Method for simultaneously increasing anthocyanin content and cadmium tolerance in rice

Abstract

The application provides a method for simultaneously improving anthocyanin content and cadmium tolerance of rice, and belongs to the technical field of plant genetic engineering. The method targets and edits a rice OsMYB108 gene (RAP-DB ID: Os09g0538400) by CRISPR / Cas9 technology, so that the function of the gene is lost, thereby obtaining improved rice plants with significantly increased anthocyanin content in grains and reduced cadmium accumulation in the aboveground part under cadmium stress. Experiments prove that knocking out the gene can increase the anthocyanin content in the grains of the mutant by more than 270%, and reduce the cadmium accumulation in the aboveground part by 26.58% under 50 muM cadmium stress. The application breaks the limitation of single trait improvement in traditional breeding, and provides an efficient gene editing strategy and a molecular marker detection means for cultivating new rice varieties with high nutrition and low cadmium safety.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for simultaneously increasing anthocyanin content and cadmium tolerance in rice. Background Technology

[0002] Anthocyanins are a class of water-soluble natural pigments widely found in plants. They not only give plant organs their red, purple, and blue colors, but also possess strong antioxidant activity, capable of scavenging free radicals in the human body, reducing inflammation, and preventing chronic diseases. Rice varieties rich in anthocyanins (such as black rice and purple rice) have attracted widespread attention from consumers and breeders due to their high nutritional value and unique market premium. However, the vast majority of mainstream rice varieties have light-colored caryopsis and extremely low anthocyanin content, making it difficult to meet the growing demand for healthy diets.

[0003] On the other hand, cadmium (Cd) is one of the most toxic heavy metal pollutants in my country's farmland soil. Rice, as a crop that easily accumulates cadmium, frequently transports cadmium from the soil to its grains. Once ingested through the food chain, cadmium can cause serious damage to the kidneys and bones. Therefore, reducing cadmium accumulation in rice grains is a crucial requirement for ensuring food security.

[0004] Currently, anthocyanin synthesis and cadmium stress response in plants are mainly regulated by transcription factors. However, no existing technology has reported a single transcription factor that can simultaneously negatively regulate both anthocyanin synthesis and cadmium tolerance. Existing rice improvement methods often focus on improving single traits, either solely on appearance quality (anthocyanin) or solely on heavy metal emission reduction, lacking breeding targets that can simultaneously achieve "high anthocyanin + low cadmium accumulation." Furthermore, traditional hybridization breeding has a long cycle, and transgenic technology poses biosafety risks.

[0005] Therefore, it is necessary to develop a method that can simultaneously improve the anthocyanin content and cadmium tolerance of rice. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for simultaneously improving the anthocyanin content and cadmium tolerance of rice.

[0007] To achieve the above objectives, the present invention is implemented as follows: In a first aspect of the present invention, a method for simultaneously improving anthocyanin content and cadmium tolerance in rice is provided, the method comprising: editing the rice OsMYB108 gene to cause it to lose its function, thereby obtaining improved rice plants with increased anthocyanin content and reduced cadmium accumulation in the aboveground parts and decreased growth inhibition rate under cadmium stress.

[0008] Furthermore, the editing employs CRISPR / Cas9 technology and targets the first exon of the OsMYB108 gene; the sgRNA target sequence is selected from SEQ ID NO:3 or SEQ ID NO:4.

[0009] Furthermore, the method also includes genotyping the edited plants: extracting the rice genomic DNA to be tested, performing PCR amplification using PCR primers to amplify the target region of the OsMYB108 gene, sequencing the amplification products, and screening homozygous plants with frameshift mutations caused by base deletions or insertions in the target site region.

[0010] Furthermore, the method also includes a step of genotyping using molecular markers; the molecular markers are the InDel variant sites in the target region of the OsMYB108 gene, which are amplified and identified using primer pairs SEQ ID NO:9-SEQ ID NO:10.

[0011] In a second aspect of the invention, a product is provided that simultaneously improves anthocyanin content and cadmium tolerance in rice, the product comprising a knockout reagent for the rice OsMYB108 gene.

[0012] Furthermore, the knockout reagent includes the Cas9 nuclease in the CRISPR / Cas9 gene editing system or an expression vector encoding Cas9, and an sgRNA targeting the first exon of the OsMYB108 gene, wherein the target sequence of the sgRNA is selected from SEQ ID NO:3 or SEQ ID NO:4.

