A breeding method for synthesizing resveratrol and pterostilbene in the endosperm of rice seeds

Abstract

The application discloses a breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm, and relates to the technical field of biology.The transgenic breeding method comprises the steps of transforming a recombinant carrier into rice callus and then cultivating the rice callus into a transgenic rice plant; the recombinant carrier comprises a gene expression cassette for expressing VvSTS protein, a gene expression cassette for expressing VvROMT protein, a gene expression cassette for expressing At4CL protein and a gene expression cassette for expressing transcription factor OsMYBP1 protein.The application realizes the synthesis of resveratrol and pterostilbene in rice endosperm, and the product can be used as raw material for producing resveratrol and pterostilbene, and has important guiding significance for new functional breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm. Background Technology

[0002] Resveratrol is a class of highly active stilbene compounds with multiple effects, including preventing and treating cardiovascular diseases, anti-oxidation, anti-inflammation, and delaying aging. It is abundant in plants such as grapes and peanuts. Pterostilbene is a methylated natural derivative of resveratrol with higher bioavailability and stability, exhibiting good physiological activities in anti-tumor activity, blood sugar regulation, and lipid metabolism improvement. As one of the major food crops, rice could potentially have its nutritional value and functionality enhanced if the synthetic pathways of resveratrol and pterostilbene could be reconstructed in its endosperm.

[0003] However, rice lacks key enzyme genes in the stilbene synthesis pathway, such as stilbene synthase and O-methyltransferase genes, and its phenylpropanoid metabolism pathway mainly leads to the synthesis of flavonoids, lacking the ability to efficiently convert substrates into resveratrol and its methylation product pterostilbene. Therefore, rice seed endosperm does not contain resveratrol and pterostilbene. Summary of the Invention

[0004] This invention proposes a breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm to solve the problems existing in the prior art.

[0005] The technical solution of the present invention is as follows:

[0006] This invention proposes a recombinant vector comprising a gene expression cassette expressing VvSTS protein, a gene expression cassette expressing VvROMT protein, a gene expression cassette expressing At4CL protein, and a gene expression cassette expressing transcription factor OsMYBP1 protein.

[0007] The gene expression cassette expressing the VvSTS protein includes the gene encoding the VvSTS protein, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0008] The gene expression cassette expressing the VvROMT protein includes the gene encoding the VvROMT protein, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0009] The gene expression cassette expressing the At4CL protein includes the gene encoding the At4CL protein, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0010] The gene expression cassette expressing the transcription factor OsMYBP1 protein includes the coding gene for the transcription factor OsMYBP1 protein, and the nucleotide sequence of the coding gene for the transcription factor OsMYBP1 protein is shown in SEQ ID NO.7.

[0011] Furthermore, the gene expression cassette expressing the VvSTS protein also includes the promoter P10 and the Tnos terminator;

[0012] The gene expression cassette expressing the VvROMT protein also includes the promoter PgluB5 and the terminator TAtubq3;

[0013] The gene expression cassette expressing the At4CL protein also includes the promoter P26 and the terminator T35;

[0014] The gene expression cassette expressing the transcription factor OsMYBP1 protein also includes the promoter P16 and the Tnos terminator.

[0015] The present invention also proposes a recombinant host cell, comprising the aforementioned recombinant vector.

[0016] Furthermore, the host cell is Agrobacterium.

[0017] The present invention also proposes the application of the recombinant vector or the recombinant host cell in the preparation of rice with the ability to synthesize resveratrol and pterostilbene in seed endosperm.

[0018] The present invention also proposes a breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm, comprising the steps of transforming the recombinant vector into rice callus tissue, or infecting rice callus tissue with the recombinant host cells, and then cultivating rice plants.

[0019] Furthermore, the recombinant vector was transformed into rice callus tissue using Agrobacterium-mediated transformation.

[0020] The present invention also proposes a method for preparing rice with high resveratrol and pterostilbene content, comprising the steps of obtaining rice seeds by planting rice, and then obtaining the rice by dehulling.

[0021] The rice was cultivated using the breeding method described above.

[0022] Furthermore, the resveratrol content of the rice is 40-82 ug / g based on the weight of the rice.

[0023] Furthermore, based on the weight of the rice, the pterostilbene content of the rice is 2~5ug / g.

