Wheat blue grain gene and its application

Abstract

This invention discloses a wheat blue-grain gene and its applications, belonging to the fields of plant molecular biology, biochemistry, genetics, and plant breeding. Using differential expression analysis between blue-grain and white-grain wheat, four genes controlling the blue-grain trait were identified: two MYB family transcription factors and two bHLH family transcription factors. Furthermore, plant recombinant expression vectors for these genes and a method for regulating anthocyanin synthesis in plants are provided. This has significant theoretical and practical implications for studying the aleurone layer pigment synthesis pathway in blue-grain wheat, its use as a screening marker in plant transformation processes, and improving the nutritional value of plants.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology, biochemistry, genetics, and plant breeding, and particularly to a gene that regulates the blue grain trait in wheat. This invention provides nucleic acid molecules and plant recombinant vectors for four wheat blue grain trait-related genes, as well as methods for applying the genes or vectors. Background Technology

[0002] Common wheat grains naturally exist in two colors: white or red, with a very small number being blue or purple. Blue or purple wheat can serve as important genetic markers for wheat breeding, especially in my country, where research on the application of blue wheat is quite extensive. For example, the "4E-ms hybrid wheat production system" successfully utilized the blue grain characteristic to effectively maintain male sterility in wheat (Zhou et al., 2006).

[0003] The endosperm of a wheat grain is surrounded by three layers from the outside in: the pericarp, the seed coat, and the aleurone layer. The color of the wheat grain is determined by the different anthocyanins accumulated in these different tissues. Purple wheat originates from the purple anthocyanins in the outermost pericarp, which develops from the maternal parent, thus exhibiting a maternal inheritance pattern. Blue wheat, on the other hand, originates from the blue anthocyanins in the aleurone layer, primarily produced by distant hybridization between common wheat and other species. Its sources are mainly two: *Thinopyrum ponticum* and wild einkorn wheat (*Triticum monococcum*). From the 1960s to the 1980s, many scholars, including Knott, Sharman, and Li Zhensheng, obtained blue wheat from the hybrids of *Thinopyrum ponticum* and common wheat, and proved that chromosome 4E (also known as 4Ag) of *Thinopyrum ponticum* carries the blue grain gene. Li Zhensheng et al., through genetic analysis, concluded that the blue grain gene is inherited stably and independently, exhibiting a significant dose-response effect, and that the blue grain trait is very similar to being controlled by a pair of genes (Li Zhensheng et al., 1982). Further GISH and FISH experiments on a series of different blue grain translocation lines located the blue grain gene within the 0.71-0.80 segment of the long arm of chromosome 4Ag (distance from the centromere) (Zheng et al., 2006). In 1990, Keppenne named the blue aleurone (Ba) gene derived from *Eriocheir sinensis*. Some foreign scholars also believe that the blue trait is controlled by two complementary genes. In 1982, Joppa et al. demonstrated that the Blaukom series derived from *Eriocheir sinensis* is formed by the substitution of chromosome 4A or 4B of common wheat with a pair of chromosome 4A from *Eriocheir sinensis*. mThe chromosome is non-homologous to chromosome 4A in tetraploid and hexaploid wheat. In 1989, Kuspiral et al. named the blue aleurone layer gene derived from wild einkorn wheat the Ba2 gene, which is located on chromosome 4A. m Near the centromere on the long arm of the chromosome (Dubcovsky et al., 1996).

[0004] To date, genes controlling red and purple grain traits in wheat have been cloned, but those controlling blue grain traits have not been reported. In 2005, Himi et al. reported that the major genes R1, R2, and R3, located on chromosomes 3AL, 3BL, and 3DL, encode three highly homologous Myb family transcription factors (Himi et al., 2005). Genetic analysis of purple wheat showed that the purple grain trait is controlled by the Pp-1 (purple pericarp) locus on the short arm of chromosome 7 and the Pp3 locus on chromosome 2AL. Pp-1 encodes a Myb family transcription factor, while Pp3 encodes a Myc family transcription factor containing a bHLH (basic helix-loop-helix) domain (Khlestkina et al., 2013; Shoeva et al., 2014).

