Application of specific expression of endosperm of ZlRc and ZlMYB1 genes of zizania sinensis in increasing flavonoid content of rice seeds

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of specific expression of endosperm of ZlRc and ZlMYB1 genes of zizania sinensis in increasing the flavonoid content of rice seeds. The purpose of the invention is to increase the flavonoid content of rice seeds through specific expression of endosperm of ZlRc and ZlMYB1 genes. Under the condition that the growth environment is consistent with that of a control rice plant, compared with the control rice, the flavonoid content of the rice seeds specifically expressed by endosperm of the ZlRc and ZlMYB1 genes is remarkably increased.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of endosperm-specific expression of ZlRc and ZlMYB1 genes in Chinese wild rice to increase the flavonoid content of rice seeds. Background Technology

[0002] Flavonoids are major secondary metabolites in plants, possessing strong antioxidant activity and health benefits. Flavonoids include seven main subclasses: flavonols, flavones, isoflavones, anthocyanins, flavanones, flavanols, and chalcones. With improved living standards and a deeper understanding of nutrition and health, people are paying increasing attention to the quality, nutritional components, and health benefits of rice. Colored rice, due to its beneficial effects on human health in areas such as antioxidation, anti-inflammation, anti-obesity, anti-diabetic, anti-cancer, and anti-aging, has received widespread attention from consumers and nutritionists. The seed coat of colored rice exhibits different colors, such as brown, red, purple, and black, due to the richness of different types of flavonoids. Purple and black seed coats are generally formed by the accumulation of anthocyanins, while brown and red seed coats are formed by the accumulation of proanthocyanidins. Although colored rice is rich in flavonoids such as proanthocyanidins or anthocyanins, these are mainly concentrated in the seed coat, with virtually no flavonoids in the endosperm.

[0003] Chinese wild rice (Zizania latifolia) belongs to the genus Zizania in the tribe Oryzeae of the family Gramineae. It has a long history of consumption and cultural significance. Besides carbohydrates, protein, vitamins, and minerals, Chinese wild rice contains flavonoids and other bioactive substances, making it a whole grain beneficial to health. Adding Chinese wild rice to one's daily diet can help prevent chronic and metabolic diseases such as diabetes, obesity, and cardiovascular disease. Previous studies have found that the flavonoid content and antioxidant activity of Chinese wild rice are higher than those of common indica rice, japonica rice, and red rice, making it a natural source of antioxidants. The applicant's team identified 34 phenolic acids and flavonoids in Chinese wild rice, and flavonoids (mainly proanthocyanidins and flavonoid glycosides) contributed more to its antioxidant activity than phenolic acids. Compared to rice, 72 types of flavonoids were upregulated and 6 types were downregulated in Chinese wild rice. Therefore, Chinese wild rice is rich in flavonoids, especially those lacking in rice.

[0004] The biosynthesis of plant flavonoids is regulated by transcription factors, mainly including MYB, basic helix-loop-helix (bHLH), WD40, and the MBW (MYB-bHLH-WD40) protein complex. In the flavonoid biosynthesis pathway, early biosynthetic genes participate in the synthesis of common precursors, while late biosynthetic genes are downstream genes in the pathway. MBW transcription factors and their protein complexes can bind to the promoters of flavonoid synthesis structural genes to activate (or inhibit) their expression, thereby regulating the flavonoid synthesis process. In rice, Rc belongs to the bHLH transcription factors that regulate flavonoid synthesis; ZlRc in *Zizania latifolia* is homologous to Rc in rice. Overexpression of the ZlRc gene changes rice seeds from colorless to brown, increases the content of phenolic compounds, antioxidant activity, and the gene expression and enzyme activity of flavonoid biosynthesis-related enzymes in rice seeds. Knocking out the OsMYB3 gene in 'Zixiangnuo 1' rice changed its seed coat from black to brown and significantly reduced the anthocyanin content to below the detection limit. The ZlMYB1 gene in *Zizania latifolia* is homologous to the OsMYB3 gene in rice. Overexpression of ZlMYB1 in 'Zixiangnuo' rice with OsMYB3 knockout changed the rice seeds from brown to black and significantly increased the anthocyanin content, antioxidant activity, and expression levels and enzyme activities of key genes involved in anthocyanin biosynthesis.

[0005] Rice is the staple food for over 65% of my country's population, accounting for approximately 40% of total grain consumption. Developing functional rice germplasm with health benefits using modern biotechnology is of great significance for improving the nutritional and health status of the rice-dependent population, promoting the transformation of my country's agricultural industrial structure, increasing farmers' grain production efficiency, and contributing to rural revitalization. Although colored rice is rich in flavonoids such as proanthocyanidins and anthocyanins, these are mainly concentrated in the seed coat, with virtually no flavonoids in the endosperm. Notably, Chinese wild rice is rich in flavonoids, especially those lacking in rice; ZlRc is a key bHLH transcription factor regulating flavonoid synthesis, and ZlMYB1 is a key MYB transcription factor regulating anthocyanin synthesis. Therefore, starting with the ZlRc and ZlMYB1 genes of Chinese wild rice, which have already completed gene function identification, gene aggregation and endosperm-specific expression were carried out to create a new rice germplasm with endosperm rich in flavonoids. This has important practical significance and application prospects for improving the dietary structure of residents and reducing dietary risk factors that cause chronic diseases. Summary of the Invention

[0006] The purpose of this invention is to increase the flavonoid content of rice seeds, and it proposes the application of endosperm-specific expression of the ZlRc and ZlMYB1 genes of *Zizania latifolia* in increasing the flavonoid content of rice seeds. After gene aggregation and endosperm-specific expression of the ZlRc and ZlMYB1 genes of *Zizania latifolia*, the flavonoid content of rice seeds significantly increased.