[0024] The beneficial effects of this invention are as follows:

[0025] The recombinant vector of this invention comprises three gene expression cassettes for specific structural proteins and one gene expression cassette for specific transcription factor proteins. Through a specific combination of these recombinant vectors, resveratrol and pterostilbene were successfully synthesized in the endosperm of rice seeds. Rice cultivated using this technology exhibits high levels of resveratrol and pterostilbene, which not only enhances the nutritional value of rice but also serves as a raw material for the production of these two substances. Furthermore, this invention provides a feasible technical pathway for the synthesis of key substances with complex metabolic pathways in crop seed endosperm; it also offers valuable insights for the breeding of novel functional crops. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A diagram illustrating the synthetic pathways for reconstructing resveratrol and pterostilbene in rice endosperm;

[0028] Figure 2 A schematic diagram of the construction of the SR recombinant vector;

[0029] Figure 3 Image showing the PCR identification results of SR transgenic rice;

[0030] The sizes of the Marker (M) stripes used in the figure, from top to bottom, are 2KB, 1KB, 0.75KB, 0.5KB, 0.25KB, and 0.1KB;

[0031] Figure 4 The image shows the results of gene expression level detection in SR transgenic rice.

[0032] Figure 5 Chromatograms for the detection of resveratrol and pterostilbene in the endosperm of SR transgenic rice seeds by HPLC-MS;

[0033] Figure 6 A secondary fragment control diagram of resveratrol standard and SR transgenic rice sample;

[0034] Figure 7 Secondary fragment control diagram of Pterostilbene standard and SR transgenic rice sample.

[0035] Figure 8 This is a standard curve of resveratrol.

[0036] Figure 9 For the standard curve of Pterocarya strychnifolia;

[0037] Figure 10 Figure showing the quantitative results of resveratrol in the endosperm of SR transgenic rice seeds;

[0038] Figure 11 Figure showing the quantitative results of pterostilbene in the endosperm of SR transgenic rice seeds;

[0039] Figure 12 SR transgenic rice ABTS + Figure showing the results of a free radical scavenging experiment;

[0040] Figure 13 The figure shows the results of the DPPH free radical scavenging experiment in SR transgenic rice. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Resveratrol and pterostilbene are natural stilbene compounds with multiple effects such as preventing and treating cardiovascular diseases, anti-oxidation, anti-inflammation, and delaying aging. If these two substances can be accumulated in the endosperm of rice seeds, it can not only improve the nutritional value of rice, but also provide a new way for large-scale, low-cost production of stilbene compounds, which will help promote the development of related industries.

[0047] Traditional breeding relies on existing intraspecific genetic variation, aggregating superior alleles through hybridization and selection. However, it cannot introduce novel metabolic pathways and key genes not present in the species itself. The rice genome lacks key enzyme genes for stilbene synthesis pathways, such as stilbene synthase and O-methyltransferase genes. This results in its phenylpropanoid metabolic pathway primarily targeting flavonoid synthesis, lacking the genetic basis for shifting substrate metabolism towards stilbene branches. Therefore, traditional breeding methods alone cannot endow rice with the ability to synthesize these exogenous bioactive components.

[0048] Based on this, the present invention obtained rice whose seed endosperm can synthesize resveratrol and pterostilbene by introducing a gene combination containing three genes expressing structural proteins and one gene expressing a transcription factor into rice, and named this rice material astragalus crystal rice ( Stilbene Rice (abbreviated as SR). Among them, two genes expressing structural proteins are from grapes ( Vitis vinifera The gene expressing the stilbene synthase (VvSTS) and resveratrol O-methyltransferase (VvROMT) was found in Arabidopsis thaliana. One gene expressing a structural protein was derived from Arabidopsis thaliana. Arabidopsis thaliana The study included coumaric acid from rice, a coenzyme A ligase gene (At4CL), and an endogenous transcription factor gene from rice (OsMYBP1). The specific steps involved fusing these genes with the rice seed promoter to construct multiple gene expression cassettes, ultimately obtaining a recombinant vector containing the genes VvSTS, VvROMT, At4CL, and OsMYBP1. This recombinant vector was then converted into breeding crops to obtain rice with seed endosperm rich in resveratrol and pterostilbene. The synthetic pathways of resveratrol and pterostilbene are described in [link to relevant documentation]. Figure 1 Metabolite detection was performed on the obtained rice seeds, and resveratrol and pterostilbene were detected in the polished rice. Quantitative analysis determined that the contents of resveratrol and pterostilbene were 40-82 ug / g and 2-5 ug / g, respectively.

