Application of zizania sinensis ZlTTG1 gene in increasing anthocyanin content of rice seeds

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a Zizizania sinensis ZlTTG1 gene in increasing the anthocyanin content of rice seeds, the nucleotide sequence of the ZlTTG1 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the ZlTTG1 gene is shown as SEQ ID NO: 2. The purpose of the invention is to increase the anthocyanin content of rice seeds through overexpression of the ZlTTG1 gene. Under the condition that the growth environment of the rice seeds is consistent with that of a control group, compared with the control group, the color of the rice seeds overexpressed with the ZlTTG1 gene becomes black, and the anthocyanin content of the rice seeds is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of the Chinese wild taro Z1TTG1 gene in increasing the anthocyanin content of rice seeds. Background Art

[0002] Flavonoid compounds, such as flavonols, anthocyanins, and proanthocyanidins, are major secondary metabolites of plants. Colored rice exhibits various colors, such as brown, red, purple, and black, due to the abundance 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 due to the accumulation of proanthocyanidins. Anthocyanidins are water-soluble flavonoid pigments with a basic structure of C6-C3-C6. These are combined with various substituents to form six major anthocyanidin classes: pelargonidin, cyanidin, peonidin, petunidin, delphinidin, and malvidin. Anthocyanidins determine the color of flowers, fruits, and leaves, imparting a rich and varied color to plants. These colors attract insect pollinators and enhance their resilience to adverse environmental conditions. Anthocyanidins are also potent free radical scavengers, scavenging free radicals and protecting plants from intense light. In addition, anthocyanins play an important role in promoting human health. Studies have shown that anthocyanins can reduce reactive oxygen species in the body; reduce the incidence of diabetes, inflammation, allergies, obesity, heart disease, etc.; inhibit the growth rate of tumors, improve the digestive system, and even have certain anti-aging and life-extending functions. Currently, anthocyanins have been widely used in food, medicine, cosmetics and other fields.

[0003] While most cultivated rice varieties have white seed coats, black rice has attracted significant attention for its sensory properties, high nutritional value, and health benefits. Genetic studies have shown that black rice originates from an acquired mutation in a bHLH transcription factor, kala4 (also known as OsB2). This mutation significantly upregulates the expression of kala4, which was previously barely expressed, in the seed coat and leaves, thereby activating the expression of genes involved in anthocyanin synthesis. Numerous regulatory proteins control the spatiotemporal expression of key anthocyanin biosynthesis genes within the anthocyanin biosynthesis pathway. Among these, three transcription factors, MYB, bHLH, and WD40 repeat proteins, are the primary regulators of flavonoid biosynthesis. MYB transcription factors can regulate anthocyanin biosynthesis genes individually or form a MYB-bHLH-WD40 (MBW) complex with bHLH and WD40 to activate the expression of structural genes. In purple-leaved rice, OsC1, the ortholog of ZmC1 in maize, is an R2R3-MYB transcription factor; OsB1 and OsB2 are homologous to the bHLH transcription factor booster 1 in maize; and OsTTG1 is a WD40 transcription factor. Researchers have demonstrated that OsC1, OsB1 / OsB2, and OsTTG1 form the MBW complex to activate the expression of anthocyanin biosynthesis genes in rice. Notably, recent studies have identified OsTTG1 as a key WD40 transcription factor regulating anthocyanin biosynthesis in rice seeds. Knocking out OsTTG1 in black rice results in a change in the seed coat from black to light brown, significantly reducing seed anthocyanin content to below the detection limit. Therefore, the complex anthocyanin biosynthesis pathway involves a series of genes and proteins involved in the formation, modification, and transport of metabolites, prompting researchers to uncover more WD40 transcription factors or MBW complexes.

[0004] Chinese wild rice (Zizania latifolia) originated in China and belongs to the same tribe, Oryzeae, in the Gramineae family, along with rice. It is distributed throughout China, Japan, Korea, and Southeast Asia. Chinese wild rice resources are abundant in China, particularly in the middle and lower reaches of the Yangtze River and in some waterways of the Huaihe River basin. Studies have shown that wild wild rice from the middle and lower Yangtze River region is a promising candidate for domestication as a cereal crop. The caryopsis of Chinese wild rice, known as Chinese wild rice, has been consumed as a cereal grain in my country for over 3,000 years. Chinese wild rice is a whole grain that contains phenolic acids, flavonoids, and other phytochemicals with excellent antioxidant properties, making it a promising functional food ingredient. Chinese wild rice contains 159 flavonoid compounds. Compared to rice, 72 are upregulated in Chinese wild rice, while 6 are downregulated. The 72 upregulated flavonoids may be related to the brown-black seed coat of Chinese wild rice seeds. The anthocyanin content in wild rice (Zodium truncatum) is reported to be as high as 258.00±17.31mg / 100g. OsTTG1 is a key WD40 transcription factor that regulates anthocyanin synthesis in rice seeds. Through colinearity analysis of the wild rice and rice genomes, a homologous gene, ZlTTG1, was cloned and identified in wild rice. ZlTTG1 is expected to be a gene source for increasing the anthocyanin content of rice seeds. Therefore, the discovery of regulatory genes involved in anthocyanin biosynthesis in wild rice has important practical significance and application prospects for the development of anthocyanin-rich functional rice varieties, improving the dietary structure of residents, and reducing dietary risk factors for chronic diseases. Summary of the Invention

[0005] The present invention aims to increase the anthocyanin content of rice seeds and proposes the use of the ZlTTG1 gene from Chinese wild rice to increase anthocyanin content in rice seeds. The present invention isolates and uses a DNA fragment of the ZlTTG1 gene. When the DNA fragment is driven by a constitutive promoter to overtranscribe the ZlTTG1 gene, the anthocyanin content of rice seeds is significantly increased.