[0007] The technical solution of this invention is:

[0008] Application of endosperm-specific expression of ZlRc and ZlMYB1 genes in increasing flavonoid content in rice seeds, wherein the ZlRc and ZlMYB1 gene endosperm-specific expression vectors are as follows: Figure 1 As shown.

[0009] Furthermore, the constructed rice endosperm-specific expression vectors of the ZlRc and ZlMYB1 genes were transferred into rice to obtain transgenic rice capable of overexpressing ZlRc and ZlMYB1.

[0010] Furthermore, the overexpression vector was transformed into Agrobacterium through chemical transformation, and independent transformants were obtained by infecting callus tissue with Agrobacterium. Through plant regeneration, transgenic rice with significantly increased seed flavonoid content was obtained.

[0011] The beneficial effects of this invention are:

[0012] (1) The present invention uses PCR technology to amplify the sequence fragments of ZlRc and ZlMYB1 from the cDNA library of Zizania latifolia, and constructs them into a rice endosperm-specific expression vector. Using this vector to transform rice, transgenic rice with significantly increased seed flavonoid content is obtained by increasing the expression level of ZlRc and ZlMYB1 genes in the rice seed endosperm.

[0013] (2) This invention provides the application of endosperm-specific expression of the ZlRc and ZlMYB1 genes from the Chinese wild rice plant in increasing the flavonoid content of rice seeds. Under the same growth environment as the control rice plants, the seeds harvested from rice plants with endosperm-specific expression of the ZlRc and ZlMYB1 genes had higher total phenolic, total flavonoid, and total proanthocyanidin contents, as well as higher DPPH free radical scavenging capacity and ABTS content compared to the control. ·+ Free radical uptake capacity. This indicates that the endosperm-specific expression of the ZlRc and ZlMYB1 genes effectively regulates the synthesis pathway of flavonoids in rice endosperm, thereby increasing the flavonoid content in transgenic rice seeds.

[0014] (3) The endosperm-specific expression of the ZlRc and ZlMYB1 genes obtained in this invention has a colorless seed coat, just like the control rice. Therefore, compared with the control, the flavonoids added in the endosperm-specific expression of the ZlRc and ZlMYB1 genes in the rice seeds are mainly enriched in the endosperm and do not cause changes in seed coat color. Attached Figure Description

[0015] Figure 1 This is a physical map of the rice endosperm-specific expression vectors for the ZlRc and ZlMYB1 genes in Example 2.

[0016] Figure 2 This is an agarose gel electrophoresis image of the transgenic rice plants identified in Example 2. Labeling information: M represents the DL2000 marker, B represents the blank control, N represents the negative control, P represents the positive control, and 1-15 are the transgenic rice plant numbers.

[0017] Figure 3 Phenotypic observation of rice seeds expressing ZlRc and ZlMYB1 genes in rice endosperm. Figure labels: (A) control rice seeds; (B) rice seeds obtained by culturing control rice seeds after endosperm-specific expression of ZlRc and ZlMYB1 genes in rice.

[0018] Figure 4 This is a comparison of the total phenolic content of one control (CK) and three ZlRc-MYB1 (ZlRc-MYB1-1, ZlRc-MYB1-2, ZlRc-MYB1-3) transgenic rice seeds from Example 4. The test results are the mean of three replicates, and the error bars represent the standard deviation (SD).

[0019] Figure 5 This is a comparison of the total flavonoid content in one control (CK) and three ZlRc-MYB1 (ZlRc-MYB1-1, ZlRc-MYB1-2, ZlRc-MYB1-3) transgenic rice seeds from Example 4. The test results are the mean of three replicates, and the error bars represent the standard deviation (SD).

[0020] Figure 6 This is a comparison of the total proanthocyanidin content between one control (CK) and three ZlRc-MYB1 (ZlRc-MYB1-1, ZlRc-MYB1-2, ZlRc-MYB1-3) transgenic rice seeds in Example 4. The test results are the mean of three replicates, and the error bars represent the standard deviation (SD).

[0021] Figure 7This is a comparison of the DPPH free radical scavenging capacity of one control (CK) and three ZlRc-MYB1 (ZlRc-MYB1-1, ZlRc-MYB1-2, ZlRc-MYB1-3) transgenic rice seeds in Example 5. The test results are the mean of three replicates, and the error bars represent the standard deviation (SD).

[0022] Figure 8 ABTS for one control (CK) and three ZlRc-MYB1 (ZlRc-MYB1-1, ZlRc-MYB1-2, ZlRc-MYB1-3) transgenic rice seeds in Example 5. ·+ Results of free radical absorption capacity comparison. The test results are the mean of three replicates, and the error bars represent the standard deviation (SD). Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The biological material used in the following examples, *Zizania latifolia*, was collected from Huai'an City, Jiangsu Province, China; the control rice was *Nipponbare* rice, with seeds sourced from Wuhan City, Hubei Province, China.

[0026] Example 1: Obtaining the ZlRc and ZlMYB1 genes

[0027] 1.1 Extraction of total RNA and preparation of cDNA from Zizania latifolia

[0028] 1.1.1 Extraction of total RNA from Chinese wild rice

[0029] Using a polysaccharide and polyphenol plant RNA extraction kit ( RNA was extracted from *Zizania latifolia* leaves using the Universal Plant TotalRNA Isolation Kit (Vazyme) and then reverse transcribed into cDNA. RNA extraction from *Zizania latifolia* leaves was performed according to the instructions for the plant RNA extraction kit; the specific experimental steps are as follows:

[0030] (1) The leaf samples of Chinese wild rice were rapidly ground into powder in liquid nitrogen. 50 mg of the ground sample was weighed and 500 μL of Bufer PRL preheated at 65 °C was added. The sample was then immediately subjected to violent vortexing for 60 s.

