Application of Rht-B1b in regulating and controlling synthesis of flavonoid compounds in wheat

Abstract

The invention discloses an application of an Rht-B1b gene in regulation and control of synthesis of flavonoid compounds in wheat. The Rht-B1b gene is used as a target in screening and regulating products synthesized by flavonoid compounds in wheat. The invention also provides application of the Rht-B1b gene, the Rht-B1b protein or related biological materials in regulation and control of synthesis of flavonoid compounds in wheat or preparation of products for regulation and control of synthesis of flavonoid compounds in wheat. By detecting the content of flavonoid compounds in wheat with the Rht-B1b gene knocked out or over-expressed, it is found that the Rht-B1b gene has the new application of regulating and controlling synthesis of the flavonoid compounds, and important gene resources are provided for functional wheat breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the use of Rht-B1b in regulating the synthesis of flavonoid compounds in wheat. Background Technology

[0002] With increasing health awareness, the demand for agricultural products and food has gradually shifted from simply "eating enough" to "eating well," "consuming nutritious food," and even "eating for health." Developing and utilizing agricultural products with specific functions has become a new trend in agricultural development in recent years. Wheat, as one of my country's important grain crops, is a vital source of nutrition for the human body. Not only are its grains rich in nutrients, but wheat seedlings (leaflets and green juice) also possess nutritional and health-promoting value (Avisar et al., 2020; Sharma et al., 2020; Adhikary et al., 2021). Currently, green juice is consumed in various forms such as tablets and freeze-dried powder. However, the functional nutritional components of wheatgrass products are still unclear. Therefore, further improving the nutritional value of wheat and expanding its utilization scope while ensuring stable grain supply is of great significance for increasing farmers' income and promoting national health.

[0003] Flavonoids are important secondary metabolites in plants, possessing strong biological and pharmacological activities (Shen et al., 2022). They offer multiple benefits to humans, including antioxidant, antibacterial, anti-inflammatory, and immune-enhancing effects, thus becoming a target for improving crop nutritional quality (Tian et al., 2022; Chen Jie and Chen Wei, 2023). Currently, screening and creation of high-flavonoid germplasm have been carried out in crops such as rice and wheat (Chen et al., 2020; Wang Yanxun et al., 2023). Related studies have shown that regulating key genes in the flavonoid synthesis pathway through molecular genetics can help further increase the flavonoid content in food and enhance its health benefits (Tao et al., 2022). Therefore, systematically elucidating the molecular mechanisms and genetic basis affecting flavonoid content is not only expected to meet the growing consumer demand for nutritional quality but also to provide genetic, germplasm resources, and theoretical foundations for the development and utilization of functional agricultural products.

[0004] Flavonoids are a collective term for various structurally similar substances in plants, and their synthesis begins in the phenylpropanoid pathway (Yonekura-Sakakibara et al., 2019). Enzymes related to the flavonoid synthesis pathway, as well as various regulatory factors that control the expression levels of genes involved in phenylpropanoid metabolism, can affect flavonoid content (Dong and Lin, 2021; Lam et al., 2023). Among these, DELLA proteins belong to the plant-specific GRAS family and are major negative regulators of the gibberellin (GA) signaling pathway. They can integrate multiple signaling pathways through direct interactions with many transcription and co-transcription factors, and the role of DELLA proteins in regulating the phenylpropanoid metabolic pathway has been reported in earlier studies. For example, in anthocyanin biosynthesis, GA signal transduction has been shown to be inhibited by the DELLA protein RGA. The expression of anthocyanin-specific genes is regulated by the conserved MBW complex (composed of MYB, bHLH, and WD40 subunits), but MBW activity is inhibited by MYBL2 and JAZ proteins. RGA can bind to MYBL2 and JAZ proteins, releasing the bHLH / MYB subunit to form an active MBW complex, thereby activating the anthocyanin biosynthesis pathway (Xie et al., 2016). Furthermore, RGA can interact with the transcription factors MYB12 / MYB111 in the flavonol biosynthesis pathway and promote the binding of MYB12 to the promoter of the downstream target gene FLS1 / CHS, thus activating the expression of genes involved in the flavonol biosynthesis pathway (Tan et al., 2019). Therefore, DELLA proteins play an important regulatory role in the biosynthesis of flavonoids.