[0006] The technical solution of the present invention is:

[0007] Application of the ZlTTG1 gene from Chinese wild rice in increasing the anthocyanin content in rice seeds, wherein the nucleotide sequence of the ZlTTG1 gene is shown in SEQ ID NO: 1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the ZlTTG1 gene is shown in SEQ ID NO: 2.

[0009] Furthermore, the constructed ZlTTG1 gene overexpression vector was transferred into rice to obtain transgenic rice capable of overexpressing ZlTTG1.

[0010] Furthermore, the overexpression vector is transformed into Agrobacterium by chemical transformation, and callus tissue is infected with Agrobacterium to obtain independent transformants, and transgenic rice with significantly increased anthocyanin content in seeds is obtained through plant regeneration.

[0011] Beneficial effects of the present invention:

[0012] (1) The present invention uses PCR technology to amplify a genomic DNA fragment containing the ZlTTG1 gene coding sequence from a Chinese wild rice cDNA library, constructs this sequence into a PC1300S-GFP overexpression vector, and uses this vector to transform rice. By increasing the expression level of the ZlTTG1 gene mRNA, transgenic rice with significantly increased seed anthocyanin content is obtained.

[0013] (2) The present invention provides the use of the ZlTTG1 gene from Chinese wild rice to increase the anthocyanin content of rice seeds. Under the same growth environment as the control plants, the seeds harvested from rice plants overexpressing the ZlTTG1 gene have higher total phenolic, total flavonoid and total anthocyanin contents, as well as higher DPPH free radical scavenging ability and ABTS activity than the control plants. ·+ This indicates that overexpression of the ZlTTG1 gene effectively regulates the synthesis pathway of anthocyanin compounds in rice, increasing the anthocyanin content in transgenic rice seeds and causing the rice seeds to change from light brown to black.

[0014] (3) Based on genome sequencing results from Chinese wild rice, the present invention cloned the gene ZlTTG1, which regulates anthocyanin synthesis, from Chinese wild rice through colinearity analysis with the rice genome. Biological function verification showed that overexpression of the ZlTTG1 gene significantly increased the anthocyanin content in transgenic rice seeds. This invention confirms the biological function of the ZlTTG1 gene and its application pathways and methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of Example 1 using ClustalΩ software (publicly available software) to compare the nucleotide sequence of the ZlTTG1 gene with the nucleotide sequence of the homologous gene OsTTG1 in rice. Figure 1 It can be seen that the nucleotide sequences of ZlTTG1 and OsTTG1 have a high degree of similarity, and several sites are conserved. Figure 1 In the present invention, OsTTG1 is a gene homologous to that in rice, and ZlTTG1 is a gene cloned by the present invention.

[0016] Figure 2 This is the physical map of the ZlTTG1 overexpression vector in Example 2.

[0017] Figure 3 This is an agarose electrophoresis gel image of the transgenic rice plant identification in Example 2. Explanation of the accompanying symbols: M represents DL2000 marker, B represents blank control, N represents negative control, P represents positive control, and 1 to 32 represent the transgenic rice plant numbers.

[0018] Figure 4 Phenotypic observation of ZlTTG1 transgenic rice seeds in Example 3. Explanation of Figure Symbols: (A) is a homozygous control rice seed obtained by culturing Zixiangnuo No. 1 with the OsTTG1 gene knocked out, and (B) is a rice seed obtained by culturing the control rice seed after the ZlTTG1 gene was transferred.

[0019] Figure 5 Comparison of total phenolic content in one control (CK) and three ZlTTG1 transgenic rice seeds (ZlTTG1-1, ZlTTG1-2, and ZlTTG1-3) from Example 4. Test results are the means of three replicates, and error bars represent standard deviations (SD).

[0020] Figure 6 Comparison of total flavonoid content in one control (CK) and three ZlTTG1 transgenic rice seeds (ZlTTG1-1, ZlTTG1-2, and ZlTTG1-3) from Example 4. Test results are the means of three replicates, and error bars represent standard deviations (SD).

[0021] Figure 7 Comparison of total anthocyanin content between one control (CK) and three ZlTTG1 transgenic rice seeds (ZlTTG1-1, ZlTTG1-2, and ZlTTG1-3) from Example 4. The test results are the means of three replicates, and the error bars represent the standard deviation (SD).

[0022] Figure 8 The results are a comparison of the DPPH radical scavenging abilities of one control (CK) and three ZlTTG1 transgenic rice seeds (ZlTTG1-1, ZlTTG1-2, and ZlTTG1-3) from Example 5. The test results are the means of three replicates, and the error bars represent the standard deviation (SD).