[0031] (2) Incubate the lysate in a 65°C water bath for 5 min, inverting it twice during the process. Centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 0.5 times the volume of the supernatant in anhydrous ethanol, and immediately mix by pipetting.

[0032] (3) Transfer the above mixture to FastPure gDNA-Filter Column II, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0033] (4) Add 500 μL of Buffer PRLPlus to FastPure gDNA-Filter Column II, centrifuge at 12000 rpm for 30 s, and collect the filtrate.

[0034] (5) Add 0.5 times the volume of anhydrous ethanol to the filtrate and immediately mix by pipetting; transfer the above mixture to FastPure RNAColumn IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0035] (6) Add 700 μL of Buffer PRW1 to FastPure RNAColumn IV, incubate at room temperature for 1 min, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0036] (7) Add 500 μL of Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12000 rpm for 30 s, discard the filtrate, and repeat this step once.

[0037] (8) Centrifuge the FastPure RNAColumn IV adsorption column at 12000 rpm for 2 min to remove the residual Buffer PRW2 in FastPure RNAColumn IV.

[0038] (9) Transfer FastPure RNAColumn IV to a new RNase-free 1.5mL centrifuge tube, add 40μL of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 2min, and centrifuge at 12000rpm for 1min.

[0039] 1.1.2 Preparation of cDNA

[0040] After RNA extraction, the RNA concentration was measured, and 2.0 μg of each sample was used as the substrate for reverse transcription. Reverse transcription was performed using a reverse transcription kit to obtain cDNA products, which were then stored at -20°C for later use.

[0041] Table 1. Reverse Transcription PCR System and Procedure

[0042]

[0043]

[0044] 1.2 Amplification of ZlRc and ZlMYB1 genes

[0045] Primers designed based on the ZlRc gene sequence are as follows:

[0046] ZlRc-F: 5'-ATGCACGCCATGGCC-3' (SEQ ID NO: 1);

[0047] ZlRc-R: 5'-TCATGGTGAGGAGAGGACAAG-3' (SEQ ID NO: 2).

[0048] Using the prepared ZlRc cDNA as a template, PCR amplification was performed using primers to obtain the target fragment ZlRc.

[0049] Primers designed based on the ZlMYB1 gene sequence are as follows:

[0050] ZlMYB1-F: 5'-ATGAAGAGAGGGGCATGGAC-3' (SEQ ID NO: 3);

[0051] ZlMYB1-R: 5'-CTACTCCGCATGAGGTTGGG-3' (SEQ ID NO: 4).

[0052] Using the prepared ZlMYB1 cDNA as a template, PCR amplification was performed using primers to obtain the target fragment ZlMYB1.

[0053] The PCR amplification system and reaction procedure are as follows:

[0054] Table 2 PCR System and Procedure

[0055]

[0056]

[0057] The PCR products of the ZlRc and ZlMYB1 genes were sequenced. The full-length ZlRc sequence was 1971 bp, and the full-length ZlMYB1 sequence was 762 bp.

[0058] Example 2: Construction and genetic transformation of rice endosperm-specific expression vectors for ZlRc and ZlMYB1 genes

[0059] 2.1 Construction of rice endosperm-specific expression vectors for ZlRc and ZlMYB1 genes

[0060] First, the amplified ZlRc gene fragment was cloned into 322D1-Glub1-T16KD (with restriction sites retained) linearized with HindIII, resulting in 322D1-Glub1-ZlRc-T16KD. Then, the amplified ZlMYB1 fragment was cloned into 322D1-GluC-TGluC (with restriction sites retained) linearized with HindIII+SalI, resulting in 322D1-GluC-ZlMYB1-TGluC. Finally, using 322D1-GluC-ZlMYB1-TGluC as a template, the GluC-ZlMYB1-TGluC fragment was amplified and cloned into pYL322d2 (with restriction sites retained) linearized with KpnI+BamHI, resulting in 322D2-GluC-MYB1-TGluC.

[0061] The 322D1-Glub1-ZlRc-T16KD vector and the recipient vector 22OT64 underwent the first round of polymerization following Professor Liu Yaoguang's multi-gene polymerization method, involving E. coli transformation, plasmid extraction, enzyme digestion, and a second E. coli transformation. Clones were selected and cultured, and positive clones were verified by colony PCR, followed by sequencing. The final positive clone was named 22OT64-Glub1-ZlRc-T16KD. The 322D2-GluC-ZlMYB1-TGluC vector and the recipient vector 22OT64-Glub1-ZlRc-T16KD underwent the second round of polymerization following Professor Liu Yaoguang's multi-gene polymerization method, involving E. coli transformation, plasmid extraction, enzyme digestion, and a second E. coli transformation. Clones were selected and cultured. Positive clones were verified by colony PCR using primers 380kpni-seqF: 5'-CTCCACCATGTTGGCAAGCT-3' (SEQ ID NO:5) and TGluC-346F: 5'-CCTCAAAAGGACCCGACATCC-3' (SEQ ID NO:6), followed by sequencing verification. This yielded rice endosperm-specific expression vectors for the ZlRc and ZlMYB1 genes. Their physical maps are shown below. Figure 1 .

[0062] The steps for extracting plasmids and transforming Agrobacterium EHA105 competent cells are as follows:

[0063] (1) Take Agrobacterium EHA105 competent cells stored at -80℃ and let them partially melt at room temperature or in the palm of your hand. When they are in an ice-water mixture, insert them into ice.