[0005] Wheat (Triticum aestivum L.) is an important food crop. The Rht-1 dwarfing gene encodes the DELLA protein and is an important genetic basis for semi-dwarf wheat breeding. In hexaploid wheat, the DELLA protein is encoded by three homologous Rht-1 genes: Rht-A1a, Rht-B1a, and Rht-D1a. The allelic variant Rht-B1b is produced by a single-base mutation of Rht-B1a near the DELLA region. The Rht-B1b mutation causes premature termination of translation of the Rht-1 encoded protein, which is then restarted through translation to produce the N-terminal truncated DELLA protein Rht-B1b. Rht-B1b is insensitive to gibberellin signaling, thus leading to semi-dwarfing of the plant, but its role in regulating wheat flavonoid content has not yet been reported. Summary of the Invention

[0006] One of the objectives of this invention is to provide the use of Rht-B1b as a target in screening products that regulate the synthesis of flavonoid compounds in wheat.

[0007] In some embodiments, the amino acid sequence encoded by the Rht-B1b gene comprises the sequence shown in SEQ ID NO.3.

[0008] In some embodiments, the flavonoid compound is selected from one or more of luteolin, sennaol, isothiazolin-7-O-glucoside, isothiazolin, luteolin O-deoxyglycoside-C-glycoside, isothiazolin, isovitexin-2”-O-glucoside, isothiazolin-2”-O-glucoside, apigenin O-deoxyglycoside-C-glycoside, isothiazolin-2”-O-rhamnoside, isovitexin, and isothiazolin.

[0009] In this invention, the use of the Rht-B1b gene as a target for screening products that regulate the synthesis of flavonoids in wheat specifically refers to: using the Rht-B1b gene or Rht-B1b protein as the target, screening products to find those that can promote or inhibit the expression level of the Rht-B1b gene, or promote or inhibit the content or activity of the Rht-B1b protein, as candidate products for the synthesis of flavonoids in wheat.

[0010] In this invention, the product regulating the synthesis of flavonoids in wheat may include molecules capable of specifically inhibiting the transcription or translation of the Rht-B1b gene, or molecules capable of specifically inhibiting the content or activity of the Rht-B1b protein, thereby reducing the expression level of the Rht-B1b gene in wheat and achieving the purpose of inhibiting the synthesis of flavonoids in wheat. Inhibiting the transcription or translation of the Rht-B1b gene, or molecules capable of specifically inhibiting the expression or activity of the Rht-B1b protein, can be achieved through gene knockout or gene silencing. Gene knockout is the inactivation of a specific target gene through changes in the DNA sequence. Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing is based on the premise of not altering the DNA sequence, resulting in the prevention or reduction of gene expression. Gene silencing can occur at two levels: one is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects; the other is post-transcriptional gene silencing, which is gene inactivation at the post-transcriptional level through specific inhibition of target RNA, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0011] Furthermore, the product may be a reagent for inhibiting or reducing Rht-B1b gene expression. The reagent for inhibiting or reducing Rht-B1b gene expression may be a reagent for knocking out the gene, such as a reagent for knocking out the gene by homologous recombination, or a reagent for knocking out the gene by CRISPR-Cas9, as prepared in the examples below. The reagent for inhibiting or reducing gene expression may contain a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0012] In this invention, the product for regulating the synthesis of flavonoid compounds in wheat may also include: molecules that can specifically promote the transcription or translation of the Rht-B1b gene, or molecules that can specifically promote the expression or activity of the Rht-B1b protein, thereby increasing the expression level of the Rht-B1b gene in wheat and achieving the purpose of promoting the synthesis of flavonoid compounds in wheat.

[0013] In this invention, the products used to regulate the synthesis of flavonoid compounds in wheat include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

[0014] In this invention, the product for regulating the synthesis of flavonoid compounds in wheat can be a single-component substance or a multi-component substance. In this invention, the form of the product is not particularly limited and can be various forms such as solid, liquid, gel, semi-fluid, or aerosol.

[0015] In this invention, the wheat includes, but is not limited to, wheat grains and wheat leaves.

