccgccgg gggacacggg caggaggagg agctaagact aagagaagcc 840 gaggccctgt cagacgacgc aagcctggag cacatcacca aggagatcga ggagttctac 900 agcctctgcg acgaaatgga cctgcaggcg ctaccactac cgctagagga cggctggacc 960 gtggacgcgt ccaatttcga ggtcccctgc tcttccccgc agccagcgcc gcctccggtg 1020 gacagggcta ccgctaacgt cgccgccgac gcctcaaggg cgcccgtcta cggctctcgc 1080 gcgaccagtt tcatggcttg gacgaggtcc tcgcagcagt cgtcgtgctc cgacgacgcg 1140 gcgccggcag tagtgccggc catcgaggag ccgcagagat tgctgaagaa agtggtggcc 1200 ggcggcggtg cttgggagag ctgtggcggc gcgacgggag cagcacagga aatgagtgcc 1260 accaagaacc acgtcatgtc ggagcgaaag cgacgagaga agctcaacga gatgttcctc 1320 gtcctcaagt cactgcttcc gtccattcac agggtgaaca aagcgtcgat cctcgccgaa 1380 acgatagcct acctcaagga gcttcagcga agggtgcaag agctggagtc cagtagggaa 1440 cctgcgtcgc gcccatccga aacgacgaca aggctaataa caaggccctc ccgtggcaat 1500 aatgagagtg tgaggaagga agtctgcgcg ggctccaaga ggaagagccc agagctcggc 1560 agagacgacg tggagcgccc cccggtcctc accatggacg ccggctccag caacgtcacc 1620 gtcaccgtct cggacaagga cgtgctcctg gaggtgcagt gccggtggga ggagctcctg 1680 atgacgcgag tgttcgacgc catcaagagc ctccatttgg acgtcctctc ggttcaggct 1740 tcagcgccag atggcttcat ggggcttaag atacgagctc agtttgctgg ctccggtgcc 1800 gtcgtgccct ggatgatcag cgaggctctt cgcaaagcta tagggaagcg gtgaaggggc 1860 agc 1863 <210> 3 <211> 638 <212> DNA <213> Oryza sativa <400> 3 ggtgtagcaa cacgacttta ttattattat tattattatt attattattt tacaaaaata 60 taaaatagat cagtccctca ccacaagtag agcaagttgg tgagttattg taaagttcta 120 caaagctaat ttaaaagtta ttgcattaac ttatttcata ttacaaacaa gagtgtcaat 180 ggaacaatga aaaccatatg acatactata attttgtttt tattattgaa attatataat 240 tcaaagagaa taaatccaca tagccgtaaa gttctacatg tggtgcatta ccaaaatata 300 tatagcttac aaaacatgac aggcttagtt tgaaaaattg caatccttat cacattgaca 360 cataaagtga gtgatgagtc ataatattat tttctttgct acccatcatg tatatatgat 420 agccacaaag ttactttgat gatgatatca aagaacattt ttaggtgcac ctaacagaat 480 atccaaataa tatgactcac ttagatcata atagagcatc aagtaaaact aacactctaa 540 agcaaccgat gggaaagcat ctataaatag acaagcacaa tgaaaatcct catcatcctt 600 caccacaatt caaatattat agttgaagca tagtagta 638 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 attctagacg agcttgatcg acgagagagc gag 33 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cgagctcgac gtgtacttgt tgtctacgca ag 32 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gctctagacg ttcagcaggc gcgtgatg 28 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 cccccggggg ctgccccttc accgcttccc t 31 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 aagcttggtg tagcaacacg actt 24 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 cgggatcccg gatctagtaa catagatgac ac 32 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 ggatccggtg tagcaacacg agtt 24 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 ggaattccga tctagtaaca tagatgacac 30 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 tacatcgaga caagcacggt caactt 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 tgccagaaac ccacgtcatg ccagtt 26 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 aactgggatg atatggagaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 cctccaatcc agacactgta 20

Claims (8)

(a) the polynucleotide of the maize C1 gene shown in SEQ ID NO: 1; (b) the polynucleotide of the maize RS gene shown in SEQ ID NO: 2; And (c) comprising regulatory sequences operably linked to the polynucleotides such that said polynucleotides are expressed in plant cells to contain high levels of flavonoids in the kernel; Expression Vectors for Transformation of Rice The expression vector of claim 1, wherein the regulatory sequence comprises a rice 13-kD prolamin gene promoter as shown in SEQ ID NO: 3 Transformed rice cells transformed with the expression vector of claim 1 Transgenic Rice Grown from Transgenic Rice Cells of Claim 3 The method of claim 4, wherein the rice is Oryza sativa japonica cv. Transformed rice characterized by Hwa-Young The method of claim 4 wherein the flavonoid is quercetin-dihydro (taksi morpholine), sorbitan di-Hydro also netin (3'- Ο - taksi methyl morpholine) and 3'- Ο - transgenic rice, characterized in that it comprises a methyl quercetin 7. The method of claim 6, dihydro-quercetin (taksi morpholine), sorbitan di-Hydro also netin (3'- Ο - taksi methyl morpholine) and 3'- Ο - level of quercetin methyl 120~180 ㎍ per gram of grains each drying 270-390 μg and 40-70 μg transgenic rice 7. The transgenic rice according to claim 6, wherein the total flavonoids are 12 mg moving per gram of dried grain.