[0023] Figure 9 ABTS of one control (CK) and three ZlTTG1 (ZlTTG1-1, ZlTTG1-2, ZlTTG1-3) transgenic rice seeds in Example 5 ·+ Comparison of free radical absorbance capacity. The test results are the means of three replicates, and the error bars represent the standard deviation (SD). DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments, providing a clear and complete description of the technical solutions in the embodiments of the present invention. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0026] The biological material used in the following examples, Zizogneospermum officinale, was collected from Huai'an City, Jiangsu Province, China; the control rice was the OsTTG1 gene-knockout rice variety Zixiangnuo No. 1, whose seeds were sourced from Wuhan City, Hubei Province, China.

[0027] Example 1 Acquisition of Z1TTG1 gene

[0028] 1.1 Extraction of total RNA and preparation of cDNA from Zizyphus chinensis

[0029] 1.1.1 Extraction of total RNA from Zizyphus chinensis

[0030] Polysaccharide and polyphenol plant RNA extraction kit ( RNA from Chinese wild rice leaves was extracted using a Universal Plant Total RNA Isolation Kit (Vazyme) and reverse transcribed into cDNA. RNA extraction from Chinese wild rice leaves was performed according to the instructions of the plant RNA extraction kit. The specific experimental steps are as follows:

[0031] (1) The leaf sample of Zizania latifolia was quickly ground into powder in liquid nitrogen. 50 mg of the ground sample was weighed and added to 500 μL of Bufer PRL preheated at 65 °C. The mixture was immediately vortexed vigorously for 60 s.

[0032] (2) Incubate the lysate in a 65°C water bath for 5 min, invert twice, and 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 and mix immediately by pipetting.

[0033] (3) Transfer the above mixture to FastPure gDNA-Filter Column II, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0034] (4) Add 500 μL of Buffer PRLPlus to FastPure gDNA-Filter Column II, centrifuge at 12,000 rpm for 30 seconds, and collect the filtrate.

[0035] (5) Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix immediately by pipetting; transfer the mixture to FastPure RNA Column IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0036] (6) Add 700 μL of Buffer PRW1 to FastPure RNA Column IV, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0037] (7) Add 500 μL of Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 s, discard the filtrate, and repeat this step once.

[0038] (8) Centrifuge the FastPure RNA Column IV adsorption column at 12000 rpm for 2 min to remove the residual Buffer PRW2 in the FastPure RNA Column IV.

[0039] (9) Transfer FastPure RNA Column IV to a new RNase-free 1.5 mL centrifuge tube, add 40 μL of RNase-free ddH2O to the center of the adsorption column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0040] 1.1.2 Preparation of cDNA

[0041] After RNA extraction, the RNA concentration was determined and 2.0 μg of RNA was used as a substrate for reverse transcription. Reverse transcription was performed using a reverse transcription kit to obtain cDNA products, which were stored at -20°C until use.

[0042] Table 1 Reverse transcription PCR system and procedure

[0043]

[0044] 1.2 Amplification of the ZlTTG1 gene

[0045] The primers designed based on the ZlTTG1 gene sequence are as follows:

[0046] ZlTTG1-F: 5'-ATGGAGCAGCCCAAGCCGCC-3' (SEQ ID NO: 3);

[0047] ZITTG1-R: 5'-TCAGACCCTGAGAAGCTGGA-3' (SEQ ID NO: 4).

[0048] Using the prepared ZlTTG1 cDNA as a template, PCR amplification was performed using primers to obtain the target fragment ZlTTG1. The PCR amplification system and reaction procedure are as follows:

[0049] Table 2 PCR system and procedure

[0050]

[0051]

[0052] The PCR product was sequenced, and the full length of the sequence was 1071 bp. The nucleotide sequence is shown in SED ID NO.1, and the amino acid sequence is shown in SED ID NO.2.

[0053] Example 2 Construction and genetic transformation of ZlTTG1 gene overexpression vector

[0054] 2.1 Construction of ZlTTG1 gene overexpression vector

[0055] The PC1300S-GFP vector was digested with restriction endonucleases KpnI and BamHI, and the digestion products were separated by agarose gel electrophoresis. The linearized PC1300S-GFP large fragment was recovered using a gel recovery kit. It was recombined with the PCR amplification product ZlTTG1, and the target gene was ligated into the vector and transformed into competent E. coli DH5α to obtain the ZlTTG1 gene overexpression vector. Its physical map is shown in Figure 2 .

[0056] The ligation product was transformed into Escherichia coli DH5α. The specific transformation steps were as follows: DH5α stored in a -80°C refrigerator was placed on ice for about 10 minutes for ice bath freezing and thawing, 10 μL of the ligation product was added to 100 μL DH5α, the mixture was pipetted and ice bathed for 30 minutes; then heat shocked in a 42°C water bath for 90 seconds, and quickly transferred to an ice bath for 2 minutes; 1 mL of LB liquid culture medium without antibiotics was added to the clean bench, and the culture was revived in a shaker at 220 rpm at 37°C for 45-60 minutes; the revived bacterial solution was centrifuged at 6000 rpm for 5 minutes, 500 μL of supernatant was removed with a pipette, and 500 μL of culture medium was retained to resuspend the precipitated bacteria. After resuspension, the plate was spread and cultured upside down in a 37°C incubator overnight, and the positive clones were picked and cultured in a shaker at 220 rpm at 37°C overnight.