[0016] This invention discovers that overexpression of the wheat Rht-B1b gene can significantly increase the content of flavonoids in wheat, while knocking out the wheat Rht-B1b gene can significantly decrease the content of flavonoids in wheat. Therefore, the Rht-B1b gene can be used as a target to screen products that regulate the synthesis of flavonoids in wheat.

[0017] The second objective of this invention is to provide the use of the Rht-B1b gene, Rht-B1b protein, or related biological materials in regulating the synthesis of flavonoids in wheat or in preparing products that regulate the synthesis of flavonoids in wheat.

[0018] The third objective of this invention is to provide the use of the Rht-B1b gene, Rht-B1b protein, or related biological materials in the cultivation of wheat or the preparation of products made from cultivated wheat.

[0019] In some embodiments, the biomaterial is at least one of the following:

[0020] a) Expression cassettes, recombinant expression vectors, or recombinant bioengineered bacteria containing the Rht-B1b gene;

[0021] b) Nucleic acid molecules that inhibit the Rht-B1b gene or reduce the content of Rht-B1b protein;

[0022] c) A recombinant vector containing the nucleic acid molecules described in b);

[0023] d) Recombinant bioengineered bacteria containing the nucleic acid molecules described in b), or bioengineered bacteria containing the recombinant expression vector described in c);

[0024] The sequence of the Rht-B1b protein is shown in SEQ ID NO.3.

[0025] In some embodiments, in b), the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA. Specifically, it is sgRNA or a DNA molecule expressing the sgRNA, whose target sequence includes the sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7.

[0026] In some embodiments, in b), inhibiting Rht-B1b gene expression and / or Rht-B1b protein content can be achieved through methods well-known in the art, such as RNA interference, homologous recombination, site-specific gene editing, and CRISPR / Cas9 editing, to reduce Rht-B1b gene expression and / or Rht-B1b protein content. Specifically, this may include editing (deleting or replacing) the exon sequence of the Rht-B1b gene in the genomic DNA of the starting plant using CRISPR / Cas9 technology. More specifically, it may include introducing the aforementioned specific sgRNA, the aforementioned DNA molecule expressing the specific sgRNA, or a vector containing the aforementioned DNA molecule expressing the specific sgRNA into the starting plant. The vector containing the aforementioned DNA molecule expressing the specific sgRNA is, for example, pBUE411-Rht-B1b prepared in the following examples.

[0027] In some embodiments, the recombinant vector refers to the introduction of the Rht-B1b gene or the nucleic acid molecule described in b) into an expression vector to form a recombinant expression vector. The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements. The expression vectors in this invention are not limited to those mentioned in the following embodiments. Methods well known to those skilled in the art can be used to construct expression vectors containing nucleotide sequences encoding stress-resistant proteins and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0028] In some embodiments, the engineered bacteria contain a recombinant expression vector as described above or have a nucleic acid molecule such as the Rht-B1b gene or b) integrated into its genome. The engineered bacteria can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; plant cells, etc. The engineered bacteria is, for example, the *Agrobacterium* strain EHA105 described in the following examples.

[0029] In some embodiments, increasing Rht-B1b gene expression and / or Rht-B1b protein content can be achieved through methods well-known in the art, such as multiple copies, promoter modification, regulatory factors, and transgenic methods, thereby increasing Rht-B1b gene expression and / or Rht-B1b protein content in wheat. Specifically, this may include introducing the Rht-B1b gene or a vector containing the Rht-B1b gene into wheat. A vector containing the Rht-B1b gene is, for example, the overexpression vector pWMB110-Rht-B1b prepared in the following examples.

[0030] The fourth objective of this invention is to provide a method for regulating the synthesis of flavonoid compounds in wheat, the method comprising one or more of the following steps:

[0031] D1) The Rht-B1b gene was introduced to increase the content of flavonoid compounds;

[0032] D2) Introduce biological materials associated with the Rht-B1b gene to increase the content of flavonoid compounds;

[0033] D3) Reduce the content of Rht-B1b protein in wheat to reduce the content of flavonoid compounds;

[0034] The amino acid sequence of Rht-B1b is shown in SEQ ID NO.3.