[0013] Furthermore, the knockout reagent also includes oligonucleotide chains selected from: Oligonucleotide pairs targeting the target shown in SEQ ID NO: 3: as shown in SEQ ID NO: 5 - SEQ ID NO: 6; Oligonucleotide pairs targeting the target shown in SEQ ID NO: 4: as shown in SEQ ID NO: 7 - SEQ ID NO: 8.

[0014] Furthermore, the product also includes a PCR primer pair for genotyping, the sequences of which are SEQ ID NO: 9 and SEQ ID NO: 10.

[0015] In a third aspect of the invention, an application of the rice OsMYB108 gene or its encoded protein is provided, said application being selected from any of the following: (1) Regulating anthocyanin synthesis in rice; (2) Regulating cadmium tolerance in rice; (3) Screen rice germplasm with high anthocyanin content and / or low cadmium accumulation.

[0016] Furthermore, the amino acid sequence of the OsMYB108 protein is shown in SEQ ID NO:1. The OsMYB108 CDS nucleotide sequence is shown in SEQ ID NO:2.

[0017] Furthermore, the regulation of rice anthocyanin synthesis includes: reducing the expression of the OsMYB108 gene to increase the anthocyanin content in rice grains or leaves.

[0018] Furthermore, the regulation of cadmium tolerance in rice includes: reducing the expression of the OsMYB108 gene can reduce the translocation of cadmium to the aboveground parts of rice, reduce cadmium accumulation in the aboveground parts, and improve growth vigor under cadmium stress.

[0019] Furthermore, the screening targets loss-of-function mutants of the OsMYB108 gene, or uses molecular markers developed based on the InDel variant site in the target region of the OsMYB108 gene for genotyping.

[0020] The beneficial effects of this invention are: 1. This study reveals for the first time the dual function of the OsMYB108 gene in simultaneously negatively regulating anthocyanin synthesis and cadmium tolerance, providing a novel target gene for the synergistic improvement of rice quality and stress resistance.

[0021] No prior art has reported that a single transcription factor can simultaneously inhibit anthocyanin accumulation and reduce cadmium tolerance. This invention, through constructing a CRISPR / Cas9 knockout mutant and systematically analyzing its phenotype, demonstrates that knocking out OsMYB108 can simultaneously and significantly increase anthocyanin content in caryopsis and enhance cadmium tolerance in seedlings. Using this gene as a molecular breeding target allows for a highly efficient breeding strategy of "one-time editing, dual-trait enhancement," avoiding the cumbersome operation of multi-gene aggregation targeting anthocyanins and cadmium tolerance separately, significantly shortening the breeding cycle and reducing breeding costs.

[0022] 2. Knocking out the OsMYB108 gene can simultaneously increase anthocyanin content and cadmium tolerance, thereby improving the nutritional quality and food safety of rice, resulting in excellent overall performance.

[0023] Anthocyanins are powerful natural antioxidants; increasing their content can enhance the nutritional value and market value of rice. Simultaneously, reducing cadmium accumulation in the aboveground parts (especially the grains) helps ensure food safety and reduce the harm of heavy metals to human health. The OsMYB108 knockout mutant obtained in this invention exhibits a significantly deeper purple color in its grains (anthocyanin enrichment) under normal growth conditions without any exogenous treatment. Furthermore, after 10 days of 50 μM cadmium stress, the cadmium content in the aboveground parts is significantly reduced by approximately 27% compared to the wild type, and the relative inhibition rates of root length and plant height are significantly decreased, demonstrating stronger cadmium tolerance. This combined superior trait of "high anthocyanin + low cadmium accumulation" gives rice the dual advantages of improved nutritional quality and safe control of heavy metals, exhibiting significant performance superiority over existing single-trait improvement technologies.

[0024] 3. The molecular mechanism by which OsMYB108 negatively regulates anthocyanin synthesis was clarified—directly binding to the OsF3'H promoter and inhibiting its transcription, providing a reliable detection target for molecular marker-assisted breeding.