[0049] The information regarding the protein, its encoding gene, and its promoter involved in this invention is as follows:

[0050] The nucleotide sequence of the gene encoding the VvSTS protein is shown in SEQ ID NO.3, and the amino acid sequence of the VvSTS protein is shown in SEQ ID NO.4.

[0051] The nucleotide sequence of the gene encoding the VvROMT protein is shown in SEQ ID NO.5, and the amino acid sequence of the VvROMT protein is shown in SEQ ID NO.6;

[0052] The nucleotide sequence of the gene encoding the At4CL protein is shown in SEQ ID NO.1, and the amino acid sequence of the At4CL protein is shown in SEQ ID NO.2;

[0053] The nucleotide sequence of the gene encoding the transcription factor OsMYBP1 protein is shown in SEQ ID NO.7.

[0054] The nucleotide sequence of the rice endosperm storage protein gene promoter P10 (hereinafter referred to as promoter P10) is shown in SEQ ID NO. 9; the nucleotide sequence of the rice endosperm storage protein gene promoter PgluB5 (hereinafter referred to as promoter PgluB5) is shown in SEQ ID NO. 10; the nucleotide sequence of the rice endosperm storage protein gene promoter P26 (hereinafter referred to as promoter P26) is shown in SEQ ID NO. 11; and the nucleotide sequence of the rice endosperm storage protein gene promoter P16 (hereinafter referred to as promoter P16) is shown in SEQ ID NO. 12.

[0055] All terminators used in this invention were purchased from the Addgene website (https: / / www.addgene.org / ), and all other substances are commercially available products unless otherwise specified.

[0056] The primer sequences involved in the following examples are shown in Table 1.

[0057] Table 1 Primer Sequences

[0058]

[0059] Example 1 Construction of recombinant vector

[0060] In this invention, OsMYBP1, At4CL, VvSTS, and VvROMT are respectively constructed into gene expression cassette vectors driven by rice seed endosperm-specific promoters. The specific steps are as follows:

[0061] (1) Cloning of four essential genes (including three structural genes At4CL, VvSTS and VvROMT and one transcription factor OsMYBP1) and rice seed endosperm expression promoters.

[0062] The promoter pGluB5 was amplified using primer pair pGluB5-F / R with rice ZH11 gDNA as a template; the promoter P16 was amplified using primer pair P16-F / R with rice ZH11 gDNA as a template; the promoter P10 was amplified using primer pair P10-F / R with rice ZH11 gDNA as a template; and the promoter P26 was amplified using primer pair P26-F / R with rice ZH11 gDNA as a template.

[0063] The At4CL gene was amplified using Arabidopsis cDNA as a template using primer pair At4CL-F / R; the VvSTS gene was amplified using grape cDNA as a template using primer pair VvROMT-F / R; the VvROMT gene was amplified using grape cDNA as a template using primer pair VvROMT-F / R; and the OsMYBP1 gene was amplified using rice ZH11 cDNA as a template using primer pair OsMYBP1-F / R.

[0064] The specific amplification method described above can be any method well known to those skilled in the art, and this invention is not particularly limited thereto. Therefore, this invention need not describe the specific amplification method in detail.

[0065] (2) Constructing gene expression cassettes

[0066] In this invention, promoters PgluB5, P16, P10, and P26 are first constructed into the pUPD2 vector to obtain primary expression elements pUPD2-promotors. Then, genes At4CL, VvSTS, VvROMT, and OsMYBP1 are constructed into primary expression elements pUPD2-genes. Finally, terminators Tnos, T35, Tact2, and TAtubq3 are constructed into primary expression elements pUPD2-terminator. Next-generation sequencing analysis is used to ensure sequence correctness. Subsequently, using the GoldenBraid vector assembly principle (e.g., Sarrion-Perdigones, Alejandro, et al. "GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules." PLOS ONE, vol.6, no.7, 2011, p. e21622.), the primary elements pUPD2-promotors, pUPD2-genes, and pUPD2-ternimators were further combined. The following gene expression cassettes were ultimately constructed:

[0067] The gene expression cassette P26::At4CL::T35 for At4CL protein expression contains the promoter P26, the gene encoding the At4CL protein, and the terminator T35.