[0057] Designed bacterial PCR primers A580-SeqR: 5'-AGAAGATGGTGCGCTCCTG-3' (SEQ ID NO: 5) and ZlTTG1-RICE: 5'-CTCGTAGAGGATGGTGG-3' (SEQ ID NO: 6). Colony PCR was performed to detect single positive ZlTTG1 clones. After electrophoresis, the PCR products were screened for positive single colonies expressing the target band and sequenced for verification.

[0058] The extracted plasmid was transformed into Agrobacterium EHA105 competent cells. The steps of the operation method are as follows:

[0059] (1) Take the competent cells of Agrobacterium tumefaciens EHA105 stored at -80℃ and let them partially thaw at room temperature or in the palm of your hand for a while. When they are in an ice-water mixture, insert them into ice.

[0060] (2) Add 5 μL of extracted plasmid to every 100 μL of Agrobacterium EHA105 competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0061] (3) Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking for 2-3 h.

[0062] (4) Centrifuge at 6000 rpm for 1 min to collect the bacteria. Take about 100 μL of the supernatant and gently blow to resuspend the bacteria. Spread the plate on an LB plate containing kanamycin antibiotics and place it upside down in a 28°C incubator for 2-3 days.

[0063] 2.3 Obtaining rice with overexpression of ZlTTG1 gene

[0064] (1) Callus preparation

[0065] Step 1: Sterilize mature rice seeds. Use appropriate tools to hull mature seeds. Discard seeds with mold spots and those with underdeveloped embryos (shrunken, brown), ensuring seed integrity and purity. After hulling, rinse the rice seeds with 75% ethanol for 1 minute, sterilize them with 0.15% HgCl₂ for 15-20 minutes, and finally rinse them 4-5 times with sterile ddH₂O. Soak the seeds overnight.

[0066] Step 2: Inducing Rice Callus: Wash the seeds overnight with sterile water, peel the embryos from the aleurone layer with a scalpel, and inoculate them onto induction medium. Inoculate 8-12 sterilized rice seeds per bottle of induction medium. Set the temperature to 30°C and incubate in the dark for 40-45 days to induce callus formation.

[0067] Step 3: Subculture of callus. Select pale yellow, granular, dry, and vigorous calli from the induced calli and transfer them to subculture medium for dark culture for 20 days. During the first subculture, be sure to remove any attached tissue (such as endosperm and buds). Calli that have been subcultured once are ready for Agrobacterium tumefaciens infection. Calli intended for transformation can be subcultured a maximum of two times. Multiple subcultures can easily lead to somatic mutations in the calli and reduce transformation efficiency.

[0068] Step 4: Pre-culture of callus tissue. From the subcultured callus, select pale yellow, granular, dry, and vigorous callus tissue and transfer it to the pre-culture medium. Inoculate each dish with 60-80 mung bean-sized callus granules. Larger callus granules can be crushed with tweezers. Pre-culture at 28°C in the dark for 3-4 days. After the pre-culture, use a small spoon to collect the small, well-proportioned, actively dividing granules into a 250mL sterile Erlenmeyer flask for Agrobacterium infection.

[0069] (2) Preparation of Agrobacterium

[0070] Step 1: Agrobacterium activation: 2 days before the experiment, streak the Agrobacterium tumefaciens strain containing the target gene on an LA plate containing the corresponding antibiotic and then incubate at 28°C for 2 days.

[0071] Step 2: Resuspend Agrobacterium. Take out the streaked plate of Agrobacterium tumefaciens and inoculate approximately one ring of Agrobacterium into 100 mL of suspension culture medium with an inoculating loop. Add 100 μL of 100 mM acetosyringone stock solution and 2 mL of 50% glucose. Place in a constant temperature shaker at 28°C and 200 rpm for 30 minutes. The concentration of the Agrobacterium suspension is approximately OD 600 =0.3 is sufficient.

[0072] (3) Agrobacterium infection and liquid co-culture

[0073] Step 1: Agrobacterium infection. Pour the prepared Agrobacterium suspension into the triangular flask containing the callus tissue until all the callus tissue is immersed, and let it stand for 10 minutes. Pour off the bacterial solution. Take a sterile small plate containing absorbent paper and filter paper, open the small plate, and invert the triangular flask containing the callus on the filter paper in the small plate. Drain as much bacterial solution as possible. Then spread the callus on the filter paper in a sterile large plate, cover it with a piece of sterile filter paper, and gently press the filter paper with tweezers to absorb the bacterial solution on the surface of the callus. Then remove the filter paper after moisture absorption. Change the filter paper up and down four times. Finally, cover the callus with a piece of filter paper, cover the large plate, and let it dry naturally for 1-2 hours.

[0074] Step 2: Co-culture. Use tweezers to transfer the dried callus particles to the co-culture medium and seal with sealing glue. Co-culture at 19°C in the dark for 3 days.

[0075] Step 3: Wash with water. Transfer the co-cultivated callus to a water washing cup and pour in sterile distilled water until the callus is completely submerged. Cover the cup and shake for 20-30 seconds. Pour out the sterile distilled water. Repeat this process for 3-4 times. Observe the cup. If the distilled water in the cup is clear, the Agrobacterium has been essentially cleaned. Otherwise, continue washing. Finally, pour out the sterile distilled water and add 500 mg / L carbenicillin. Let it stand for 30 minutes. Pour out the sterile distilled water containing 500 mg / L carbenicillin.

