Application of SiMC1 gene in regulation and control of anabolism of millet carotenoid

Abstract

The invention discloses application of a SiMC1 gene in regulation and control of anabolism of millet carotenoid, and belongs to the technical field of genetic engineering breeding. According to the invention, the SiMC1 gene for regulating and controlling metabolism of carotenoid in millet is screened, and the nucleotide sequence of the SiMC1 gene is shown as SEQ ID NO. 1. Experimental results show that compared with a wild plant (WT), the millet yellowness and the carotenoid content of the SiMC1 gene knockout strain (KO) are remarkably increased, the SiMC1 gene negatively regulates synthesis of millet carotenoid, and the SiMC1 gene is a potential efficient millet breeding gene and can be used for improving the appearance character and the eating quality of millet. Gene modification targets and theoretical basis are provided for screening and cultivating high-quality millet varieties, and new millet germplasm which has both appearance and taste and is rich in natural antioxidant nutritional ingredients is expected to be obtained.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, and in particular to the application of the SiMC1 gene in regulating the biosynthesis and metabolism of carotenoids in millet. Background Technology

[0002] Millet (Setaria italica) is an important coarse grain crop in my country. Its hulled product, millet, is rich in carotenoids, giving it a bright yellow color. Millet quality is a comprehensive trait, including appearance quality, cooked taste quality, and nutritional quality. Millet appearance quality refers to its physical characteristics and is the most direct and primary selection indicator for consumers when evaluating millet quality. It is also an important direction for millet quality breeding. Among these, the color of the millet grain greatly influences consumer preference and is a key indicator for evaluating millet quality, as well as a major direction for millet quality breeding.

[0003] Carotenoids directly affect the beige color of millet. They are widely found in animals, plants, and microorganisms in nature and are a class of terpenoids composed of isoprene. Their polyene backbone contains a variable number of conjugated double bonds, exhibiting antioxidant activity. Carotenoids are a collective term for two major pigments: 40-carbon hydrocarbons (carotene) and their oxidation derivatives (lutein). They mainly include α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene.

[0004] The overall pathway of carotenoid biosynthesis in higher plants is relatively well understood. The entire process involves five reactions: condensation, dehydrogenation, cyclization, hydroxylation, and epoxidation, involving related enzyme genes such as PSY, PDS, ZDS, LCYE, LCYB, CHYb, CYP97, ZEP, CCD, and NCED. It is also a complex process involving the regulation of numerous transcription factors, and current research on the transcriptional regulatory mechanisms of carotenoid biosynthesis is still very limited. In recent years, many transcription factors involved in regulating the structural genes of plant carotenoid metabolism have been identified, including members of the bHLH, bZIP, MADS-box, NAC, MYB, and ERF families. However, these transcription factors mostly exhibit species-specific regulatory characteristics and lack universal regulatory patterns in the plant kingdom. Therefore, further research is needed to identify and clarify the key genes regulating carotenoid metabolism in millet. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the SiMC1 gene in regulating the biosynthesis and metabolism of millet carotenoids, in order to solve the problems existing in the prior art. This invention provides an application of the SiMC1 gene in regulating the color of millet grains, so as to increase the carotenoid content of millet grains and thus improve the quality of millet, thereby providing a molecular mechanism and theoretical basis for screening and breeding high-quality millet varieties.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an application of the SiMC1 protein in any of the following:

[0008] (1) Application in regulating the synthesis of carotenoids in millet;

[0009] (2) Application in increasing the carotenoid content of millet;

[0010] (3) Application in the cultivation of transgenic millet with increased carotenoid content;

[0011] (4) Application in regulating the beige color of millet;

[0012] The amino acid sequence of the SiMC1 protein is shown in SEQ ID NO.2.

[0013] Furthermore, the expression level of the SiMC1 protein was downregulated in millet, which promoted the synthesis of millet carotenoids, increased the carotenoid content of millet, and made the millet rice turn yellow.

[0014] The present invention also provides an application of the gene encoding the above-mentioned SiMC1 protein in any of the following:

[0015] (1) Application in regulating the synthesis of carotenoids in millet;

[0016] (2) Application in increasing the carotenoid content of millet;

[0017] (3) Application in the cultivation of transgenic millet with increased carotenoid content;

[0018] (4) Application in regulating the beige color of millet;

[0019] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0020] Furthermore, downregulating the expression level of the encoding gene in millet promotes the synthesis of millet carotenoids, increases the carotenoid content of millet, and turns the millet rice yellow.