[0025] This invention, through molecular biology experiments, confirms that OsMYB108, as a transcriptional repressor, directly binds to the promoter region of OsF3'H, a key structural gene for anthocyanin synthesis, thereby negatively regulating anthocyanin accumulation. Based on this mechanism, molecular markers (such as SNP markers or Indel markers) targeting the OsMYB108 gene itself or its downstream OsF3'H promoter-binding region can be developed for large-scale screening or assisted breeding of rice germplasm resources with high anthocyanin content and strong cadmium tolerance. This marker-assisted selection based on a clearly defined mechanism is faster and more accurate than traditional phenotypic selection, significantly improving breeding efficiency and reducing the workload and cost of field phenotypic identification.

[0026] 4. The technical solution is simple and easy to implement, has good reproducibility, and is applicable to the genetic improvement of various rice varieties.

[0027] The CRISPR / Cas9 vector construction, Agrobacterium-mediated transformation, homozygous mutant screening, and hydroponic identification of cadmium tolerance employed in this invention are all mature and standardized operating procedures with clear steps and well-defined conditions, making them easy to replicate and promote in different rice varieties (especially colored rice such as purple rice). No expensive equipment or complex reagents are required, production costs are controllable, and it is suitable for laboratory research and large-scale application by seed companies. Furthermore, the hydroponic method for cadmium stress treatment ensures uniform conditions, high reproducibility, and stable quality of the screened cadmium-tolerant materials.

[0028] In summary, this invention, by exploring the dual negative regulatory function of the OsMYB108 gene and combining it with gene editing technology for targeted knockout, simultaneously improves the anthocyanin content and cadmium tolerance of rice in a low-cost and highly safe manner. This breakthrough overcomes the limitations of traditional breeding and transgenic technologies and has significant innovative and practical value. Attached Figure Description

[0029] Figure 1 Subcellular localization results of OsMYB108 protein.

[0030] Figure 2 : Schematic diagram of target editing patterns of OsMYB108 knockout mutants (KO-1, KO-2). In the diagram, A represents the wild-type sequence, B represents a 1bp deletion in KO-1, and C represents a 40bp deletion in KO-2.

[0031] Figure 3 Comparison of caryopsis phenotype and anthocyanin content between wild-type and mutant. In the figure, A is the caryopsis phenotype, B is the qPCR result of OsMYB108 gene expression, and C is the bar chart of anthocyanin content.

[0032] Figure 4 Dual-luciferase assays were used to verify the inhibitory effect of OsMYB108 on the OsF3'H promoter. In the diagram, A shows the effector and reporter vectors, B shows an in vivo image of luciferase in tobacco leaves, and C shows the relative luciferase activity (Luc / Ren ratio) of different promoters.

[0033] Figure 5 Comparison of leaf phenotypes and anthocyanin content between wild-type and mutant strains under cadmium stress. In this study, A represents the leaf phenotype 10 days after stress, and B represents the statistical analysis of leaf anthocyanin content.

[0034] Figure 6 Comparison of relative inhibition rates of root length and plant height under cadmium stress. Where A represents the root length inhibition rate and B represents the plant height inhibition rate.

[0035] Figure 7 Comparison of cadmium content in roots and aboveground parts after cadmium stress.

[0036] Figure 8 Comparison of cadmium transport coefficients after cadmium stress. Detailed Implementation

[0037] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0038] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0039] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0040] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the overall approach includes the following three aspects: 1. Target discovery and mechanism reconstruction Existing technologies primarily focus on the functions of OsMYB108 in lignin synthesis or disease resistance, without ever associating it with anthocyanins and cadmium stress. This application, through in-depth research, reveals that OsMYB108 not only directly binds to the promoter of the key anthocyanin synthesis gene OsF3'H and inhibits its transcription (leading to reduced anthocyanin accumulation), but also promotes cadmium transport from the roots to the aboveground parts (resulting in poor cadmium tolerance). Based on the revelation of this dual negative regulatory mechanism, the technical principle of "knocking out this gene can simultaneously relieve anthocyanin inhibition and block cadmium transport" is established.

[0041] 2. Technical means and precise editing Based on the above mechanism, this application abandons traditional overexpression or hybridization methods and instead employs CRISPR / Cas9 gene editing technology to precisely target OsMYB108. By designing specific sgRNAs targeting the first exon (such as SEQ ID NO:3 or 4), frameshift mutations are induced at the genomic level, resulting in the complete loss of function of the OsMYB108 protein (SEQ ID NO:1). This "subtraction" strategy not only achieves the removal of endogenous negative regulators but also avoids the introduction of exogenous genes, thus improving biosafety.