[0035] The fifth objective of this invention is to provide a method for cultivating wheat, the method comprising one or more of the following steps:

[0036] D1) Introduce the Rht-B1b gene to breed wheat with increased flavonoid content;

[0037] D2) Introduce biological materials associated with the Rht-B1b gene to cultivate wheat with increased flavonoid content;

[0038] D3) Reduce the content of Rht-B1b protein in wheat to cultivate wheat with reduced flavonoid content;

[0039] The amino acid sequence of Rht-B1b is shown in SEQ ID NO.3.

[0040] In some embodiments, the introduction can be the transformation of the vector into wheat by any known transformation method, such as chemical transformation, genetic transformation, or electroporation transformation. The nucleic acid molecule that introduces the Rht-B1b gene, inhibits the Rht-B1b gene, or reduces the Rht-B1b protein content can be a single copy or multiple copies. The introduction can be the integration of the foreign gene into the wheat chromosome, or it can be expressed extrachromosomally by a recombinant expression vector.

[0041] This invention discovers that the Rht-B1b gene is involved in the synthesis of flavonoids. Overexpression or knockout of the Rht-B1b gene can regulate flavonoid synthesis, thereby increasing or decreasing the flavonoid content in wheat. Overexpression of expression cassettes, recombinant expression vectors, or recombinant bioengineered bacteria containing the Rht-B1b gene in wheat significantly increases the flavonoid content. Knockout of the Rht-B1b gene in wheat significantly decreases the flavonoid content. Therefore, the Rht-B1b gene plays an important biological role in flavonoid synthesis in wheat and in wheat breeding. Attached Figure Description

[0042] Figure 1This is a statistical chart showing the content of flavonoid compounds in the young leaves of Rht-B1b gene knockout, Rht-B1b gene overexpression, and wild-type Fielder wheat in Example 2. A is a chromatogram of flavonoid compounds in the young leaves of wild-type Fielder, rht1-bb, and Rht-B1b-OE wheat. B is a bar chart of flavonoid compound content in the young leaves of wild-type Fielder, rht1-bb, and Rht-B1b-OE wheat. rht1-bb represents Rht-B1b gene knockout, and Rht-B1b-OE represents Rht-B1b gene overexpression. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0044] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, 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 application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0046] In this invention, the nucleotide sequence of the protein-coding region of the Rht-B1b gene is shown in SEQ ID NO.1, the full-length sequence of the Rht-B1b gene is shown in SEQ ID NO.2, and the protein encoded by the Rht-B1b gene is an N-terminally truncated DELLA protein composed of 555 amino acids, the amino acid sequence of which is shown in SEQ ID NO.3, specifically:

[0047] SEQ ID NO.1:

[0048]

[0049] SEQ ID NO.2:

[0050]

[0051] SEQ ID NO.3:

[0052] MAMGMGGVGAGAAPDDSFATHLATDTVHYNPTDLSSWVESMLSELNAPPPPLPPAPQLNASTSSTVTGGGYFDLPPSVDSSCSTYALRPIPSPAVAPADLSADSVVRDPKRMRTGGSSTSSSSSSLGGGGARSSVV EAAPPVAAAAGAPALPVVVVDTQEAGIRLVHALLACAEAVQQENFSAAEALVKQIPLLAASQGGAMRKVAAYFGEALARRVFRFRPQPDSSLLDAAFADLLHAHFYESCPYLKFAHFTANQAILEAFAGCRRVHVVDFG IKQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQYRGLVAATLADLEPFMLQPEGEEDPNEEPEVIAVNSVFEMHRLLAQPGALEKVLGTVRAVRPRIVTVVEQEANHNSGTFL DRFTESLHYYSTMFDSLEGGSSGGPSEVSSGAAAAPAAAGTDQVMSEVYLGRQICNVACEGAERTERHETLGQWRNRLGNAGFETVHLGSNAYKQASTLLALFAGGDGYKVEEKEGCLTLGWHTRPLIATSAWRLAAP

[0053] Example 1: Cloning of the wheat Rht-B1b encoding gene, construction of gene knockout engineered bacteria and gene overexpression engineered bacteria.