[0076] Step 4: Callus screening. After the callus is dry, use tweezers to transfer the callus particles to the screening medium, seal them with sealing glue, and place them in a dark culture room for screening and culture for 20 days (first screening S1). Select dry calli without Agrobacterium contamination from the S1 medium and transfer them to the S2 medium. After 20 days of dark culture, observe whether fresh, tender yellow resistant calli grow. If no resistant calli are found, continue to transfer the dish for S3 screening and culture. Generally, japonica rice varieties can grow resistant calli after two screenings, that is, in the S2 stage.

[0077] Step 5: Differentiation culture. Pick small pieces of light yellow, dense, dry, and vigorously growing resistant callus. Pick only one resistant callus from each group. Be careful not to pick callus with Agrobacterium. Place 3-4 small pieces of resistant callus evenly in each bottle of differentiation medium, because callus cells will continue to grow on the differentiation medium. Placing them too densely will easily cause different callus pieces to grow together and be indistinguishable. Culture under 28℃ light for 30-40 days, with a light cycle of 16h light / 8h dark. After the seedlings are differentiated to be 3-5cm high, rooting culture can be carried out. During the light culture period, the contaminated materials with bacteria should be cleaned up in time.

[0078] Step 6: Plant rooting. Use forceps to remove the differentiated seedlings from the differentiation medium and place them in a sterilized, empty dish. Select only one healthy seedling from each callus. Use scissors to clean the seedlings, removing any dead or yellowed leaves and any roots growing on the differentiation medium. Place the seedlings in rooting tubes, with one seedling per tube. Rooting incubate in a light-sensitive chamber for 15-20 days. Transplant after new roots have fully developed.

[0079] Step 7: Transplant the plants. Remove the film sealing the rooting tubes, add some tap water, and continue hardening the seedlings in a light-controlled incubator for 3-4 days. During hardening, small leaf samples can be collected for transgenic testing. Remove the transformed seedlings from the rooting tubes, clean the culture medium from the roots, and transplant them into a pot or bucket with prepared soil.

[0080] (4) Preparation of reagents and culture medium used in transformation:

[0081] 1) Abbreviations of reagents and solutions:

[0082] The abbreviations of the plant hormones used in the culture medium of the present invention are as follows: 6-Benzylaminopurine (6-BA), 6-benzyladenine; Indole-3-acetic acid (IAA), indole-3-acetic acid; Napthalene acetic acid (NAA), naphthylacetic acid; 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenoxyacetic acid; Kinetin (KT), 6-glycosylaminopurine.

[0083] 2) Main solution formula:

[0084] MS max Stock solution (10x): Dissolve 16.5 g NH4NO3, 1.7 g KH2PO4, 19.0 g KNO3, 3.7 g MgSO4·7H2O, and 3.32 g CaCl2 or 4.4 g CaCl2·2H2O one by one, and then adjust the volume to 1000 mL at room temperature.

[0085] MS min Stock solution (100x): 2.23 g MnSO4·4H2O, 0.86 g ZnSO4·7H2O, 0.083 g KI, 0.62 g H3BO3, 0.025 g Na2MoO4·2H2O, 0.0025 g CoCl2·6H2O, 0.0025 g CuSO4·5H2O, dissolve them one by one, and then adjust the volume to 1000 mL at room temperature.

[0086] N 6max Stock solution (10x): 28.3g KNO3, 4.63g (NH4)2SO4, 4.0g KH2PO4, 1.85g MgSO4·7H2O, 1.25g CaCl2 or 1.66g CaCl2·2H2O, dissolve them one by one, and then adjust the volume to 1000mL at room temperature.

[0087] N 6min Stock solution (100x): Dissolve 0.08 g KI, 0.16 g H3BO3, 0.15 g ZnSO4·7H2O, 0.44 g MnSO4·4H2O or 0.3335 g MnSO4·H2O one by one, and then adjust the volume to 1000 mL at room temperature.

[0088] Fe 2+EDTA stock solution (100x): Add approximately 300 mL of dH2O and 2.78 g of FeSO4·7H2O to one reagent bottle. To another reagent bottle, add approximately 300 mL of dH2O and heat to 70°C, then add 3.73 g of Na2·EDTA·2H2O. Once dissolved, cool the solution to room temperature, combine the solutions in both bottles, and then add dH2O to make the volume 1000 mL. Store at 4°C in dark.

[0089] Vitamin stock solution (100x): 0.1 g niacin, 0.1 g nicotinamide thiamine, 1 g pyridoxine hydrochloride, 10 g inositol, 0.2 g glycine. Add dH2O to 1000 mL and store at 4°C.

[0090] AA max Stock solution (10x): 29.50 g KCl, 2.50 g MgSO4·7H2O, 1.50 g NaH2PO4, 1.50 g CaCl2·2H2O, add dH2O to 1000 mL, and store at room temperature in the dark.

[0091] AA min Stock solution (100x): 1.0 g MnSO4·H2O, 0.2 g ZnSO4·7H2O, 0.0025 g CuSO4·5H2O, 0.3 g H3BO3, 0.075 g KI, 0.0025 g CoCl2·6H2O, 0.025 g NaMoO4·2H2O, add dH2O to make up to 1000 mL, and store at room temperature in the dark.

[0092] 6-BA stock solution (1 mg / mL): To 100 mg of 6-BA, add 1.0 mL of 1N KOH and shake until the 6-BA is dissolved. Then add dH2O to make up to 100 mL and store at room temperature.