[0005] Anthocyanins, which determine the color of wheat grains, are water-soluble secondary metabolites—flavonoid compounds—widely distributed in the roots, stems, leaves, flowers, fruits, and seeds of higher plants. Anthocyanin synthesis in plants is a branch of the flavonoid biosynthesis pathway. The main synthases in this pathway include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and dihydroflavonol-4-reductase (DFR) (Gong et al., 2011). These genes are all structural genes involved in anthocyanin synthesis. The regulation of anthocyanin synthesis is mainly accomplished by three types of transcription factors: MYB, bHLH, and WD40. In most species, anthocyanin synthesis is directly regulated and activated by a protein complex composed of these three transcription factors; however, in a few species, anthocyanin synthesis can be activated by a single regulatory factor.

[0006] Blue-grained wheat is an ideal morphological feature for wheat marker traits and wheat cytogenetics research, and it is also an important basic material for wheat chromosome engineering research. This invention utilizes differential expression analysis between blue-grained and white-grained wheat to identify four genes controlling the blue-grained trait in wheat: two MYB family transcription factors and two bHLH family transcription factors. This invention contributes to the study of the aleurone layer pigment synthesis pathway in blue-grained wheat and can be used as a selection marker in plant transformation processes. Furthermore, expressing these genes in plants can increase anthocyanin synthesis, thereby improving the nutritional value of the plants. Summary of the Invention

[0007] All references mentioned in this article are incorporated herein by way of citation.

[0008] 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 invention pertains. Unless otherwise specified, the techniques used or mentioned herein are standard techniques well known to one of ordinary skill in the art. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0009] This invention provides a blue grain gene with anthocyanin synthesis regulation function. The blue grain genes are named ThMYB1, ThMYB2, ThR1, and ThR2, and can turn the tissue or organ at their expression site blue. The genomic nucleotide sequence of the ThMYB1 gene from start codon to stop codon is shown in SEQ ID NO:1, its coding region (CDS) nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence encoded by its CDS is shown in SEQ ID NO:3. The genomic nucleotide sequence of the ThMYB2 gene from start codon to stop codon is shown in SEQ ID NO:4, its coding region (CDS) nucleotide sequence is shown in SEQ ID NO:5, and the amino acid sequence encoded by its CDS is shown in SEQ ID NO:6. The genomic nucleotide sequence of the ThR1 gene from start codon to stop codon is shown in SEQ ID NO:7, its coding region (CDS) nucleotide sequence is shown in SEQ ID NO:8, and the amino acid sequence encoded by its CDS is shown in SEQ ID NO:9. The genomic nucleotide sequence of the ThR2 gene from start codon to stop codon is shown in SEQ ID NO:10, the nucleotide sequence of its coding region (CDS) is shown in SEQ ID NO:11, and the amino acid sequence encoded by its CDS is shown in SEQ ID NO:12.

[0010] Those skilled in the art should understand that the blue grain gene described in this invention also includes nucleotide or protein sequences that are highly homologous to the nucleotide or protein sequences of the ThMYB1, ThMYB2, ThR1, and ThR2 genes and have the same function of regulating anthocyanin synthesis in plants. The highly homologous homologous genes with anthocyanin synthesis regulatory function include DNA sequences that, under stringent conditions, can hybridize with DNA having the sequences shown in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, or 11, or nucleotide sequences whose encoded amino acid sequences have more than 85% similarity to the protein amino acid sequences shown in SEQ ID NO: 3, 6, 9, or 12. The “strict conditions” used herein are well-known and include hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA, preferably at a temperature of 53°C-60°C and a hybridization time of 12-16 hours, followed by washing with a washing solution containing 0.5 × SSC and 0.1% SDS, preferably at a temperature of 62°C-68°C and a washing time of 15-60 minutes.

[0011] The aforementioned homologous genes also include DNA sequences that share at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity with the full length of the sequences shown in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, or 11, and that have the function of regulating anthocyanin synthesis in plants, and can be isolated from any plant. The percentage of sequence similarity can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment, the Smith-Waterman local alignment, the Pearson and Lipman similarity search, and the Karlin and Altschul algorithm. This is well known to those skilled in the art.

[0012] The present invention also provides an expression cassette containing the DNA sequence of the blue grain gene disclosed in the present invention, wherein the nucleotide sequence of the blue grain gene is selected from one of the following groups of sequences:

[0013] (a) A nucleotide sequence as shown in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10 or 11;

[0014] (b) The nucleotide sequence that encodes an amino acid sequence as shown in SEQ ID NO: 3, 6, 9 or 12;

[0015] (c) A DNA sequence capable of hybridizing with the sequence described in (a) or (b) under stringent conditions; or

[0016] (d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a)-(c) and having anthocyanin synthesis regulatory function; or

[0017] (e) A DNA sequence complementary to any of the sequences described in (a)-(d).