[0021] The present invention also provides an application of a knockout vector that targets the gene encoding the SiMC1 protein;

[0022] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1;

[0023] The application is any one of the following:

[0024] (1) Application in regulating the synthesis of carotenoids in millet;

[0025] (2) Application in increasing the carotenoid content of millet;

[0026] (3) Application in the cultivation of transgenic millet with increased carotenoid content;

[0027] (4) Application in regulating the beige color of millet.

[0028] The present invention also provides the use of engineered bacteria comprising the above-described knockout vector in any of the following:

[0029] (1) Application in regulating the synthesis of carotenoids in millet;

[0030] (2) Application in increasing the carotenoid content of millet;

[0031] (3) Application in the cultivation of transgenic millet with increased carotenoid content;

[0032] (4) Application in regulating the beige color of millet.

[0033] The present invention also provides a method for promoting the synthesis of carotenoids in millet, comprising the step of downregulating the expression level of the gene encoding the SiMC1 protein in millet to promote the synthesis of said carotenoids in millet;

[0034] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0035] The present invention also provides a method for increasing the carotenoid content of millet, comprising the step of downregulating the expression level of the gene encoding SiMC1 protein in millet to increase the carotenoid content of the millet;

[0036] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0037] This invention also provides a breeding method for transgenic millet with increased carotenoid content, comprising the following steps:

[0038] The gene encoding the SiMC1 protein was knocked out in millet cells, the millet cells were then cultured, and millet plants were regenerated using the millet cells to obtain transgenic millet with increased carotenoid content.

[0039] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0040] The present invention also provides a method for turning millet rice yellow, comprising the step of downregulating the expression level of the gene encoding SiMC1 protein in millet to turn the millet rice yellow;

[0041] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0042] The present invention discloses the following technical effects:

[0043] This invention, through analysis of the millet beige phenotype, identified the SiMC1 gene as a key gene influencing its phenotype. Further analysis revealed that the SiMC1 gene is a repressor of SiPSY1, a key rate-limiting enzyme gene in carotenoid metabolism. SiMC1 knockout lines (KO) showed significantly increased beige color and carotenoid content. This indicates that SiMC1 directly affects millet beige color by inhibiting SiPSY1 gene expression and reducing carotenoid synthesis. Therefore, SiMC1 is a potential high-quality millet breeding gene that can be used to improve the appearance and eating quality of millet.

[0044] This invention clarifies the function of SiMC1 in the carotenoid metabolism pathway of millet. First, a genome-wide association study (GWAS) of a natural population was used to locate genes affecting the color of millet beige. Weighted co-expression network analysis, yeast one-hybrid analysis, and dual-luciferase reporter gene analysis revealed that SiMC1 regulates millet carotenoid metabolism by inhibiting the expression of the SiPSY1 gene. Through transgenic technology, the SiMC1 gene was edited (knocked out), confirming that SiMC1 is a negative regulator of millet beige color. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the carotenoid content in gene-knockout and wild-type millet. The results showed that knocking out the SiMC1 gene significantly increased the content of millet beige color, total carotenoids, lutein, zeaxanthin, and β-carotene. Using molecular biology and genetic breeding techniques, it is hoped that by regulating the endogenous expression level of the SiMC1 gene, new millet germplasm with improved appearance, taste, and rich in natural antioxidant nutrients can be obtained. Attached Figure Description

[0045] 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.

[0046] Figure 1 A genome-wide association analysis (GWAS) result for millet beige, a core germplasm resource of Chinese millet.

[0047] Figure 2 Figure showing the results of the co-expression network analysis of the SiMC1 gene and genes in the carotenoid metabolism pathway;

[0048] Figure 3The figure shows the results of yeast one-hybrid analysis of the promoters of the SiMC1 and SiPSY1 genes.

[0049] Figure 4 The image shows the results of dual-luciferase reporter gene analysis; where a represents the vector cassette and b represents the LUC / REN ratio.