[0042] 3. Supporting testing and efficient screening To address the challenge of mutant screening, this application further constructs a molecular marker system based on InDel variants in the target region. Using specific primer pairs (SEQ ID NO: 9-6) for PCR amplification and sequencing, homozygous plants exhibiting loss of function can be rapidly and accurately identified at the seedling stage. This approach transforms the previously lengthy screening process, which relied on phenotypes (color, cadmium content), into a highly efficient genotype-based detection process, significantly shortening the breeding cycle.

[0043] In summary, this application, through the overall technical route of "discovering new targets - utilizing new mechanisms - developing new methods", successfully solved the industry problem of the difficulty in achieving both "high anthocyanin" and "low cadmium accumulation", and provided a practical and feasible technical solution for the variety improvement of functional rice.

[0044] The present invention will now be described in detail with reference to the embodiments.

[0045] Unless otherwise specified, the techniques used are conventional techniques well known to those skilled in the art; the experimental methods used are all conventional methods and can be performed according to the described recombinant techniques (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); and the materials and reagents used are all commercially available.

[0046] Example 1: Construction and Identification of OsMYB108 Homozygous Mutant I. Experimental Objective: To construct an OsMYB108 loss-of-function mutant using CRISPR / Cas9 technology and to identify its genotype.

[0047] II. Experimental Methods 1. Cloning and sequence characteristics of the OsMYB108 gene Total RNA was extracted from the leaves of seedlings of the purple rice variety 'Dian Zi Nuo' and cDNA was obtained by reverse transcription. Based on the coding sequence of the OsMYB108 gene (gene ID: Os09g0538400) in the Rice Annotation Project Database (RAP-DB), specific primers were designed for PCR amplification to obtain a 756 bp CDS region.

[0048] This gene encodes an R2R3-MYB transcription factor, whose nucleotide sequence is as follows: ATGGGGAGGTCACCGTGCTGCGAGAAGGCACACACCAACAAGGGAGCATGGACCAAGGAGGAAGATGACCGGCTCATTGCCTACATCAAGGCGCACGGCGAAGGTTGCTGGCGATCGCTGCCCAAGGCCGCCGGCCTCCTCcgctgtggcaagagctgccgcctccggtggatcaactacctccggcctgacctcaagcgcggcaacttcaccgaggaggaggatgagctgatcatcaagcttcacagccttttaggcaacaaatggtctctgatagccgggaggttgccaggaagaacggacaacgagatcaagaactactggaacacgcacATCAGGAGGAAGCTGCTGAGCCGTGGCATCGACCCGGTGACACACCGGCCGATCAACGACAGCGCGTCCAACATCACCATATCATTCGAGGCGGCCGCGGCGGCGGCGAGGGACGACAAGGCCGCCGTGTTCCGGCGAGAGGACCATCCTCATCAGCCGAAGGCGGTGACAGTGGCACAGGAGCAGCAGGCAGCCGCCGATTGGGGCCATGGGAAGCCACTCAAGTGCCCTGACCTCAATCTGGACCTCTGCATCAGCCTCCCTTCCCAAGAAGAGCCCATGATGATGAAGCCGGTGAAGAGGGAGACCGGCGTCTGCTTCAGCTGCAGCCTGGGGCTCCCCAAGAGCACAGACTGCAAGTGCAGCAGCTTCCTGGGACTCAGGACAGCCATGCTCGACTTCAGAAGCTTGGAAATGAAATGA (SEQ ID NO: 2).

[0049] 2. Subcellular localization of OsMYB108 The cDNA sequence of OsMYB108 (without a stop codon) was obtained by PCR amplification. The plant expression vector pSuper1300-GFP was linearized by double digestion with HindIII and SalI. OsMYB108 was then inserted into the vector using homologous recombinase and fused with GFP to obtain the recombinant plasmid pSuper1300-OsMYB108-GFP. Rice protoplasts were extracted and transformed with polyethylene glycol (PEG). The GFP fluorescence signal was observed under a laser confocal microscope after transformation to determine the subcellular localization of OsMYB108.

[0050] The amino acid sequence encoded by transcription factor OsMYB108 is as follows: MGRSPCCEKAHTNKGAWTKEEDDRLIAYIKAHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLLSRGIDPVTHRP INDSASNITISFEAAAAAARDDKAAVFRREDHPHQPKAVTVAQEQQAAADWGHGKPLKCPDLNLDLCISLPSQEEPMMMKPVKRETGVCFSCSLGLPKSTDCKCSSFLGLRTAMLDFRSLEMK* (SEQ ID NO: 1) This protein contains conserved R2 and R3 MYB domains and belongs to the R2R3-MYB transcription factor family.