[0093] KT stock solution (1 mg / mL): To 100 mg of KT, add 1.0 mL of 1N KOH and shake until the KT is dissolved. Then add dH2O to make up to 100 mL and store at room temperature.

[0094] 2,4-D stock solution (1 mg / mL): To 100 mg of 2,4-D, add 1.0 mL of 1N KOH and shake for 5 minutes. Then add 10 mL of dH2O and shake until the 2,4-D is dissolved. Dose up to 100 mL with dH2O and store at room temperature.

[0095] 100 mM acetosyringone stock solution: 0.196 g acetosyringone, 10 mL dimethyl sulfoxide, divided into 1.5 mL centrifuge tubes, stored at 4°C.

[0096] IAA stock solution (1 mg / mL): To 100 mg of IAA, add 1.0 mL of 1N KOH and shake until the IAA dissolves. Then, dilute to 100 mL with dH2O and store at room temperature in the dark.

[0097] NAA stock solution (1 mg / mL): Add 1.0 mL of 1N KOH to 100 mg of NAA and shake until the NAA dissolves. Then, dilute to 100 mL with dH2O and store at room temperature in the dark.

[0098] 1N KOH stock solution: Dissolve 5.6 g KOH in 100 mL dH2O and store at room temperature.

[0099] 0.15% HgCl2: Dissolve 1.5 g HgCl2 partially or completely in 1 mL of anhydrous ethanol, then dilute to 1000 mL with dH2O. Stir for 4-8 hours and store at room temperature.

[0100] 3) Culture medium formulation for rice genetic transformation:

[0101] Induction medium: N 6max Stock solution (10x) 100 mL, N 6min Stock solution (100x) 10mL, Vitamin stock solution (100x) 10mL, Fe 2+ -EDTA stock solution (100x) 10 mL, 2,4-D stock solution (1 mg / mL) 2.5 mL, hydrolyzed casein 0.6 g, proline 0.3 g, sucrose 30 g, plant gel (Phytagel) 3 g, adjust the pH to 5.9, and add dH2O to 1000 mL.

[0102] Subculture medium: N 6max Stock solution (10x) 100 mL, N 6min Stock solution (100x) 10mL, Vitamin stock solution (100x) 10mL, Fe 2+ -EDTA stock solution (100x) 10 mL, 2,4-D stock solution (1 mg / mL) 2.0 mL, hydrolyzed casein 0.6 g, proline 0.5 g, sucrose 30 g, plant gel (Phytagel) 3 g, adjust the pH to 5.9, and add dH2O to 1000 mL.

[0103] Pre-culture medium: N 6max Stock solution (10x) 12.5 mL, N 6min Stock solution (100x) 1.25mL, Vitamin stock solution (100x) 2.5mL, Fe 2+-EDTA stock solution (100x) 25 mL, 2,4-D stock solution (1 mg / mL) 0.75 mL, 100 mM acetosyringone stock solution 300 μL, 50% glucose solution 5 mL, hydrolyzed casein 0.15 g, sucrose 5 g, agarose 1.75 g, adjust the pH to 5.4, and add dH2O to 250 mL.

[0104] Co-culture medium: N 6max Stock solution (10x) 12.5 mL, N 6min Stock solution (100x) 1.25mL, Vitamin stock solution (100x) 2.5mL, Fe 2+ -EDTA stock solution (100x) 25 mL, 2,4-D stock solution (1 mg / mL) 0.75 mL, 100 mM acetosyringone stock solution 300 μL and 50% glucose solution 5 mL, hydrolyzed casein 0.2 g, sucrose 5 g, agarose 1.75 g, adjust the pH to 5.4, and add dH2O to 250 mL.

[0105] Suspension medium: N 6max Stock solution (10x) 5mL, N 6min Stock solution (100x) 0.5mL, Vitamin stock solution (100x) 1mL, Fe 2+ -EDTA stock solution (100x) 0.5 mL, 2,4-D stock solution (1 mg / mL) 0.2 mL, 100 mM acetosyringone stock solution 100 μL and 50% glucose solution 2 mL, hydrolyzed casein 0.08 g, sucrose 2 g, adjust the pH to 5.4, and add dH2O to 100 mL.

[0106] Screening medium: N 6max Stock solution (10x) 25 mL, N 6min Stock solution (100x) 2.5mL, Vitamin stock solution (100x) 2.5mL, Fe 2+ -EDTA stock solution (100x) 2.5 mL, 2,4-D stock solution (1 mg / mL) 0.625 mL, 400 μL of 400 mg / mL carbenicillin, 250 μL of 50 mg / mL hygromycin B, 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 250 mL.

[0107] Differentiation medium: MS max Stock solution (10x) 100 mL, MS min Stock solution (100x) 10mL, Vitamin stock solution (100x) 10mL, Fe 2+-EDTA stock solution (100x) 10 mL, 6-BA stock solution (1 mg / mL) 2.0 mL, KT stock solution (1 mg / mL) 2.0 mL, IAA stock solution (1 mg / mL) 0.2 mL, NAA stock solution (1 mg / mL) 0.2 mL, sucrose 30 g, hydrolyzed casein 1 g, plant gel (Phytagel) 3 g, adjust the pH to 6.0, and add dH2O to 1000 mL.