[0050] Figure 5 Map of the SiMC1 gene knockout vector;

[0051] Figure 6 The image shows the PCR molecular detection results of the SiMC1 gene knockout positive lines; where M is the DNA Marker; WT is the wild-type Ci846 plant; and 1-23 are plants incorporating the knockout vector.

[0052] Figure 7 Figure 1 shows the analysis results of beige color, carotenoids, and major components of millet from SiMC1 gene knockout positive lines; where a represents the knockout sequence analysis; b represents the beige phenotype observation; and c represents b... * Value; d is the total carotenoid content; e is the lutein content; f is the zeaxanthin content; g is the β-carotene content. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Example 1

[0059] 1. SiMC1 gene localization

[0060] This invention uses 317 core resources of Chinese millet as materials, employs a non-contact colorimeter to statistically analyze the beige color of millet, combines existing resequencing results, and uses Tassel software to perform genome-wide association analysis, with the results expressed as log-log 10 (p) > 6 is the screening criterion, and candidate genes are screened in combination with linkage disequilibrium (about 50 kb).

[0061] like Figure 1 As shown, genome-wide association analysis results indicate that the yellowness value of millet beige is significantly associated with the chr6 region (34080216 bp - 35282842 bp, 1.14 Mb) on chromosome 6. The SiMC1 gene was among the 173 candidate genes identified within this region.

[0062] The nucleotide sequence of the SiMC1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the expressed SiMC1 protein is shown in SEQ ID NO.2.

[0063] SEQ ID NO.1:

[0064] ATGAGGTACATTCAGGAGCTTGAGCACAAGGTGCAGGTATTGCAGACGGAGGCTACGACACTCTCAGCACAGTTGACAATGCTGCAGAGGGACTCAGCTGGACTGGCCACTCAGAACAATGAGTTGAAAATCAGGCTGCAAGCAATGGAACAACAAGCACAGCTGAGAGATGCCCTGAATGAAGCGTTAACCGGCGAGGT CCAGCGTCTGAAGCTCGCCACTGGTGAGATAAGCGATGGCCGCATGCCAAAGAGCCTACAGCAGCAGATGAACTCCCAGATGCTCCAGCTCCAGCAGCTGCAAATACAGCAGCAGCAGCAGCAGCAGGCTCCGCAGCAGCAGCAGCAGCAGCACCAGCACCAGCAACAGCACCAGCAGCAGCCTCAGAAATCGGCATAG.

[0065] SEQ ID NO.2:

[0066] MRYIQELEHKVQVLQTEATTLSAQLTMLQRDSAGLATQNNELKIRLQAMEQQAQLRDALNEALTGEVQRLKLATGEISDGRMPKSLQQQMNSQMLQLQQLQIQQQQQQQAPQQQQQHQHQQQHQQQPQKSA.

[0067] 2. Analysis of SiMC1 transcriptional regulation

[0068] We used weighted co-expression network (WGCNA) analysis on publicly available RNA-seq data from the NCBI website to identify carotenoid metabolism pathway genes highly associated with the SiMC1 gene. The results are as follows: Figure 2 As shown, the SiMC1 gene has a high degree of association with multiple structural genes in the carotenoid metabolic pathway. A total of 12 carotenoid structural genes have a connectivity with the SiMC1 gene higher than 0.2, among which SiPSY1, SiPSY2, and SiLCYE have relatively high association.

[0069] Using the yeast one-hybrid method, we discovered the carotenoid metabolism pathway gene regulated by the SiMC1 protein. The SiMC1 sequence was transferred into the pGADT7 vector as a prey bait, and the 2000 bp promoter upstream of the SiPSY1 gene was cloned and transferred into pBait-AbAi as a bait vector.

[0070] The restriction enzyme sites used are SmaⅠ and XhoⅠ, and the nucleotide sequences of the homologous arm primers F and R are shown in SEQ ID NO. 3-4, respectively:

[0071] F: 5'-GCTTGAATTCGAGCTCGGTACCCGGG-3', SEQ ID NO.3;

[0072] R: 5'-ACATACAGAGCACATGCCTCGAG-3', SEQ ID NO.4.

[0073] like Figure 3 As shown, the results indicate that 800 ng / ml AbA concentration inhibits the self-activation of the SiPSY1 promoter and can interact with SiMC1, further verifying the regulatory role of SiMC1 on the SiPSY1 gene.