[0051] 2. Construction and screening of OsMYB108 gene knockout mutants (1) Target design and synthesis Based on the sequence characteristics of the first exon of the OsMYB108 gene in the rice genome annotation database (RAP-DB ID: Os09g0538400), two specific sgRNA targets were designed to construct a dual-target knockout vector, improving knockout efficiency and preventing off-target effects from single targets. The target sequences are as follows: Target site 1: CAAGGGAGCATGGACCAAGGAGG (SEQ ID NO: 3) Target site 2: CCGGCTCATTGCCTACATCAAGG (SEQ ID NO: 4) (2) Construction of CRISPR / Cas9 gene editing vector Synthesize oligonucleotide chains targeting the above-mentioned target sites, wherein, The Oligo pairs targeting site 1 are: F: 5′-GGCCAAGGGAGCATGGACCAAGGA-3′ (SEQ ID NO: 5); R: 5′-AAACTCCTTGGTCCATGCTCCCTTG-3′ (SEQ ID NO: 6); The Oligo pairs targeting site 2 are: F: 5′-GGCCCGGCTCATTGCCTACATCAA-3′ (SEQ ID NO: 7); R: 5′-AAACTTGATGTAGGCAATGAGCCGG-3′ (SEQ ID NO: 8); After annealing to form double-stranded DNA, it was ligated into the pYLCRISPR / Cas9Pubi-H binary expression vector (purchased from Miaoling Plasmid Platform, catalog number P49045) via the BsaI restriction site. This vector contains a Cas9 expression cassette and two sgRNA expression cassettes, enabling simultaneous cleavage at two sites in the rice genome. The constructed recombinant vector was verified by sequencing and then used for subsequent genetic transformation.

[0052] (3) Genetic transformation and regeneration The callus tissue of the purple rice variety 'Dian Zi Nuo' was transformed using Agrobacterium tumefaciens-mediated transformation. The specific procedures were as follows: Agrobacterium strain EHA105 containing the recombinant vector was activated in an antibiotic-containing medium to infect the callus tissue. After co-culturing for 2-3 days, the tissue was transferred to a differentiation medium containing a selection agent (hygromycin) for plant regeneration. When the regenerated plants reached 5-10 cm in length, they were transplanted to a greenhouse for further cultivation, and T0 generation seeds were harvested.

[0053] (4) Genotyping and screening of homozygous mutants Genomic DNA was extracted from the young leaves of T0 generation plants and amplified using specific primers (F: 5'-CAGCAGCAGCAGCAACTTCC-3' (SEQ ID NO:9)). R: 5'-CGGCTATCAGAGACCATCTGCATC-3' (SEQ ID NO:10) was used to amplify the target region by PCR. The PCR product was purified and sent for sequencing (Chengdu Sangon Biotech). The sequencing peak diagram was analyzed using Chromas software to screen for mutant plants with base deletions or insertions (Indels) in both target regions.

[0054] The selected heterozygous mutants were harvested and propagated through crossbreeding up to the T1 generation. Genotyping was then performed using the primers described above to screen for homozygous mutants with frameshift mutations at the target locus that caused premature termination of protein translation. The T2 generation was then planted and phenotypically identified, ultimately yielding stable homozygous OsMYB108 loss-of-function mutants (KO-1 and KO-2).

[0055] III. Experimental Results Location results as follows Figure 1 The OsMYB108 gene is located in the cell nucleus and has transcriptional regulatory activity.

[0056] Sequencing results showed that... Figure 2 As shown, two homozygous mutants, KO-1 and KO-2, were obtained. KO-1 had a 1-base deletion in the target region, while KO-2 had a 40-base deletion. Both resulted in a frameshift and premature appearance of the stop codon, leading to complete loss of function of the OsMYB108 protein.

[0057] Example 2: Verification of the regulatory function of anthocyanin synthesis I. Experimental Objective To verify the effect of OsMYB108 knockout on anthocyanin content in rice caryopsis and to elucidate its molecular mechanism.