[0054] 1.1. Cloning of the wheat Rht-B1b encoding gene

[0055] The nucleotide sequence of the wheat Rht-B1b encoding gene was obtained from the EnsemblPlants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). Primers for amplifying the full length of the Rht-B1b gene were designed based on this nucleotide sequence. The primers used are:

[0056] TaRht-B1b-F: 5'-ATCATGAAGCGCGAGTACCAG-3' (SEQ ID NO. 4);

[0057] TaRht-B1b-R: 5'-CGGCGCGGCCAGGCGCCAT-3' (SEQ ID NO. 5).

[0058] Polymerase chain reaction (PCR) was performed using cDNA from Fielder wheat plants as a template, employing a 2× Hieff... Gold Master Mix high-fidelity enzyme premix (YEASEN, Shanghai, China) was used in a 20 μL mixing system. The reaction was performed on a C1000 Touch PCR instrument (Bio-Rad, USA) using the following program: Step 1: Denaturation at 98℃ for 3 min; Step 2: 98℃ for 10 sec, 70-55℃ for 20 sec, 72℃ for 80 sec, decreasing by 1℃ for 15 cycles; Step 3: 98℃ for 10 sec, 55℃ for 20 sec, 72℃ for 1 min, 20 cycles; Step 4: Extension at 72℃ for 5 min. The PCR products were then detected on a 1.2% agarose gel, and the target fragment was extracted from the gel. Subsequently, the target fragment was directly cloned into a T-Vector pMD using the TA cloning method. TM The sample was placed in a 19(Simple) vector and sequenced.

[0059] Sequencing revealed the full-length sequence of the Rht-B1b gene as shown in SEQ ID NO.2, the nucleotide sequence of the encoded protein as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein as shown in SEQ ID NO.3.

[0060] 1.2. Construction of Rht-B1b gene knockout engineered bacteria

[0061] Based on the genome sequence (SEQ ID NO.2), target sequences “GGCGCTCGGGTACAAGGTGC” (SEQ ID NO.6) and “GTGGGTGGCGAAGCTGTCGTCGG” (SEQ ID NO.7) were selected, and complementary primers were synthesized according to these target sequences:

[0062] TaRht-B1b-CRISPR-1F:ATATATGGTCTCTGGCGGCGCTCGGGTACAAGGTGCGTT (SEQ IDNO.8);

[0063] TaRht-B1b-CRISPR-1R:ATTATTGGTCTCTAAACACGACAGCTTCGCCACCCAC (SEQ IDNO.9);

[0064] TaRht-B1b-CRISPR-2F:TGGCGCTCGGGTACAAGGTGCGTTTTAGAGCTAGAAATAGC (SEQ IDNO.10);

[0065] TaRht-B1b-CRISPR-2R:AACACGACAGCTTCGCCACCCACGCTTCTTGGTGCC (SEQ ID NO. 11).

[0066] Using the pCBC-MT1T2 vector as a template, Rht-B1b-CRISPR-2F / 2R and Rht-B1b-CRISPR-1F / 1R primers were added at a ratio of 1:20, and KOD One was used. TM PCR amplification was performed using PCR Master Mix (TOYOBO, Shanghai, China). The reaction program was as follows: Step 1: Denaturation at 94℃ for 3 min; Step 2: Cycle 30 times with 94℃ for 15 sec, 60℃ for 30 sec, and 68℃ for 1 min; Step 3: Extension at 68℃ for 5 min. The obtained PCR products were then detected on a 1.2% agarose gel and purified. Subsequently, the purified PCR fragments were incorporated into the pBUE411 vector using the Golden Gate reaction. The 15 μL reaction mixture consisted of: 2 μL of PCR product (~100 ng / μL), 2 μL of pBUE411 vector (~100 ng / μL), 1.5 μL of 10×T4 DNA Ligase buffer, 1.5 μL of 10×BSA, 1 μL of Bsa I, 1 μL of T4 DNA Ligase, and 6 μL of ddH2O. The ligation product was obtained by reacting at 37℃ for 5 hours, 50℃ for 5 minutes, and 80℃ for 10 minutes. This ligation product was then transformed into competent *E. coli* cells, and positive monoclonal colonies were screened to obtain the gene-editing vector (gene knockout vector) pBUE411-Rht-B1b. The recombinant vector was then introduced into *Agrobacterium* strain EHA105 using a heat shock transformation method, resulting in *Agrobacterium* engineered bacteria containing the gene-editing vector pBUE411-Rht-B1b, which was used for subsequent genetic transformation.