[0108] Rooting medium: MS max Stock solution (10x) 50 mL, MS min Stock solution (100x) 5mL, Vitamin stock solution (100x) 10mL, Fe 2+ -Add 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.

[0109] 2.4 Identification of ZlTTG1 gene overexpressing plants

[0110] Cut the leaves of the transformed plants, extract DNA using the CTAB method, and perform PCR detection using the specific primers for the screening marker gene. The operation method is as follows:

[0111] (1) Take 1-2g of freshly transformed rice leaves, place them in a mortar pre-cooled with liquid nitrogen, add liquid nitrogen and grind them into powder, and transfer them to a 2mL centrifuge tube; (2) Add 600μL CTAB separation buffer, invert the centrifuge tube, mix well, and place it in a 65℃ water bath for 30min, shaking it gently every 3-4min to mix well; (3) Add an equal volume of chloroform:isoamyl alcohol solution with a volume ratio of 24:1, invert the centrifuge tube, 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 DNA to the DNA precipitate. Wash with 70% ethanol, invert the centrifuge tube upside down to mix evenly, centrifuge at 12000 rpm for 5 minutes, pour out 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 use.

[0112] PCR detection was performed using the selection marker gene specific primers hpt472-F and hpt472-R, where hpt472-F: 5'-GAAGTGCTTGACATTGGGGAGT-3' (SEQ ID NO: 7) and hpt472-R: 5'-AGATGTTGGCGACCTCGTATT-3' (SEQ ID NO: 8). Agarose gel electrophoresis of ZlTTG1 transgenic rice plants is shown in the figure. Figure 3 .

[0113] Example 3 Phenotypic Observation of ZlTTG1 Gene Overexpression and Control Rice Seeds

[0114] ZlTTG1 transgenic rice and control rice (Zixiangnuo No. 1 rice with the OsTTG1 gene knocked out) were cultured under the same greenhouse conditions and given the same regeneration conditions such as culture medium and culture temperature. The materials were grown under normal water and fertilizer conditions until they were normally fruitful and ripe. The grains were separated from the ears and dried, and the grain husks were manually removed. After the grains were husked, the seeds of the ZlTTG1 transgenic rice plants showed a significant difference in seed coat color compared to the control rice seeds. The seeds of the ZlTTG1 transgenic rice plants were black, while the seeds of the control plants were light brown. Figure 4 shown.

[0115] Example 4 Determination of total phenolic, total flavonoid and total anthocyanin contents in ZlTTG1 transgenic and control rice seeds

[0116] ZlTTG1 transgenic and control rice seeds were freeze-dried to constant weight after husking and then ground through a 100-mesh sieve. The methods for extracting total phenolic compounds from seeds and determining their total phenolic and flavonoid contents were adapted 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)." The methods for extracting anthocyanins from seeds and determining their total anthocyanin content were adapted from the article "Measurement of anthocyanins and other phytochemicals in purple wheat."

[0117] (1) Extraction of total phenolic compounds from seeds

[0118] 0.2 g (accuracy 0.0001) of ZlTTG1 transgenic and control rice seed powder was weighed, added to 5 mL of methanol, and ultrasonically extracted at 50°C for 80 min. The mixture was centrifuged in a low-speed centrifuge at 4°C and 3000 rpm for 10 min. The supernatant was filtered through a 0.22 μm polar membrane 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 in a thermostatic shaker at 30°C and 220 rpm for 4 h. The mixture was centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was collected into a 40 mL glass centrifuge tube. The pH of the supernatant was adjusted to 1.5-2.0 with 6 mol / L HCl, and bound phenols were extracted three times with 30 mL of ethyl acetate. The resulting ethyl acetate mixture was rotary evaporated to dryness at 35°C, reconstituted by ultrasonication with 5 mL of methanol, and filtered through a 0.22 μm polar membrane filter to obtain the bound phenolic extract, which was stored at 4°C. For measurement, equal amounts (1 mL) of free phenol and bound phenol extracts were mixed to obtain a total phenolic compound solution.

[0119] (2) Determination of total phenolic content in seeds

[0120] The total phenol content was determined using the folin-phenol colorimetric method. To 250 μL of a three-fold diluted folin-ciocalteu solution, 250 μL of the sample solution was added and mixed. After reacting at room temperature for 5 minutes, 1 mL of ultrapure water and 250 μL of 20% Na2CO3 were added. The mixture was thoroughly mixed and reacted in the dark for 30 minutes. The mixture was centrifuged at 3000 rpm at 4°C for 10 minutes. 200 mL of the supernatant was placed in a 96-well plate and its 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 a standard. The total phenol content in each sample was expressed as milligrams of gallic acid equivalent per 100 g of rice seed powder (mgGAE / 100 g).

[0121] (3) Determination of total flavonoid content in seeds

[0122] The reaction was performed in a 96-well plate. 50 μL of the sample extract was added to 10 μL of a 5% NaNO₂ aqueous solution, mixed, and allowed to react at room temperature for 5 minutes. 10 μL of a 10% AlCl₃ aqueous solution was added, mixed, and allowed to react at room temperature for 1 minute. 100 μL of a 0.5 M NaOH solution was added, allowed to react for 10 minutes, and the absorbance at 510 nm was measured. Each sample was measured in triplicate, using anhydrous methanol as a blank control. A standard curve was established using catechin (C) as a standard. The total flavonoid content of each sample was expressed as milligrams of catechin equivalent per 100 g of rice seed powder (mg CE / 100 g).