[0068] The gene expression cassette P10::VvSTS::Tnos for expressing VvSTS protein contains the promoter P10, the gene encoding the VvSTS protein, and the Tact2 terminator.

[0069] The gene expression cassette Pglub5::VvROMT::TAtubq3 for expressing VvROMT protein contains the promoter Pglub5, the gene encoding the VvROMT protein, and the terminator TAtubq3.

[0070] The gene expression cassette P16::OsMYBP1::Tnos for expressing the transcription factor OsMYBP1 protein contains the promoter P16, the gene encoding the transcription factor OsMYBP1 protein, and the Tnos terminator.

[0071] In addition, an expression cassette, Pnos::Hyg::Tnos, was constructed with the promoter Pnos, the selection marker gene Hyg (NCBI accession number AUO29335.1), and the terminator Tnos, for screening transgenic positive materials. The nucleotide sequence of Pnos is shown in SEQ ID NO.13.

[0072] (3) Assembling SR recombinant vectors for the specific synthesis of resveratrol and pterostilbene from rice seed endosperm

[0073] Construction of SR recombinant vectors: such as Figure 2 The diagram shows the construction of the SR recombinant vector. The gene expression cassette P26::At4CL::T35 expressing At4CL protein and the gene expression cassette P10::VvSTS::Tnos expressing VvSTS protein were combined into the same vector and sequenced to obtain intermediate vector 1. This intermediate vector 2 was then combined with the gene expression cassette Pnos::Hyg::Tnos and the gene expression cassette P16::OsMYBP1::Tnos expressing the transcription factor OsMYBP1 protein. Finally, this intermediate vector 2 was combined with the gene expression cassette Pglub5::VvROMT::TAtubq3 expressing VvROMT protein and sequenced to obtain the SR recombinant vector.

[0074] Example 2: Transformation and Detection of SR Recombinant Vectors in Rice

[0075] Genetic transformation of rice: Mature seeds of rice variety Zhonghua 11 (ZH11) were induced to form callus under dark conditions at 28℃ (method reference "Nishimura et al. 2006, A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc., 1:2796-2802"). The SR recombinant vector was transformed into Agrobacterium EHA105, streaked, and Agrobacterium EHA105 was added to a suspension medium containing 100 μmol / L acetylsyleugenol and 50% glucose and cultured until the OD value reached 0.4. Fresh, light yellow, and vigorously growing granular embryogenic callus was selected, and the bacterial solution was poured into the callus to ensure full infiltration. After the bacterial suspension on the surface of the callus was blotted dry with sterile filter paper, it was transferred to a co-culture medium and cultured for 2 days. Then, it was transferred to a selection medium containing 50 mg / L hygromycin. After resistant callus grew, it was transferred to a differentiation medium to differentiate and produce seedlings, thus obtaining 14 SR transgenic rice plants (SR-1 to SR-14). The formulations of the co-culture medium, selection medium, and differentiation medium are shown in Tables 2, 3, and 4, respectively.

[0076] Table 2 Co-culture medium formulation

[0077]

[0078] Table 3 Screening medium formulation

[0079]

[0080] Table 4 Differentiation medium formulation

[0081]

[0082] Leaves of SR transgenic rice were collected, and DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, as follows: Fresh rice leaf tissue, 2-3 cm long, was placed in a 2 mL centrifuge tube containing 3 steel balls and ground into powder. The milling program was set to a frequency of 30 Hz and a time of 30 s. 800 μL of CTAB extraction buffer was added to the 2 mL centrifuge tube containing the rice leaf powder, and the mixture was shaken for 15 s. The tube was then placed at 65°C to allow CTAB to fully lyse the leaf tissue. The sample was mixed every 5 min, and this process was repeated 3 times. An equal volume of chloroform / isoamyl alcohol (chloroform to isoamyl alcohol volume ratio of 24:1) was added, and the mixture was shaken and allowed to stand until the DNA separated from the organic phase. Then, the mixture was centrifuged at high speed. The upper aqueous phase was aspirated into 500 μL of isopropanol, and the mixture was agitated. After about 10 min, the DNA precipitated to the bottom of the tube. The DNA precipitate was washed with a total of 600 μL of 75% ethanol in two separate applications. After discarding the supernatant, the tube was allowed to air dry until the DNA at the bottom became colorless and transparent. The DNA was then dissolved in sterile water and stored at -20°C for later use. Using the DNA extracted by the CTAB method as a template, the exogenous gene was detected by PCR amplification.