[0067] 1.3. Construction of engineered bacteria overexpressing the Rht-B1b gene

[0068] Using the recombinant T-vector plasmid containing the Rht-B1b gene fragment SEQ ID NO.1 obtained in Example 1.1 as a template, amplification was performed using the forward primer Rht-B1b-110-F and the reverse primer Rht-B1b-110-R to obtain a PCR product with a homologous recombination arm. The vector pWMB110 was digested with the restriction endonuclease BamHI, and the digested product and the PCR product were ligated (ligation conditions: 50℃ for 20 min) to obtain the recombinant vector pWMB110-Rht-B1b. The recombinant vector pWMB110-Rht-B1b was introduced into Agrobacterium strain EHA105 using a heat shock transformation method, resulting in an engineered Agrobacterium strain containing the overexpression vector pWMB110-Rht-B1b, which was used for subsequent genetic transformation.

[0069] Rht-B1b-110-F:5'-TCTAGAGGATCCCCGGGATGGCCATGGGGATGGGCGGCG-3'(SEQ IDNO.12)

[0070] Rht-B1b-110-R:5'-GAGCTCTCTAGAACTAGTCCCGGGGTACCGCCCTTATCAT-3'(SEQ IDNO.13)

[0071] Example 2: Obtaining wheat with Rht-B1b gene knockout and overexpression, and detecting the content of flavonoid compounds in young leaves.

[0072] 2.1. Obtaining wheat with Rht-B1b gene knockout and overexpression

[0073] Fielder, a hexaploid spring wheat variety with good growth status, was selected as the recipient. Genetic transformation was carried out by embryo removal. Agrobacterium tumefaciens engineered bacteria containing the gene editing vector pBUE411-Rht-B1b obtained in Example 1 and Agrobacterium tumefaciens containing the overexpression vector pWMB110-Rht-B1b were used for infection and differentiation, and then screened to obtain the corresponding genetically transformed plants.

[0074] 2.2. Identification of Rht-B1b gene knockout and Rht-B1b gene overexpression wheat

[0075] Rht-B1b gene knockout wheat: DNA was extracted from genetically transformed plants of Rht-B1b gene knockout wheat. The endogenous Rht1 gene of transgenic wheat was amplified using primers Rht1-AA (forward and reverse), Rht1-BB (forward and reverse), and Rht1-DD (forward and reverse). The PCR products were sequenced to verify the results. The results showed that the Rht-B1b gene had undergone base deletion, thus obtaining transgenic plants with Rht-B1b gene loss of function, named rht1-bb.

[0076] Rht1-AA forward primer: CTCGCTGCTTTGCTCTCTCT (SEQ ID NO.14)

[0077] Rht1-AA reverse primer: TAGGTGCTGCAGGAGGAGTC (SEQ ID NO.15)

[0078] Rht1-BB forward primer: AGGCAAGCAAAAGCTTGAGA (SEQ ID NO.16)

[0079] Rht1-BB reverse primer: AGGACGATGAGGATGACGAC (SEQ ID NO.17)

[0080] Rht1-DD forward primer: CAAAAGCTTCGCGCAATTAT (SEQ ID NO.18)

[0081] Rht1-DD reverse primer: GAGACGAGGAGGAGGAGGAT (SEQ ID NO.19)

[0082] Rht-B1b gene overexpression wheat: RNA was extracted from genetically transformed wheat plants overexpressing the Rht-B1b gene using RNAiso Plus, and 1 μg RNA, oligo-dT18 primers, and... III Reverse Transcriptase System (Yeasen, Shanghai, China) for reverse transcription of cDNA. Using... Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green Master Mix (Yeasen, Shanghai, China). The wheat β-Actin gene was used as an internal control, and 2... -ΔΔCt The relative expression level of TaRht-B1b was calculated using a method to obtain positive plants with Rht-B1b-OE. The primers used were: forward and reverse primers for Rht-B1b-qRT and forward and reverse primers for β-Actin-qRT.