[0123] (4) Extraction of seed anthocyanins and determination of total anthocyanin content

[0124] 0.5g of rice flour was extracted three times with acidified methanol, and the resulting supernatant was the anthocyanin extract. Before determining the total anthocyanin content, two solutions were prepared: Solution A: pH 1.0 solution (1.49% potassium chloride aqueous solution, pH adjusted with HCl); Solution B: pH 4.5 solution (1.64% sodium acetate aqueous solution, pH adjusted with HCl). 200μL of sample was added to 1.8mL of Solution A and Solution B, respectively, and the absorbance values ​​at wavelengths of 520nm and 700nm were measured, respectively. The total anthocyanin content was expressed as equivalents of cyanidin-3-O-glucoside (mg C3GE / 100g), and the calculation formula was as follows:

[0125] Anthocyanin monomer content (mg / L) = A × MW × DF × 1000 / (ε × L)

[0126] Wherein, A refers to the absorbance value, and the calculation method is A=(A 520nm -A 700nm )pH 1.0 -(A 520nm -A 700nm )pH 4.5 ; MW refers to the molecular mass of cyanidin-3-O-glucoside (449.2 g / mol); DF refers to the dilution factor; ε refers to the molar absorptivity of cyanidin-3-O-glucoside (26900 L / (cm×mol)); L refers to the path length of light (1 cm); 1000 refers to the conversion factor from milliliters to liters.

[0127] Test results such as Figure 5 – Figure 7 As shown, the total phenolic content, total flavonoid content, and total anthocyanin content of ZlTTG1 transgenic rice seeds were significantly higher than those of control rice seeds. The total phenolic content and total flavonoid content of ZlTTG1 transgenic rice seeds were 2.65 times and 2.11 times that of the control rice seeds, respectively. Furthermore, while the anthocyanin content of the control rice seeds was zero, the average total anthocyanin content of ZlTTG1 transgenic rice seeds was 86.55 mg C3GE / 100 g.

[0128] Example 5: DPPH free radical scavenging ability and ABTS of ZlTTG1 transgenic and control rice seeds ·+ Free radical absorbance capacity assay

[0129] Seed DPPH free radical scavenging ability, ABTS ·+The free radical absorbance capacity test method was 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).

[0130] (1) Determination of seed DPPH radical scavenging ability: 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 mixture was reacted in the dark at 30°C for 30 min, and the absorbance at 517 nm was measured using a microplate reader. Methanol was used as a blank control, and a methanol solution of water-soluble vitamin E (TE) was used as a standard. The measurement was repeated three times for each sample. The DPPH radical scavenging ability was expressed as the equivalent micromoles of water-soluble vitamin E per 100 g of rice seed powder (μmol TE / 100 g).

[0131] (2) Seed ABTS ·+ Free radical absorbance capacity determination: 1.1 mg / mL ABTS methanol solution and 0.68 mg / mL potassium persulfate aqueous solution were mixed in equal amounts and incubated overnight in a dark room to obtain ABTS ·+ Reagent, dilute with methanol, adjust the absorbance to 0.700 ± 0.020. The reaction was carried out in a 96-well plate, and 50 μL of sample was added to 150 μL of ABTS. ·+ The mixture was mixed and reacted in the dark at 30°C for 30 minutes, and the absorbance at 734 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, and ABTS ·+ The free radical absorbance capacity was expressed as the equivalent micromoles of water-soluble vitamin E per 100 g of rice seed powder (μmol TE / 100 g).

[0132] Test results such as Figure 8 and Figure 9 As shown, it can be seen that the DPPH free radical scavenging ability and ABTS ·+ The free radical absorption capacity of ZlTTG1 transgenic rice seeds was significantly higher than that of the control rice seeds. ·+ The free radical absorption capacity was 1.61 times and 1.70 times that of the control rice seeds, respectively.

[0133] In the present invention, the total phenol content, total flavonoid content and total anthocyanin content of ZlTTG1 transgenic rice seeds are significantly higher than those of the control rice seeds, and its DPPH free radical scavenging ability and ABTS ·+ The free radical absorption capacity of rice seeds was significantly higher than that of the control rice seeds. Overexpression of the ZlTTG1 gene had the most significant effect on increasing the total anthocyanin content of the control rice seeds, indicating that overexpression of the ZlTTG1 gene in rice has an effective regulatory effect on the synthesis pathway of anthocyanin compounds, effectively increasing the anthocyanin content in rice seeds.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of the Z1TTG1 gene of Chinese wild rice in increasing the anthocyanin content of rice seeds, characterized in that: The nucleotide sequence of the ZlTTG1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the ZlTTG1 gene is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that The constructed ZlTTG1 gene overexpression vector was transferred into rice to obtain transgenic rice capable of overexpressing ZlTTG1.

4. The use according to claim 3, characterized in that The ZlTTG1 gene sequence is constructed into an overexpression vector, and the overexpression vector is transformed into rice, thereby increasing the expression level of ZlTTG1 gene mRNA to obtain transgenic rice with significantly increased seed anthocyanin content.

5. The use according to claim 4, characterized in that The overexpression vector is transformed into Agrobacterium by chemical transformation, and callus tissue is infected with Agrobacterium to obtain independent transformants, and the transgenic rice is obtained by plant regeneration.

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