[0064] (2) Add 5 μL of extracted plasmid to every 100 μL of Agrobacterium-infected EHA105 cells, mix well by hand, and incubate on ice for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min.

[0065] (3) Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 hours.

[0066] (4) Centrifuge at 6000 rpm for 1 min to collect the bacteria. Take about 100 μL of supernatant, gently pipette and resuspend the bacterial block, spread it on an LB plate containing kanamycin antibiotic, and incubate upside down in a 28℃ incubator for 2-3 days.

[0067] 2.2 Obtaining rice with endosperm-specific expression of ZlRc and ZlMYB1 genes

[0068] (1) Callus preparation

[0069] Step 1: Sterilize mature rice seeds. Use suitable tools to remove the husks from the mature seeds, discarding seeds with mold spots or underdeveloped embryos (shriveled, brown), ensuring the integrity and cleanliness of the seeds. After hulling, wash the rice seeds with 75% ethanol for 1 minute, then sterilize with 0.15% HgCl2 for 15-20 minutes, and finally wash 4-5 times with sterile ddH2O. Soak overnight for the final step.

[0070] Step 2: Induction of rice callus. Wash the seeds with sterile water overnight, peel the embryo along the aleurone layer with a scalpel, and inoculate it onto the induction medium. Inoculate 8-12 sterilized rice seeds into each bottle of induction medium, set the temperature to 30℃, and incubate in the dark for 40-45 days to induce callus formation.

[0071] Step 3: Subculture of callus tissue. Select pale yellow, granular, dry, and highly viable callus tissue from the induced callus and transfer it to a subculture medium for dark culture for 20 days. During the first subculture, be sure to remove any other tissues (such as endosperm, buds, etc.) attached to the callus tissue. Callus tissue that has been subcultured once can be infected with Agrobacterium tumefaciens. Callus tissue used for transformation should be subcultured a maximum of two times. Multiple subcultures can easily lead to somatic cell mutations in the callus tissue and reduce transformation efficiency.

[0072] Step 4: Callus pre-culture. From the subcultured callus, select pale yellow, granular, dry, and highly viable callus tissue and transfer it to pre-culture medium. Inoculate each dish with 60-80 callus granules the size of mung beans. Larger callus granules can be crushed with forceps. Pre-culture at 28℃ in the dark for 3-4 days. After pre-culture, collect the healthy, actively dividing small granules with a small spoon into 250mL sterile Erlenmeyer flasks for Agrobacterium infection.

[0073] (2) Preparation of Agrobacterium

[0074] Step 1: Agrobacterium activation. Two days before the experiment, streak Agrobacterium tumefaciens strain containing the target gene onto LA plates containing the corresponding antibiotics, and then incubate at 28°C for 2 days.

[0075] Step 2: Agrobacterium resuspension. Take an Agrobacterium tumefaciens streak plate and inoculate approximately one loopful of Agrobacterium into 100 mL of suspension medium. Add 100 μL of 100 mM acetylsylgenone stock solution and 2 mL of 50% glucose. Incubate at 28°C and 200 rpm for 30 min on a shaker until the Agrobacterium suspension concentration is approximately OD0.05. 600 =0.3 is sufficient.

[0076] (3) Agrobacterium infection and liquid co-culture

[0077] Step 1: Agrobacterium infection. Pour the prepared Agrobacterium suspension into an Erlenmeyer flask containing callus tissue until all callus tissue is submerged, and let it stand for 10 minutes. Discard the bacterial suspension. Take a sterile petri dish containing absorbent paper and filter paper, open the petri dish, and invert the Erlenmeyer flask containing callus onto the filter paper in the petri dish to drain as much bacterial suspension as possible. Then spread the callus on the filter paper in a sterile large dish, cover it with a sterile filter paper, and gently press the filter paper with tweezers to absorb the bacterial suspension on the surface of the callus. Remove the absorbent filter paper, and repeat this process four times on each side. Finally, cover the callus with another filter paper, cover the large petri dish, and let it air dry for 1-2 hours.

[0078] Step 2, Co-culturing. Use tweezers to transfer the dried callus particles onto the co-culture medium and seal with sealing adhesive. Co-culture at 19°C in the dark for 3 days.

[0079] Step 3: Washing. Transfer the co-cultured callus tissue to a washing cup, pour in sterile distilled water until the callus tissue is completely submerged, cover and shake for 20-30 seconds, then discard the sterile distilled water. Repeat this washing process 3-4 times. Observe the process; if the distilled water in the washing cup is clear, it indicates that the Agrobacterium has been basically cleaned; otherwise, continue washing. Finally, discard the sterile distilled water, add sterile distilled water containing 500 mg / L carbenicillin, and let stand for 30 minutes. Discard the sterile distilled water containing 500 mg / L carbenicillin.

[0080] Step 4: Callus screening. After the callus dries, use tweezers to transfer the callus particles to screening medium, seal with sealing adhesive, and place in a dark incubator for 20 days of screening culture (first screening, S1). Select dried callus free from Agrobacterium contamination from the S1 medium and transfer it to S2 medium. Incubate in the dark for 20 days and observe whether fresh, tender yellow resistant callus grows. If no resistant callus has yet appeared, continue transferring to plates for S3 screening culture. Generally, resistant callus can grow in japonica rice varieties after two screenings, i.e., the S2 stage.