[0083] Rht-B1b-qRT forward primer: CCAGGTCATGTCCGAGGTGT (SEQ ID NO.20)

[0084] Rht-B1b-qRT reverse primer: CCTCCTTCTCCTCCACCTTGTA (SEQ ID NO.21)

[0085] β-Actin-qRT forward primer: ACAGTGTCTGGATCGGTGGC (SEQ ID NO.22)

[0086] β-Actin-qRT reverse primer: GTGGACAATGCCGGGACCAG (SEQ ID NO.23)

[0087] 2.3. Cultivation of wheat with Rht-B1b gene overexpression and Rht-B1b gene knockout

[0088] The metabolism of flavonoids in leaves is highly sensitive to environmental factors; therefore, uniform culture conditions and seedling growth are crucial for subsequent compound detection. Rht-B1b gene overexpression and Rht-B1b gene knockout homozygous wheat plants were cultivated: Rht-B1b-OE and rht1-bb. Thirty plump, intact, dry seeds from each line were soaked for 24 hours and placed in a 4°C cold storage for 7 days to ensure consistent germination. Once the seeds showed signs of sprouting, they were planted in 128-well seedling trays, with four seeds per well and 24 seeds per variety. The seedling trays were then placed on a cultivation rack at a constant temperature of 25°C, with a photoperiod of 12 hours / day, 12 hours / dark, and watered every two days. Flavonoid content was determined when the wheat seedlings reached the one-leaf-one-heart stage (approximately 7 days).

[0089] 2.4. Extraction of flavonoids from young wheat leaves after Rht-B1b gene overexpression and Rht-B1b gene knockout

[0090] The extraction steps for flavonoids from young wheat leaves are as follows: 0.01 g of young wheat leaf sample was accurately weighed, flash-frozen in liquid nitrogen, and then homogenized using a tissue homogenizer (50 Hz, 30 s, twice); 1 mL of 80% methanol was added and thoroughly mixed by shaking; the sample was ultrasonically incubated in a water bath at 90% power for 30 min; centrifuged at 12000 rpm for 15 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube and incubated at 4℃ overnight; the sample was further centrifuged at 12000 rpm for 15 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube for analysis.

[0091] 2.5. Determination of flavonoid content in young wheat leaves after Rht-B1b gene overexpression and Rht-B1b gene knockout

[0092] The flavonoid content in young wheat leaves was determined using a Thermo UltiMate 3000 high-performance liquid chromatograph with an Accucore column. TM C18 (2.1×100mm, 2.6μm). The specific detection steps are as follows: sample loading volume was 10μL, column temperature was 30℃, flow rate was 0.8mL / min, detection wavelength was 340nm, detection time was 20min, and the mobile phase consisted of chromatographically pure acetonitrile solution containing 0.1% (v / v) formic acid (mobile phase A) and pure aqueous solution containing 0.1% (v / v) formic acid (mobile phase B), using gradient elution. The gradient elution parameters are set as shown in Table 1. To eliminate the influence of individual differences, six biological replicates were performed for each sample group.

[0093] Table 1 Liquid phase setup parameters

[0094]

[0095] The results of flavonoid content in young leaves are as follows Figure 1 As shown.

[0096] from Figure 1 It was found that, compared with wild-type Fielder, knocking out the Rht-B1b gene significantly reduced the content of 10 flavonoid compounds, including peak1 isopropenin-7-O-glucoside (reduced by 58%), peak2 isopropenin (reduced by 24%), peak3 luteolin O-deoxyglucoside-C-glucoside (reduced by 41%), peak4 isopropenin (reduced by 63%), peak5 isovitrin-2”-O-glucoside (reduced by 65%), peak6 isocytisine-2”-O-glucoside (reduced by 76%), peak7 apigenin O-deoxyglucoside-C-glucoside (reduced by 51%), peak8 isocytisine-2”-O-rhamnoside (reduced by 52%), peak9 isovitrin (reduced by 61%), and peak10 isocytisine (reduced by 75%).