[0074] Using a dual-luciferase reporter gene assay, SiMC1 was ligated into the pGreenII-62-SK vector, while the yellow rice SiPSY1 promoter (Pro::SiPSY1-H) and white rice SiPSY1 promoter (Pro::SiPSY1-B) were ligated into the pGreenII-0800-LUC vector, respectively. The vector cassette is shown below. Figure 4 As shown in Figure a.

[0075] Using a protoplast preparation and transformation kit (Beijing Cooler Master Technology Co., Ltd.), several rice seedlings cultured in the dark for one week were taken, cut into small segments, and enzymatically digested for 4 hours. After filtration, the segments were washed twice with W5 solution, centrifuged for 4 minutes, resuspended, incubated on ice for 30 minutes, centrifuged for 4 minutes, and resuspended in MMG solution to determine the protoplast quality. Protoplasts were collected by centrifugation at 600 rpm for 4 minutes.

[0076] Take 100 μL of protoplast suspension, add 10 μL of plasmid, add an equal volume of PEG solution, let stand at room temperature for 10 min, stop the reaction by diluting with 1 mL of W5, wash 1-2 times with 1 mL of W5, and finally add 100 μL of W5 solution to adhere to the wall and incubate in the dark at 28℃ for 18-24 h.

[0077] Protoplasts were collected by centrifugation, and 100 μL of 1×Cell Lysis Buffer was added. The mixture was allowed to stand at room temperature, and the cell lysis products were pipetted and transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 12000 g for 2 min at room temperature, and the supernatant was collected. 100 μL of Luciferase Substrate / Renilla, equilibrated to room temperature, was added to a detection tube or microplate. 20 μL of the cell lysis supernatant was transferred to the wells of the detection tube or microplate, and the mixture was quickly mixed. The Firefly luciferase (LUC) / Renilla luciferase (REN) reporter gene activity was immediately detected using a fluorescence detector. The LUC / REN ratio was calculated to determine the regulatory mechanism of the SiMC1 protein on the SiPSY1 gene.

[0078] like Figure 4 As shown in Figure b, the results indicate that the SiMC1 protein can directly act on and inhibit the activity of the SiPSY1 gene promoter. The inhibitory effect of SiMC1 on the SiPSY1 gene promoter activity in white rice is significantly higher than that in yellow rice.

[0079] 3. Construction of SiMC1 knockout vector

[0080] The SiMC1 sequence was cloned using cDNA from Jingu 21 as a template. The nucleotide sequences of primers SiMC1-F and SiMC1-R are shown in SEQ ID NO.5-6.

[0081] SiMC1-F: 5'-AAAGAGCGCAAAATGAGG-3', SEQ ID NO.5;

[0082] SiMC1-R: 5'-CATGCTCAATCAGCTTCTGC-3', SEQ ID NO. 6.

[0083] PCR system: 20 μL nuclease-free water, 25 μL Biorun Pfu PCR Mix, 2 μL forward primer, 2 μL reverse primer, 1 μL DNA template, total volume 50 μL.

[0084] PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 23 s, 30 cycles; 7℃ for 10 min, 16℃ for 30 min.

[0085] Electrophoresis was performed on a 1.5% agarose gel at 5 V / cm for 20 min under UV light. The electrophoretic fragment with a length of 386 bp was excised, recovered from the gel, dissolved in 30 μL of water, and sequenced. After verification, the fragment was stored for vector ligation.

[0086] The SiMC1 coding region sequence of the Ci846 millet variety was obtained from the Millet Genome Database (http: / / 111.203.21.71:8000 / index.html). A gene knockout vector was constructed using CRISPR / Cas9 technology. The structure of the knockout vector is shown below. Figure 5 As shown; the target fragment obtained through the online target prediction website (http: / / crispor.tefor.net / ) is: CATTCAGGAGCTTGAGCACAAGG (SEQ ID NO.7).

[0087] The restriction enzyme sites selected were BsaⅠ and Eco31Ⅰ. The nucleotide sequences of the target fragment amplification primers SiMC1-CR-F and SiMC1-CR-F are shown in SEQ ID NO.8-9.