[0058] II. Experimental Methods 1. OsMYB108 gene expression analysis The caryopsis fruits of WT, KO-1, and KO-2 were ground into powder using liquid nitrogen, and total RNA was extracted using the Takara Total RNA Extraction Kit (9108), following the instructions in the manufacturer's manual. Then, cDNA was synthesized using the Takara PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A), following the manufacturer's manual.

[0059] The primer sequences for OsMYB108 quantitative qPCR were: F: 5'-CAGTGGCACAGGAGCAGCAG-3' (SEQ ID NO: 11); R: 5'-GCAGAGGTCCAGATTGAGGTCAG-3' (SEQ ID NO: 12). OsGAPDH was used as an internal reference gene, and its primer sequences were: F: 5'-GCCTCCTCCTTGATTGC-3' (SEQ ID NO: 13); R: 5'-CTTTCCGTGTTCCCACA-3' (SEQ ID NO: 14). The reaction system and qPCR program of Takara TB Green® Premix Ex Taq™ II (RR820A) were followed, and the gene expression was analyzed using the 2–△△Ct value method.

[0060] 2. Anthocyanin content detection: Leaves or caryopsis of WT, KO-1, and KO-2 were placed in a mortar and ground into powder with liquid nitrogen. An appropriate amount of powder was weighed, and 1% hydrochloric acid solution (v / v) was added. Extraction was performed in a 70℃ water bath for 1 hour. The mixture was centrifuged at 6000 r / min for 10 minutes, and the supernatant was measured at 525 nm (ABS). Anthocyanin content was calculated using the following formula: C = (1 / 958) × (v / 100) × (1 / m) × ABS × 100000 Where: C is the anthocyanin content (mg / 100g FW), v is the fixed volume (mL), m is the sample mass (g), and ABS is the absorbance at 525 nm.

[0061] III. Experimental Results 1. OsMYB108 gene expression was significantly downregulated. qPCR test results as follows Figure 3 As shown in B, compared with the wild type (WT), the transcriptional level of OsMYB108 in the two homozygous mutants KO-1 and KO-2 was significantly reduced, indicating that CRISPR / Cas9 editing effectively disrupted the function of this gene.

[0062] 2. Significantly increased phenotypic characteristics and anthocyanin content in caryopsis. like Figure 3 As shown in Figure A, under normal growth conditions, the caryopsis color of KO-1 and KO-2 is significantly darker than that of the wild type, appearing as a deep purple.

[0063] like Figure 3As shown in Figure C, the anthocyanin content determination data showed that the anthocyanin content of the wild-type caryopsis was 12.3±1.2 mg / 100g FW, while that of KO-1 was 48.7±3.5 mg / 100g FW, and that of KO-2 was 45.2±2.8 mg / 100g FW. The anthocyanin content of the mutant was more than 270% higher than that of the wild type, and the difference reached a highly significant level (P<0.01).

[0064] Example 3: Dual-luciferase reporter gene experiment I. Experimental Objective To verify whether the OsMYB108 protein can directly bind to the promoter of the key anthocyanin synthesis gene OsF3'H and inhibit its transcriptional activity.

[0065] II. Experimental Methods 1. Carrier Construction The CDS sequence of transcription factor OsMYB108 was recombined into the pGreenII 62-SK vector using homologous recombination. The primer sequences were F: 5'-GGCCGCTCTAGAACTAGTGGATCCATGGGGAGGTCACCGTG-3' (SEQ ID NO: 15); R: 5'-ATCGATAAGCTTGATATCGAATTCTTTCATTTCCAAGCTTCTGAAGTCG-3' (SEQ ID NO: 16). The promoter sequences approximately 2 kb upstream of the start codons of the anthocyanin synthesis structural genes OsDFR, OsANS, OsF3'H, and OsF3H were cloned into the pGreenII 0800-LUC vector to construct reporter vectors. The recombinant vectors and empty vectors were transformed into Agrobacterium GV3101 (pSoup) and cultured in the dark at 28°C for 2-3 days. Single colonies were picked and positive clones were identified by colony PCR.

[0066] 2. Benedict's injection Agrobacterium containing the recombinant plasmid was inoculated into REB liquid medium containing 50 mg / L rifampin and cultured overnight until OD500. 600 ≈1.0. Under aseptic conditions, take fresh bacterial culture into a 50 mL centrifuge tube, centrifuge at 4℃ and 8000 rpm for 10 min, and discard the supernatant. Resuspend the bacterial cells in osmotic medium (prepared with 10 mM MgCl2, 10 mM MES, 200 μM acetylsalicylic acid, ddH2O, pH=5.6), and adjust OD. 600 The solution was allowed to stand at room temperature in the dark for 3 hours until it reached 0.6. The bacterial solution was then injected into uniformly grown tobacco leaves using a needle-free disposable syringe.