[0097] from Figure 1 It was found that, compared with wild-type Fielder, overexpression of the Rht-B1b gene significantly increased the content of flavonoid compounds, including: peak3 luteolin O-deoxyglucoside-C-glucoside, an increase of 40%; peak4 isothiazolin, an increase of 143%; peak6 isocytisine-2”-O-glucoside, an increase of 91%; peak8 isocytisine-2”-O-rhamnoside, an increase of 43%; and peak10 isocytisine, an increase of 113%.

[0098] The above studies indicate that the Rht-B1b gene plays an important role in regulating the synthesis and content of flavonoids in wheat.

Claims

1. The use of the Rht-B1b gene as a target in screening products that regulate the synthesis of flavonoid compounds in wheat.

2. The use as described in claim 1, characterized in that, The amino acid sequence encoded by the Rht-B1b gene includes the sequence shown in SEQ ID NO.3; And / or, the flavonoid compound is selected from one or more of luteolin, sennaol, isothiazolin-7-O-glucoside, isothiazolin, luteolin O-deoxyglycoside-C-glycoside, isothiazolin, isovitexin-2”-O-glucoside, isothiazolin-2”-O-glucoside, apigenin O-deoxyglycoside-C-glycoside, isothiazolin-2”-O-rhamnoside, isovitexin, and isothiazolin; And / or, the regulation of flavonoids in wheat refers to increasing or decreasing the content of flavonoids in wheat by increasing or decreasing the level of the Rht-B1b gene in wheat; And / or, the product that regulates the synthesis of flavonoids in wheat refers to a molecule that can inhibit the transcription or translation of the Rht-B1b gene, or can inhibit the expression or activity of the Rht-B1b protein, so as to inhibit the synthesis of flavonoids in wheat. And / or, the product that regulates the synthesis of flavonoids in wheat refers to a molecule that can promote the transcription or translation of the Rht-B1b gene, or can promote the expression or activity of the Rht-B1b protein, so as to promote the synthesis of flavonoids in wheat.

3. Use of the Rht-B1b gene, Rht-B1b protein, or related biological materials in regulating the synthesis of flavonoids in wheat or in preparing products that regulate the synthesis of flavonoids in wheat.

4. Use of the Rht-B1b gene, Rht-B1b protein, or related biological materials in the breeding of wheat or the preparation of products made from cultivated wheat.

5. The use as described in claim 3 or 4, characterized in that, The biomaterial is at least one of the following: a) Expression cassettes, recombinant expression vectors, or recombinant bioengineered bacteria containing the Rht-B1b gene; b) Nucleic acid molecules that inhibit the Rht-B1b gene or reduce the content of Rht-B1b protein; c) A recombinant vector containing the nucleic acid molecules described in b); d) Recombinant bioengineered bacteria containing the nucleic acid molecules described in b), or bioengineered bacteria containing the recombinant expression vector described in c); The sequence of the Rht-B1b protein is shown in SEQ ID NO.

3.

6. The use as described in claim 5, characterized in that, b) The nucleic acid molecule is sgRNA or a DNA molecule expressing the sgRNA, and its target sequence includes the sequences shown in SEQ ID NO.6 and SEQ ID NO.

7.

7. A method for regulating the synthesis of flavonoid compounds in wheat, characterized in that, The method includes one or more of the following steps: D1) The Rht-B1b gene was introduced to increase the content of flavonoid compounds; D2) Introduce biological materials associated with the Rht-B1b gene to increase the content of flavonoid compounds; D3) Reduce the content of Rht-B1b protein in wheat to reduce the content of flavonoid compounds; The amino acid sequence of Rht-B1b is shown in SEQ ID NO.

3.

8. A method for cultivating wheat, characterized in that, The method includes one or more of the following steps: D1) Introduce the Rht-B1b gene to breed wheat with increased flavonoid content; D2) Introduce biological materials associated with the Rht-B1b gene to cultivate wheat with increased flavonoid content; D3) Reduce the content of Rht-B1b protein in wheat to cultivate wheat with reduced flavonoid content; The amino acid sequence of Rht-B1b is shown in SEQ ID NO.3.

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