[0088] SiMC1-CR-F: 5'-CAGTGGTCTCATGCACATTCAGGAGCTTGAGCACAGTTTCAG-3', SEQ IDNO.8;

[0089] SiMC1-CR-R: 5'-CAGTGGTCTCAAAACTTGCTTGCAGCCTGATTTTCTGCACC-3', SEQ ID NO.9.

[0090] Enzyme digestion at 37℃ for 60 min yielded the linearized vector.

[0091] Enzyme digestion system for constructing the knockout vector: 12 μL nuclease-free water, 2 μL 10× buffer, 1 μL BsaⅠ, 1 μL Eco31Ⅰ, 4 μL empty vector, total volume 20 μL.

[0092] Homologous recombination was performed at 37℃ for 30 min to obtain the ligation product. The recombination reaction system consisted of 2 μL of nuclease-free water, 10 μL of Biorun 2×easy Clone Mix, 14 μL of rDNAG, 4 μL of linearized vector, and a total volume of 20 μL.

[0093] 5 μL of the recombinant ligation product was transformed into Escherichia coli DH5α competent cells (according to the standard method for transformation of competent cells by VidiBio), plated on an antibiotic resistance plate (SiMC1 gene knockout: Kan), and cultured at 37℃ for 12 h. PCR identification was performed, and positive strains were transformed into Agrobacterium EHA105 strain and stored at -80℃ for later use.

[0094] The nucleotide sequences of the identification primers are shown in SEQ ID NO.10-11:

[0095] The primers for identifying the SiMC1 gene knockout vector are as follows:

[0096] F3: 5'-ACCGGTAAGGCGCGCCGTAGT-3', SEQ ID NO.10;

[0097] R3: 5'-GCGATTAAGTTGGGTAACGCCAGGG-3', SEQ ID NO. 11.

[0098] 4. Agrobacterium-mediated transformation of millet and positive identification

[0099] Select grains free of mold and with normal bud openings. Disinfect with 75% alcohol for 3 minutes, then rinse with sterile water; disinfect with NaClO solution for 30 minutes, then rinse with sterile water 4-5 times. Inoculate the disinfected grains onto induction medium, incubate at 28°C under light for 7 days, then incubate in the dark for 7 weeks. Transfer the induced new hard callus to subculture medium and incubate in the dark at 28°C for 3-5 days. Collect Agrobacterium in the infection solution to prepare OD. 600 Agrobacterium resuspension (0.5 g / L) was used to pick callus into Erlenmeyer flasks. The Agrobacterium resuspension was added, and after 30 min of incubation, the bacterial suspension was discarded. The callus was then inoculated onto co-culture medium and co-cultured at 22°C for 48-72 h. The co-cultured callus was then inoculated onto selection medium and cultured in the dark at 28°C for 30 days, changing the medium every 15 days. Positive callus was inoculated onto differentiation medium and cultured under light at 28°C for 7-10 days. After shoot differentiation, the positive callus was inoculated onto rooting medium and cultured under light at 28°C for 10-15 days.

[0100] When the transgenic millet reached the three-leaf stage, healthy leaves were selected, and genomic DNA was extracted using the CTAB method. The transgenic plants were then identified by PCR using the same primers as the vector identification primers (SEQ ID NO. 10-11). Wild-type Ci846 (WT) plants were used as controls.

[0101] PCR identification results as follows Figure 6 As shown in the electrophoresis results, all transgenic lines were identified as positive.

[0102] 5. Phenotypic and Carotenoid Content Determination of Transgenic Lines

[0103] We selected SiMC1 positive transgenic lines with good and consistent growth, as well as wild-type Ci846 (WT) lines. After harvesting and hulling, we selected millet of uniform size and color as experimental materials.

[0104] like Figure 7As shown in Figure a, two homozygous knockout lines were identified by sequencing analysis of the knockout lines and their wild-type plants. The genome editing of SiMC1 resulted in a single base "C" and a 210 bp Indel deletion in the first exon of KO-11 and KO-15, respectively, causing frameshift mutations in amino acids and premature termination of translation.

[0105] like Figure 7 As shown in b and c, through phenotypic observation and beige b * Value analysis revealed that the mature grains of gene knockout lines KO-11 and KO-15 showed a higher b content. * The value is significantly increased compared to the wild type.