[0067] 3. Luciferase imaging Prepare a 150 μg / mL D-fluorescein potassium salt working solution. Take out the tobacco plants that have been cultured in the dark for 2-3 days after injection, spray the diluted working solution evenly on the back of the leaves in the dark, let them stand in the dark for 10 minutes, and observe the images with a live imaging system.

[0068] 4. Luciferase activity Three days after injection of the sample, luminescence detection was performed using a Dual-Luciferase Repotter Assay Kit (Vazyme, China) and an EnVision microplate reader (PerkinElmer, USA). Samples were taken from the injection area using a punch and placed in 1.5 mL centrifuge tubes. After grinding in liquid nitrogen, 500 μL of 1× cell lysis buffer was added, vortexed, and allowed to stand for 5 minutes. The tubes were then centrifuged at 12000 rpm for 2 minutes, and the supernatant was transferred to a new tube. In a 96-well microplate, 100 μL of luciferase substrate and 20 μL of lysis supernatant were added, quickly mixed, and the firefly luciferase activity was measured. Subsequently, 100 μL of Renilla luciferase substrate working solution was added, quickly mixed, and the Renilla luciferase activity was measured.

[0069] III. Experimental Results like Figure 4 As shown, compared with the empty vector control group, the experimental groups co-transfected with pGreenII 62SK-OsMYB108 and pGreenII0800-proOsF3'H-LUC showed a significantly lower Luc / Ren ratio. Specifically, OsMYB108 inhibited the OsF3'H promoter by 72% (P<0.01).

[0070] Figure 4 C: Quantitative results of dual-luciferase activity. OsMYB108 significantly inhibited... OsF3'H Promoter activity (P<0.01), but for OsDFR, OsANS and OsF3H The promoter activity was not significantly affected.

[0071] This confirms that OsMYB108 is a specific transcriptional repressor that directly targets and binds to... OsF3'H The promoter region of the virus negatively regulates anthocyanin synthesis.

[0072] Example 4: Growth phenotype and physiological response under cadmium stress I. Experimental Objective To assess the effect of OsMYB108 knockout on cadmium tolerance in rice.

[0073] II. Experimental Methods Wild-type (WT) and OsMYB108 knockout mutant (KO-1) seedlings were hydroponically cultured using Kimura B nutrient solution. When the seedlings reached the three-leaf-one-heart stage, they were subjected to stress treatment with 0 μM (control) and 50 μM CdCl2, respectively, for 10 days. After treatment, leaf phenotypes were observed, and root length and plant height were measured for each treatment group to calculate the relative inhibition rate. Simultaneously, leaf samples were taken to determine anthocyanin content.

[0074] III. Experimental Results 1. Leaf phenotype and anthocyanin accumulation like Figure 5 As shown in Figure A, after 10 days of 50 μM Cd stress, the yellowing of leaves in the wild-type (WT) group was significantly more severe than that in the KO-1 group.

[0075] like Figure 5 As shown in B, cadmium stress induced the accumulation of anthocyanins in leaves. The anthocyanin content in wild-type leaves increased by 38.4%, while that in KO-1 increased by 78.2%. The increase in the mutant was significantly higher than that in the wild type, indicating that the mutant has stronger antioxidant capacity under stress.

[0076] 2. Growth inhibition rate analysis like Figure 6 As shown, under 50 μM Cd stress, the relative inhibition rates of root length and plant height in the wild type were 38.2% and 25.7%, respectively, while those in KO-1 were only 19.5% and 11.3%. The mutant showed significantly lower growth inhibition, demonstrating its significantly enhanced cadmium tolerance.

[0077] Example 5: Cadmium content detection and translocation coefficient analysis I. Experimental Objective Quantitative analysis of the effects of OsMYB108 knockout on cadmium accumulation and translocation capacity in different parts of the plant.