[0083] Identification of SR transgenic rice: Primers F1 and R1 were used to detect the At4CL protein coding gene, amplifying a 1810 bp fragment; primers F2 and R2 were used to detect the VvSTS protein coding gene, amplifying a 1294 bp fragment; primers F3 and R3 were used to detect the OsMYBP1 protein coding gene, amplifying a 480 bp fragment; and primers F4 and R4 were used to detect the VvROMT protein coding gene, amplifying a 1196 bp fragment. Twelve positive plants were identified, representing a positive rate of 86%. The identification results are as follows: Figure 3 As shown.

[0084] Example 3: Detection of gene expression levels in SR transgenic rice

[0085] Quantitative PCR was performed on SR transgenic rice samples that tested positive for DNA. The method was as follows: Seeds 15 days after flowering were used to analyze the expression levels of relevant genes introduced into the rice endosperm. Seeds were ground into powder under liquid nitrogen conditions, and seed RNA was extracted using a Trizol reagent kit, followed by reverse transcription to cDNA using a reverse transcription kit. RT-PCR was performed on endogenous and exogenous genes, including the encoding genes for VvSTS, VvROMT, At4CL, and OsMYBP1 proteins. The encoding gene for the rice endogenous Actin protein (detailed nucleotide sequence of which is shown in SEQ ID NO. 14) was used as an internal control, with ZH11 seeds used as a control. The detection primers are as follows: F5 and R5 are used to detect the encoding gene of At4CL protein; F6 and R6 are used to detect the encoding gene of VvSTS protein; F7 and R7 are used to detect the encoding gene of transcription factor OsMYBP1 protein; F8 and R8 are used to detect the encoding gene of VvROMT protein; and Actin-F and Actin-R are used to detect the encoding gene of rice endogenous Actin protein.

[0086] Test results as follows Figure 4 As shown, the results indicate that, compared with the wild-type control rice Zhonghua 11 (ZH11), the introduced exogenous genes were expressed in all families of SR transgenic rice. Compared with the wild type, the genes encoding At4CL, VvSTS, and VvROMT proteins achieved high-fold heterologous expression in SR transgenic rice lines 8 (SR-8) and 13 (SR-13), and the expression level of the transcription factor OsMYBP1 protein in these two families was approximately 4–12 times higher than that in the wild-type control.

[0087] Example 4: Detection of resveratrol and pterostilbene in the endosperm of SR transgenic rice seeds

[0088] To detect the accumulation of the target metabolites resveratrol and pterostilbene in the endosperm of mature SR transgenic rice seeds, polished rice grains and panicles of SR transgenic rice with four exogenous genes were ground into powder under liquid nitrogen treatment. 0.1 g of the powder was added to 70% methanol at a material-to-liquid ratio of 1 g:8000 mL. After mixing, the sample was vortexed for 1 min and placed on ice. After extraction, the sample was centrifuged. The supernatant was then filtered through a microporous membrane, and the filtered sample was stored in a liner-lined metabolic flask for subsequent HPLC-MS analysis. The metabolic level detection results showed that, compared with ZH11, resveratrol and pterostilbene significantly accumulated in the endosperm of SR transgenic rice seeds. This was confirmed by comparison with the secondary chromatograms of the standards. The detection results are as follows: Figures 5-7 As shown in the figure. The results indicate that the four exogenous genes introduced in this invention play an important role in the specific synthesis of resveratrol and pterostilbene in rice seed endosperm.

[0089] Example 5: Quantitative analysis of resveratrol and pterostilbene in the endosperm of SR transgenic rice seeds

[0090] By constructing standard curves for the target products resveratrol and pterostilbene, the endosperm of SR transgenic rice seeds was quantitatively analyzed. The specific procedure is as follows: Resveratrol (purity ≥9%) and pterostilbene (purity ≥98%) standards were accurately weighed and dissolved in methanol to a volume of 1 mg / mL. The stock solution was then serially diluted with methanol to obtain graded concentration standard working solutions, with concentrations increasing from 1 ppb, 5 ppb, 10 ppb, 50 ppb, 100 ppb, 500 ppb, and 1000 ppb (covering the expected concentration range of the samples). Finally, the standard curves for resveratrol and pterostilbene were obtained through calculation, as shown below. Figures 8-9 As shown. It was determined that the absolute contents of the target products resveratrol and pterostilbene were relatively high in the SR-8 family, at 82 ug / g and 5 ug / g, respectively, while the absolute contents of the target products were relatively low in the SR-13 family, at 40 ug / g and 2 ug / g, respectively. This indicates that rice with seed endosperm rich in resveratrol and pterostilbene was successfully prepared. Figures 10-11 As shown.