[0081] Step 5: Differentiation Culture. Select small, pale yellow, dense, dry, and vigorously growing resistant callus fragments, choosing only one resistant callus fragment per cluster. Avoid selecting callus fragments contaminated with Agrobacterium. Evenly distribute 3-4 small resistant callus fragments in each bottle of differentiation medium. Since callus cells will continue to grow on the differentiation medium, placing them too densely can cause different callus fragments to grow together and become indistinguishable. Culture at 28℃ for 30-40 days, with a light cycle of 16 hours of light / 8 hours of darkness. Once the differentiated seedlings are 3-5 cm tall, they can be used for rooting culture. During the light culture period, promptly remove any contaminated material showing signs of bacterial growth.

[0082] Step 6: Plant Rooting. Using forceps, remove the differentiated seedlings from the differentiation medium and place them in sterilized empty petri dishes. Take only one healthy seedling from each callus fragment. Clean the seedling with scissors, removing dead or yellowed leaves and roots growing from the differentiation medium. Inoculate one seedling into each rooting tube. Culture in a light-filled culture room for 15-20 days until the new roots are fully developed, then transplant.

[0083] Step 7: Plant Transplanting. Remove the sealing film from the rooting tube, add some tap water, and continue hardening off the seedlings in the light culture chamber for 3-4 days. During hardening off, leaf samples can be taken for transgenic positivity testing. Remove the transformed seedlings from the rooting tube, wash off the attached culture medium from the roots, and transplant them into pots or buckets with prepared soil.

[0084] (4) Preparation of reagents and culture media used in the transformation:

[0085] 1) Abbreviations for reagents and solutions:

[0086] The abbreviations for the plant hormones used in the culture medium in this invention are as follows: 6-Benzylaminopurine (6-BA); Indole-3-acetic acid (IAA); Napthalene acetic acid (NAA); 2,4-Dichlorophenoxyacetic acid (2,4-D); Kinetin (KT).

[0087] 2) Main solution formulation:

[0088] MS max Stock solution (10x): Dissolve 16.5g NH4NO3, 1.7g KH2PO4, 19.0g KNO3, 3.7g MgSO4·7H2O, and 3.32g CaCl2 or 4.4g CaCl2·2H2O one by one, and then bring the volume to 1000mL at room temperature.

[0089] MS min Stock solution (100x): Dissolve 2.23g MnSO4·4H2O, 0.86g ZnSO4·7H2O, 0.083g KI, 0.62g H3BO3, 0.025g Na2MoO4·2H2O, 0.0025g CoCl2·6H2O, and 0.0025g CuSO4·5H2O one by one, and then bring the volume to 1000mL at room temperature.

[0090] N 6max Stock solution (10x): Dissolve 28.3g KNO3, 4.63g (NH4)2SO4, 4.0g KH2PO4, 1.85g MgSO4·7H2O, and 1.25g CaCl2 or 1.66g CaCl2·2H2O one by one, and then bring the volume to 1000mL at room temperature.

[0091] N 6min Stock solution (100x): Dissolve 0.08g KI, 0.16g H3BO3, 0.15g ZnSO4·7H2O, 0.44g MnSO4·4H2O or 0.3335g MnSO4·H2O one by one, and then bring the volume to 1000mL at room temperature.

[0092] Fe 2+ -EDTA stock solution (100x): Add approximately 300 mL of dH2O and 2.78 g of FeSO4·7H2O to one reagent bottle; add approximately 300 mL of dH2O to another reagent bottle and heat to 70°C, then add 3.73 g of Na2·EDTA·2H2O; after both are dissolved, allow the solution to cool to room temperature, mix the solutions in the two bottles, then add dH2O to bring the volume to 1000 mL and store at 4°C protected from light.

[0093] Vitamin stock solution (100x): 0.1g nicotinic acid, 0.1g nicotinic thiamine, 1g pyridoxine hydrochloride, 10g inositol, 0.2g glycine, add dH2O to a final volume of 1000mL, store at 4℃.

[0094] AA maxStock solution (10x): 29.50g KCl, 2.50g MgSO4·7H2O, 1.50g NaH2PO4, 1.50g CaCl2·2H2O, add dH2O to make up to 1000mL, store at room temperature and protected from light.

[0095] AA min Stock solution (100x): 1.0g MnSO4·H2O, 0.2g ZnSO4·7H2O, 0.0025g CuSO4·5H2O, 0.3g H3BO3, 0.075g KI, 0.0025g CoCl2·6H2O, 0.025g NaMoO4·2H2O, add dH2O to bring the volume to 1000mL, store at room temperature protected from light.

[0096] 6-BA stock solution (1 mg / mL): Add 100 mg 6-BA to 1.0 mL of 1N KOH and shake until 6-BA dissolves. Then add dH2O to bring the volume to 100 mL and store at room temperature.

[0097] KT stock solution (1 mg / mL): Add 1.0 mL of 1N KOH to 100 mg KT and shake until KT dissolves. Then add dH2O to bring the volume to 100 mL and store at room temperature.

[0098] 2,4-D stock solution (1 mg / mL): Add 100 mg 2,4-D to 1.0 mL of 1N KOH and shake for 5 min. Then add 10 mL of dH2O and shake until 2,4-D dissolves. Make up to 100 mL with dH2O and store at room temperature.

[0099] 100mM acetylsyl syringone stock solution: 0.196g acetylsyl syringone, 10mL dimethyl sulfoxide, dispensed into 1.5mL centrifuge tubes and stored at 4℃.

[0100] IAA stock solution (1 mg / mL): Add 100 mg IAA to 1.0 mL of 1N KOH and shake until the IAA dissolves. Then, bring the volume up to 100 mL with dH2O and store at room temperature away from light.

[0101] NAA stock solution (1 mg / mL): Add 100 mg NAA to 1.0 mL of 1N KOH and shake until NAA dissolves. Then, bring the volume up to 100 mL with dH2O and store at room temperature away from light.