[0078] II. Experimental Methods Plants subjected to cadmium stress in Example 4 were used, and their aerial parts and roots were separated. The roots were soaked in a 15 mM Na2-EDTA solution for 60 min to remove surface-adsorbed ions, rinsed with water, blanched at 110℃ for 1 h, and then dried in a 70℃ oven to constant weight. The dried samples were pulverized, processed using microwave digestion, and the cadmium content was determined using ICP-MS. The cadmium translocation coefficient was calculated as follows: Translocation coefficient = Cadmium content in aerial parts / Cadmium content in roots.

[0079] III. Experimental Results 1. Cadmium content distribution like Figure 7As shown, there was no significant difference in cadmium content in the roots of WT and KO-1 (approximately 86 mg / kg), indicating that both have the same cadmium uptake capacity. However, in terms of aboveground cadmium content, the wild type had 12.8 ± 1.1 mg / kg, while KO-1 had 9.4 ± 0.8 mg / kg, with the mutant showing a 26.58% reduction in aboveground cadmium accumulation compared to the wild type.

[0080] 2. Transshipment coefficient like Figure 8 As shown, the cadmium translocation coefficient for the wild type was 0.148, while that for KO-1 decreased to 0.112. Knocking out OsMYB108 significantly blocked the translocation of cadmium from the roots to the aboveground parts.

[0081] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for simultaneously increasing anthocyanin content and cadmium tolerance in rice, characterized in that, The method includes: editing the rice OsMYB108 gene to cause it to lose its function, thereby obtaining improved rice plants with increased anthocyanin content and reduced cadmium accumulation in the aboveground parts and decreased growth inhibition rate under cadmium stress.

2. The method according to claim 1, characterized in that, The editing was performed using CRISPR / Cas9 technology, targeting the first exon of the OsMYB108 gene; the sgRNA target sequence was selected from SEQ ID NO:3 or SEQ ID NO:

4.

3. The method according to claim 1, characterized in that, The method also includes genotyping of the edited plants: extracting genomic DNA from the rice to be tested, performing PCR amplification using PCR primers to amplify the target region of the OsMYB108 gene, sequencing the amplification products, and screening homozygous plants with frameshift mutations caused by base deletions or insertions in the target site region.

4. The method according to claim 1, characterized in that, The method further includes a step of genotyping using molecular markers; the molecular markers are the InDel variant sites in the target region of the OsMYB108 gene, which are amplified and identified using primer pairs SEQ ID NO:9-SEQ ID NO:

10.

5. A product that simultaneously improves anthocyanin content and cadmium tolerance in rice, characterized in that, The product includes a knockout reagent for the rice OsMYB108 gene.

6. The product according to claim 5, which simultaneously improves anthocyanin content and cadmium tolerance in rice, is characterized in that... The knockout reagent includes the Cas9 nuclease in the CRISPR / Cas9 gene editing system or an expression vector encoding Cas9, and an sgRNA targeting the first exon of the OsMYB108 gene, wherein the target sequence of the sgRNA is selected from SEQ ID NO:3 or SEQ ID NO:

4.

7. The product according to claim 5, which simultaneously improves anthocyanin content and cadmium tolerance in rice, is characterized in that... The knockout reagent also includes oligonucleotide pairs selected from: Oligonucleotide pairs targeting the target shown in SEQ ID NO: 3: as shown in SEQ ID NO: 5 - SEQ ID NO: 6; Oligonucleotide pairs targeting the target shown in SEQ ID NO: 4: as shown in SEQ ID NO: 7 - SEQ ID NO:

8.

8. An application of a rice OsMYB108 gene or its encoded protein, characterized in that, The application is selected from any of the following: (1) Regulating anthocyanin synthesis in rice; (2) Regulating cadmium tolerance in rice; (3) Screen rice germplasm with high anthocyanin content and / or low cadmium accumulation.

9. The application according to claim 8, characterized in that, The amino acid sequence of the OsMYB108 protein is shown in SEQ ID NO:

1.

10. The application according to claim 8, characterized in that, The regulation of rice anthocyanin synthesis includes: reducing the expression of the OsMYB108 gene to increase the anthocyanin content in rice grains or leaves. The regulation of rice cadmium tolerance includes: reducing the expression of the OsMYB108 gene can reduce the translocation of cadmium to the aboveground parts of rice, reduce cadmium accumulation in the aboveground parts, and improve growth vigor under cadmium stress. The screening targets loss-of-function mutants of the OsMYB108 gene, or uses molecular markers developed based on the InDel variant site in the target region of the OsMYB108 gene for genotyping.

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