[0106] Extraction and determination of carotenoids from millet: Millet was ground into powder (50 mg), and 0.5 mL of solution containing 0.01% BHT (g·mL⁻¹) was used. -1 Extraction was performed using an extractant (ethanol:n-hexane:acetone = 1:1:1 by volume), the supernatant was collected by centrifugation, concentrated, and then redissolved in 100 μL of dichloromethane for LC-MS / MS detection of the content of each carotenoid component.

[0107] Chromatographic conditions: Column, C 30 (3 μm, 100 mm id × 2.0 mm, YMC); Mobile phase A: solvent containing 0.1% formic acid and 0.01% BHT (methanol:acetonitrile = 1:3 by volume); Mobile phase B: MTBE containing 0.01% BHT; Gradient elution program: 0 min, A:B = 100:0; 3 min, A:B = 100:0; 5 min, A:B = 30:70; 9 min, A:B = 5:95; 10 min, A:B = 100:0; 11 min, A:B = 100:0. The flow rate was fixed at 0.8 mL / min, the column temperature at 28℃, and the injection volume at 2 μL.

[0108] Mass spectrometry conditions: Atmospheric pressure chemical ionization source (APCI), temperature: 350℃, curtain gas (CUR): 25.0 psi.

[0109] like Figure 7As shown in the data, the carotenoid content in the grains of the SiMC1 gene knockout lines was significantly increased. Compared with the wild type, the average contents of total carotenoids, lutein, zeaxanthin, and β-carotene in KO-11 grains increased by 11.25%, 9.48%, 22.36%, and 21.39%, respectively, while the average contents of total carotenoids, lutein, zeaxanthin, and β-carotene in KO-15 grains increased by 12.31%, 9.68%, 27.63%, and 13.95%, respectively. This further demonstrates that the transcription factor SiMC1 negatively regulates the accumulation of carotenoids in millet, thereby affecting the beige color.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An application of a SiMC1 protein in any of the following: (1) Application in regulating the synthesis of carotenoids in millet; (2) Application in increasing the carotenoid content of millet; (3) Application in the cultivation of transgenic millet with increased carotenoid content; (4) Application in regulating the beige color of millet; The amino acid sequence of the SiMC1 protein is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, Downregulating the expression level of the SiMC1 protein in millet promotes the synthesis of millet carotenoids, increases the carotenoid content of millet, and turns the millet rice yellow.

3. The use of a gene encoding the SiMC1 protein as described in claim 1 in any of the following: (1) Application in regulating the synthesis of carotenoids in millet; (2) Application in increasing the carotenoid content of millet; (3) Application in the cultivation of transgenic millet with increased carotenoid content; (4) Application in regulating the beige color of millet; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, Downregulating the expression of the encoding gene in millet promotes the synthesis of millet carotenoids, increases the carotenoid content of millet, and turns millet rice yellow.

5. An application of a knockout vector, characterized in that, The knockout vector targets the gene encoding the SiMC1 protein; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating the synthesis of carotenoids in millet; (2) Application in increasing the carotenoid content of millet; (3) Application in the cultivation of transgenic millet with increased carotenoid content; (4) Application in regulating the beige color of millet.

6. The use of an engineered bacterium comprising the knockout vector of claim 5 in any of the following: (1) Application in regulating the synthesis of carotenoids in millet; (2) Application in increasing the carotenoid content of millet; (3) Application in the cultivation of transgenic millet with increased carotenoid content; (4) Application in regulating the beige color of millet.

7. A method for promoting the synthesis of carotenoids from millet, characterized in that, The step includes downregulating the expression level of the gene encoding the SiMC1 protein in millet to promote the synthesis of carotenoids in millet. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

8. A method for increasing the carotenoid content of millet, characterized in that, The method includes the step of downregulating the expression level of the gene encoding the SiMC1 protein in millet to increase the carotenoid content of the millet. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

9. A breeding method for transgenic millet with increased carotenoid content, characterized in that, Includes the following steps: The gene encoding the SiMC1 protein was knocked out in millet cells, the millet cells were then cultured, and millet plants were regenerated using the millet cells to obtain transgenic millet with increased carotenoid content. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

10. A method for turning millet from beige to yellow, characterized in that, The method includes the step of downregulating the expression level of the gene encoding the SiMC1 protein in millet to make the millet rice turn yellow. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

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