[0102] 1N KOH stock solution: 5.6g KOH, dissolved in 100mL dH2O, stored at room temperature.

[0103] 0.15% HgCl2: Dissolve 1.5g HgCl2 partially or completely in 1mL of anhydrous ethanol, then bring the volume to 1000mL with dH2O. Stir for 4-8 hours and store properly at room temperature.

[0104] 3) Culture medium formulation for rice genetic transformation:

[0105] Induction medium: N 6max 100 mL of stock solution (10x) and N 6min 10 mL of stock solution (100x), 10 mL of Vitamin stock solution (100x), Fe 2+ Prepare 10 mL of EDTA stock solution (100x), 2.5 mL of 2,4-D stock solution (1 mg / mL), 0.6 g of hydrolyzed casein, 0.3 g of proline, 30 g of sucrose, and 3 g of Phytagel. Adjust the pH to 5.9 and add dH2O to 1000 mL.

[0106] Subculture medium: N 6max 100 mL of stock solution (10x) and N 6min 10 mL of stock solution (100x), 10 mL of Vitamin stock solution (100x), Fe 2+ Prepare 10 mL of EDTA stock solution (100x), 2.0 mL of 2,4-D stock solution (1 mg / mL), 0.6 g of hydrolyzed casein, 0.5 g of proline, 30 g of sucrose, and 3 g of Phytagel. Adjust the pH to 5.9 and add dH2O to 1000 mL.

[0107] Pre-culture medium: N 6max 12.5 mL of stock solution (10x), N 6min 1.25 mL of stock solution (100x), 2.5 mL of Vitamin stock solution (100x), Fe 2+ Prepare 25 mL of EDTA stock solution (100x), 0.75 mL of 2,4-D stock solution (1 mg / mL), 300 μL of 100 mM acetylsylgenone stock solution, 5 mL of 50% glucose solution, 0.15 g of hydrolyzed casein, 5 g of sucrose, and 1.75 g of agarose. Adjust the pH to 5.4 and add dH2O to a final volume of 250 mL.

[0108] Co-culture medium: N 6max 12.5 mL of stock solution (10x), N 6min 1.25 mL of stock solution (100x), 2.5 mL of Vitamin stock solution (100x), Fe 2+Prepare 25 mL of EDTA stock solution (100x), 0.75 mL of 2,4-D stock solution (1 mg / mL), 300 μL of 100 mM acetylsylgenone stock solution, 5 mL of 50% glucose solution, 0.2 g of hydrolyzed casein, 5 g of sucrose, and 1.75 g of agarose. Adjust the pH to 5.4 and add dH2O to a final volume of 250 mL.

[0109] Suspension culture medium: N 6max 5 mL of stock solution (10x), N 6min 0.5 mL of stock solution (100x), 1 mL of Vitamin stock solution (100x), Fe 2+ Prepare 0.5 mL of EDTA stock solution (100x), 0.2 mL of 2,4-D stock solution (1 mg / mL), 100 μL of 100 mM acetylsylgenone stock solution, 2 mL of 50% glucose solution, 0.08 g of hydrolyzed casein, and 2 g of sucrose. Adjust the pH to 5.4 and add dH2O to 100 mL.

[0110] Screening medium: N 6max 25 mL of stock solution (10x), N 6min 2.5 mL of stock solution (100x), 2.5 mL of Vitamin stock solution (100x), Fe 2+ Prepare 2.5 mL of EDTA stock solution (100x), 0.625 mL of 2,4-D stock solution (1 mg / mL), 400 μL of carbenicillin (400 mg / mL), 250 μL of hygromycin B (50 mg / mL), 5 mL of 50% glucose solution, 0.15 g of hydrolyzed casein, 7.5 g of sucrose, and 1.75 g of agarose. Adjust the pH to 6.0 and add dH2O to a final volume of 250 mL.

[0111] Differentiation medium: MS max 100 mL of stock solution (10x) and MS min 10 mL of stock solution (100x), 10 mL of Vitamin stock solution (100x), Fe 2+ - 10 mL of EDTA stock solution (100x), 2.0 mL of 6-BA stock solution (1 mg / mL), 2.0 mL of KT stock solution (1 mg / mL), 0.2 mL of IAA stock solution (1 mg / mL), 0.2 mL of NAA stock solution (1 mg / mL), 30 g of sucrose, 1 g of hydrolyzed casein, and 3 g of Phytagel. Adjust the pH to 6.0 and add dH2O to 1000 mL.

[0112] Rooting medium: MS max 50 mL of stock solution (10x) and MS min5 mL of stock solution (100x), 10 mL of Vitamin stock solution (100x), Fe 2+ - Prepare 10 mL of EDTA stock solution (100x), 20 g of sucrose, and 3 g of Phytagel. Adjust the pH to 5.8 and add dH2O to 1000 mL.

[0113] 2.3 Identification of rice plants with endosperm-specific expression of ZlRc and ZlMYB1 genes

[0114] Cut leaves from the transformed plants, extract DNA using the CTAB method, and perform PCR detection using gene-specific primers. The operation method is as follows: (1) Take 1-2g of fresh transformed rice leaves, put them in a mortar pre-cooled with liquid nitrogen, add liquid nitrogen and grind them into powder, then transfer them to a 2mL centrifuge tube; (2) Add 600μL of CTAB separation buffer, invert the centrifuge tube and mix well, place it in a 65℃ water bath for 30min, and gently shake it every 3-4min; (3) Add an equal volume of chloroform:isoamyl alcohol solution with a volume ratio of 24:1, invert the centrifuge tube and mix well, centrifuge at 12000rpm for 15min, and transfer the supernatant to a new 1.5mL centrifuge tube; (4) Add 0.6 times the volume of isopropanol, mix gently, place it at -20℃ to precipitate DNA for 1h, centrifuge at 12000rpm for 15min, and discard the supernatant; (5) Add 700μL of CTAB separation buffer to the DNA precipitate. Wash with 70% ethanol, invert the centrifuge tube, mix well, centrifuge at 12000 rpm for 5 min, discard the supernatant, and place the DNA precipitate in a clean bench to dry naturally; (6) Dissolve the DNA in ddH2O and store at -20℃ for later use.