[0091] Example 6: Verification of the antioxidant effect of SR transgenic rice

[0092] (1) ABTS + Free radical scavenging experiment

[0093] Experimental Procedure: The ABTS free radical scavenging rate was tested using a Solarbio Science Kit (BC4770) according to the instructions in the kit's manual, to characterize the antioxidant capacity of SR transgenic rice seeds. Results are as follows: Figure 12 As shown, the results indicate that the radical scavenging rate of ZH11 (as a control) was 24.37%, the radical scavenging rate of the SR-8 family was 43.79%, and the radical scavenging rate of the SR-13 family was 39.82%.

[0094] (2) DPPH free radical scavenging experiment

[0095] Experimental Procedure: DPPH radical scavenging rate was tested using a Solarbio Science Kit (BC4755) according to the instructions to characterize the antioxidant capacity of SR transgenic rice seeds. Results are as follows: Figure 13As shown, the results indicate that in the DPPH radical scavenging experiment, the radical scavenging rate of the control ZH11 was approximately 11.16%, the radical scavenging rate of the SR-8 family was approximately 23.73%, and the radical scavenging rate of the SR-13 family was approximately 22.23%.

[0096] Both of the above antioxidant experiments consistently demonstrate that the SR transgenic rice of the present invention has strong antioxidant capacity.

[0097] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant vector, characterized in that, This includes gene expression cassettes expressing VvSTS protein, VvROMT protein, At4CL protein, and OsMYBP1 transcription factor protein. The gene expression cassette expressing the VvSTS protein includes the gene encoding the VvSTS protein, a P10 promoter, and a Tnos terminator. The nucleotide sequence of the gene encoding the VvSTS protein is shown in SEQ ID NO.

3. The gene expression cassette expressing VvROMT protein includes the gene encoding VvROMT protein, a PgluB5 promoter, and a TAtubq3 terminator. The nucleotide sequence of the gene encoding VvROMT protein is shown in SEQ ID NO.

5. The gene expression cassette expressing the At4CL protein includes the gene encoding the At4CL protein, a P26 promoter, and a T35 terminator. The nucleotide sequence of the gene encoding the At4CL protein is shown in SEQ ID NO.

1. The gene expression cassette for expressing the transcription factor OsMYBP1 protein includes the coding gene for the transcription factor OsMYBP1 protein, the P16 promoter, and the Tnos terminator. The nucleotide sequence of the coding gene for the transcription factor OsMYBP1 protein is shown in SEQ ID NO.

7.

2. A recombinant host cell, characterized in that, The recombinant vector according to claim 1 is wherein the host cell is Agrobacterium.

3. The use of a recombinant vector as described in claim 1 or a recombinant host cell as described in claim 2 in the preparation of rice with the ability to synthesize resveratrol and pterostilbene in seed endosperm.

4. A breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm, characterized in that, The method includes the steps of transforming the recombinant vector of claim 1 into rice callus tissue, or infecting rice callus tissue with the recombinant host cell of claim 2, and then cultivating it into rice plants.

5. The breeding method for synthesizing resveratrol and pterostilbene in rice seed endosperm according to claim 4, characterized in that, The recombinant vector was transformed into rice callus tissue using Agrobacterium-mediated transformation.

6. A method for preparing rice with high resveratrol and pterostilbene content, characterized in that, This includes the steps of obtaining rice seeds by planting rice, and then obtaining rice through dehulling. The rice is bred using the breeding method described in claim 4 or 5.

7. The method for preparing rice with high resveratrol and pterostilbene content according to claim 6, characterized in that, The resveratrol content of the rice is 40-82 ug / g based on the weight of the rice.

8. The method for preparing rice with high resveratrol and pterostilbene content according to claim 6, characterized in that, The pterostilbene content of the rice is 2-5 ug / g based on the weight of the rice.

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