[0115] ZlRc identification was performed using PCR detection with ZlRc-584bpF and ZlRc-584bpR, where ZlRc-584bpF: 5'-GAAACAGAGAACGACGACGA-3' (SEQ ID NO:7) and ZlRc-584bpR: 5'-GGTGTTTGGGCTTCCTTGTA-3' (SEQ ID NO:8). Agarose gel images for ZlRc identification are shown below. Figure 2 A.

[0116] ZlMYB1 was identified using PCR detection with ZlMYB1-R and ZlMYB1-749bpR, where ZlMYB1-R: 5'-CTACTCCGCATGAGGTTGGG-3' (SEQ ID NO:4) and ZlMYB1-749bpR: 5'-CATGGACGAGCAAGGAAGAC-3' (SEQ ID NO:9). Agarose gel images for ZlMYB1 identification are shown below. Figure 2 B.

[0117] Example 3: Observation of endosperm-specific expression of ZlRc and ZlMYB1 genes in rice and phenotypic comparison with control rice seeds.

[0118] The ZlRc and ZlMYB1 genes in rice endosperm-specific expression were cultured under the same greenhouse conditions as the control rice, with the same culture medium, temperature, and regeneration conditions. The materials were grown under normal water and fertilizer conditions until normal grain filling and yellow ripening. After separating the grains from the panicles, they were dried, and the husks were manually removed. After removing the husks, the ZlRc and ZlMYB1 genes in the rice endosperm-specific expression showed no significant difference from the control rice seed coat; both were colorless. Figure 3 As shown.

[0119] Example 4: Rice endosperm-specific expression of ZlRc and ZlMYB1 genes and determination of total phenols, total flavonoids and total proanthocyanidins in control rice seeds.

[0120] The ZlRc and ZlMYB1 genes obtained after grain hulling showed endosperm-specific expression in rice compared to control rice seeds. After freeze-drying to constant weight, the rice seeds were ground and passed through a 100-mesh sieve. The extraction methods for total phenolic compounds in the seeds, as well as the methods for detecting the content of total phenols, total flavonoids, and total proanthocyanidins, were derived from the article "Comparison of the contents of phenolic compounds including flavonoids and antioxidant activity of rice (Oryza sativa) and Chinese wild rice (Zizania latifolia)".

[0121] (1) Extraction of total phenolic compounds from seeds

[0122] 0.2 g (accuracy 0.0001) of rice seed powder specifically expressing ZlRc and ZlMYB1 genes in rice endosperm and as a control were weighed separately. 5 mL of methanol was added, and the mixture was ultrasonically extracted at 50 °C for 80 min. The mixture was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm polar filter to obtain the free phenolic extract. 5 mL of 4 mol / L NaOH solution was added to the remaining filter residue, and the mixture was hydrolyzed at 30 °C and 220 rpm for 4 h. The mixture was centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was collected in a 40 mL glass centrifuge tube. The pH of the supernatant was adjusted to 1.5–2.0 with 6 mol / L HCl, and the bound phenols were extracted three times with 30 mL of ethyl acetate. The ethyl acetate mixture obtained from the three extractions was evaporated to dryness at 35°C using a rotary evaporator. Then, 5 mL of methanol was added for ultrasonic redissolution. The solution was filtered through a 0.22 μm polar filter to obtain the bound phenol extract, which was stored at 4°C. For measurement, equal volumes (1 mL) of free phenol and the bound phenol extract were mixed to obtain a total phenol compound solution.

[0123] (2) Determination of total phenolic content in seeds

[0124] Total phenol content was determined using the Folin-Ciocalteu colorimetric method. 250 μL of sample solution was added to 250 μL of a three-fold diluted Folin-Ciocalteu solution and mixed thoroughly. After reacting at room temperature for 5 min, 1 mL of ultrapure water and 250 μL of 20% Na₂CO₃ were added, mixed thoroughly, and reacted in the dark for 30 min. The mixture was then centrifuged at 3000 rpm for 10 min at 4℃. 200 mL of the supernatant was transferred to a 96-well plate, and the absorbance at 725 nm was measured using a microplate reader. Each sample was measured three times. Anhydrous methanol was used as a blank control, and a standard curve was established using gallic acid (GA) as the standard. The total phenol content of each sample is expressed as milligrams of gallic acid equivalent per 100 g of rice seed powder (mgGAE / 100g).

[0125] (3) Determination of total flavonoid content in seeds

[0126] The reaction was carried out in a 96-well plate. 50 μL of sample extract was added to 10 μL of 5% NaNO₂ aqueous solution, mixed, and reacted at room temperature for 5 min. 10 μL of 10% AlCl₃ aqueous solution was added, mixed, and reacted at room temperature for 1 min. 100 μL of 0.5M NaOH solution was added, and the reaction was carried out for 10 min. The absorbance at 510 nm was then measured. Each sample was measured three times. Anhydrous methanol was used as a blank control, and a standard curve was established using catechin (C) as a standard. The total flavonoid content in each sample was expressed as milligrams of catechin equivalent per 100g of rice seed powder (mg CE / 100g).

[0127] (4) Determination of total proanthocyanidin content in seeds

[0128] The reaction was carried out in 96-well plates. 20 μL of sample was added to 100 μL of vanillin methanol solution (30 g / mL, w / v), followed by 100 μL of sulfuric acid methanol solution (30% concentrated sulfuric acid, v / v). The reaction was carried out at room temperature in the dark for 5 min, and the absorbance at 500 nm was measured using a microplate reader. Each sample was measured three times. Anhydrous methanol was used as a blank control, and a standard curve was established using catechin (C) as a standard. The total phenolic content of each sample was expressed as milligrams of catechin equivalent per 100 g of rice seed powder (mg CE / 100g).

[0129] Test results as follows Figure 4 – Figure 6 As shown, the total phenolic, total flavonoid, and total proanthocyanidin contents of rice seeds specifically expressing the ZlRc and ZlMYB1 genes were significantly higher than those of control rice seeds. The total phenolic, total flavonoid, and total proanthocyanidin contents of rice seeds specifically expressing the ZlRc and ZlMYB1 genes were 1.20–1.22 times, 1.27–1.45 times, and 1.35–1.57 times those of control rice seeds, respectively.

[0130] Example 5: Endosperm-specific expression of ZlRc and ZlMYB1 genes in rice compared with DPPH free radical scavenging ability and ABTS in control rice seeds. ·+ Free radical absorption capacity determination

[0131] Seed DPPH free radical scavenging ability, ABTS ·+ The method for detecting free radical absorption capacity is derived from the article "Comparison of the contents of phenolic compounds, including flavonoids and antioxidant activity of rice (Oryza sativa) and Chinese wild rice (Zizanialatifolia)".

[0132] (1) Determination of DPPH free radical scavenging capacity of seeds: The reaction was carried out in a 96-well plate. 50 μL of sample was added to 150 μL of 0.5 mM DPPH methanol solution. After mixing, the reaction was carried out in the dark at 30℃ for 30 min, and the absorbance at 517 nm was measured using a microplate reader. Methanol was used as a blank control, and water-soluble vitamin E (TE) methanol solution was used as a standard. Each sample was measured three times. The DPPH free radical scavenging capacity was expressed as the equivalent of micromoles of water-soluble vitamin E (μmolTE / 100g) in 100g of rice seed powder.

[0133] (2) Seed ABTS·+ Free radical absorption capacity determination: Equal volumes of 1.1 mg / mL ABTS methanol solution and 0.68 mg / mL potassium persulfate aqueous solution were mixed and incubated overnight in a dark room to obtain ABTS. ·+ The reagents were diluted with methanol, and the absorbance was adjusted to 0.700 ± 0.020. The reaction was carried out in a 96-well plate, with 50 μL of sample added to 150 μL of ABTS. ·+ In solution, the mixture was reacted in the dark at 30°C for 30 min, and the absorbance at 734 nm was measured using an ELISA reader. Methanol was used as a blank control, and water-soluble vitamin E (TE) methanol solution was used as a standard. Each sample was measured three times. ABTS ·+ Free radical absorption capacity is expressed as the equivalent number of micromoles of water-soluble vitamin E (μmol TE / 100g) in 100g of rice seed powder.

[0134] Test results as follows Figure 7 and Figure 8 As shown, this demonstrates the rice seed DPPH free radical scavenging ability and ABTS of rice genes specifically expressed in rice endosperm by the ZlRc and ZlMYB1 genes. ·+ ZlTTG1 transgenic rice seeds showed significantly higher free radical scavenging capacity than control rice seeds. The DPPH free radical scavenging capacity and ABTS of the ZlTTG1 transgenic rice seeds were also significantly higher. ·+ The free radical absorption capacity was 1.53-1.66 times and 1.26-1.50 times that of the control rice seeds, respectively.

[0135] In this invention, rice seeds specifically expressing the ZlRc and ZlMYB1 genes in the rice endosperm exhibited significantly higher total phenolic, total flavonoid, and total proanthocyanidin contents than control rice seeds. Furthermore, their DPPH free radical scavenging ability and ABTS levels were significantly higher. ·+ The free radical uptake capacity was significantly higher than that of the control rice seeds. This indicates that the endosperm-specific expression of the ZlRc and ZlMYB1 genes effectively regulates the synthesis pathway of flavonoids in the rice endosperm, increasing the flavonoid content in transgenic rice seeds. However, compared to the control, the increased flavonoids in the endosperm-specific expression of the ZlRc and ZlMYB1 genes were mainly enriched in the endosperm and did not cause any change in seed coat color.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of endosperm-specific expression of the ZlRc and ZlMYB1 genes in increasing the flavonoid content of rice seeds, characterized in that... Construct rice endosperm-specific expression vectors for the ZlRc and ZlMYB1 genes.

2. The application according to claim 1, characterized in that, The constructed rice endosperm-specific expression vectors of the ZlRc and ZlMYB1 genes were transferred into rice to obtain transgenic rice that could overexpress ZlRc and ZlMYB1.

3. The application according to claim 2, characterized in that, The ZlRc and ZlMYB1 gene sequences were constructed into a rice endosperm-specific expression vector, and the overexpression vector was transferred into rice. By increasing the expression levels of the ZlRc and ZlMYB1 genes in the rice seed endosperm, transgenic rice with significantly increased seed flavonoid content was obtained.

4. The application according to claim 3, characterized in that, The overexpression vector was chemically transformed into Agrobacterium, and independent transformants were obtained by infecting callus tissue with Agrobacterium. The transgenic rice was obtained through plant regeneration.

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