[0018] Specifically, the blue grain gene in the aforementioned expression cassette can also be operatively linked to a promoter capable of driving its expression. This promoter includes, but is not limited to, constitutive promoters, inducible promoters, tissue-specific promoters, or spatiotemporally specific expression promoters. The gene expression of the constitutive promoter described in this invention is not tissue- or time-specific, and external factors have almost no effect on the expression of exogenous genes initiated by the constitutive promoter. The constitutive promoters include, but are not limited to, CaMV35S, FMV35S, rice actin (Actin1) promoter, and maize ubiquitin promoter. The tissue-specific promoters described in this invention, in addition to containing the usual promoter elements, also possess enhancer and silencer characteristics. The advantage of this type of promoter is that it can initiate gene expression in specific plant tissues, avoiding unnecessary expression of exogenous genes, thereby saving the overall energy consumption of the plant. The tissue-specific promoters include, but are not limited to, the LTP2 seed-specific expression promoter, the END2 seed-specific expression promoter, and the aleurone layer-specific expression promoter. The inducible promoters described in this invention refer to promoters that, under the stimulation of certain specific physical or chemical signals, can significantly enhance… Gene Transcriptional promoters, currently isolated inducible promoters include, but are not limited to, stress-induced expression promoters, light-induced expression promoters, heat-induced expression promoters, wound-induced expression promoters, fungal-induced expression promoters, and symbiotic bacterial-induced expression promoters.

[0019] The expression cassette of the present invention may further include a selection gene, which can be used to screen plants, plant tissue cells, or vectors containing the expression cassette. The selection gene includes, but is not limited to, antibiotic resistance genes, herbicide resistance genes, or fluorescent protein genes. Specifically, the selection genes include, but are not limited to: chloramphenicol resistance genes, hygromycin resistance genes, streptomycin resistance genes, zizomycin resistance genes, sulfonamide resistance genes, glyphosate resistance genes, glufosinate resistance genes, bar genes, red fluorescent protein genes (DsRED), mCherry genes, cyan fluorescent protein genes, yellow fluorescent protein genes, luciferase genes, and green fluorescent protein genes.

[0020] The present invention also discloses a method for increasing the anthocyanin content in plants. The method involves co-expressing the ThMYB1 or ThMYB2 gene provided by the present invention with any bHLH transcription factor in plant tissues and organs, thereby increasing the anthocyanin content in plant tissues and organs.

[0021] The bHLH transcription factor can be isolated from any plant, including but not limited to the ThR1 and ThR2 genes provided in this invention, and the ZmR and ZmB genes from maize (Ahmed N, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize C1 and B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1):29-34.).

[0022] The methods described above for increasing anthocyanin content in plants can be used to increase the anthocyanin content of any tissue or organ in a plant. Specifically, if you want to increase the anthocyanin content in all tissues of a plant as a whole, you can use constitutive promoters to express both the ThMYB1 or ThMYB2 gene and the bHLH transcription factor. If you only want to increase the anthocyanin content in a specific tissue or organ, you can use promoters specifically expressed in that tissue or organ to express both the ThMYB1 or ThMYB2 gene and the bHLH transcription factor.

[0023] The present invention also discloses a method for increasing the anthocyanin content in plants. The method involves co-expressing the ThR1 or ThR2 gene provided by the present invention with any MYB transcription factor in plant tissues and organs, thereby increasing the anthocyanin content in plant tissues and organs.

[0024] The MYB transcription factors can be isolated from any plant, including but not limited to the ThMYB1 and ThMYB2 genes provided in this invention, and the ZmC1 gene from maize (Ahmed N, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize C1 and B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1):29-34.).

[0025] The methods described above for increasing anthocyanin content in plants can be used to increase the anthocyanin content of any tissue or organ in a plant. Specifically, if you want to increase the anthocyanin content in all tissues of a plant as a whole, you can use constitutive promoters to express both the ThR1 or ThR2 gene and the MYB transcription factor. If you only want to increase the anthocyanin content in a specific tissue or organ, you can use promoters specifically expressed in that tissue or organ to express both the ThR1 or ThR2 gene and the MYB transcription factor.

[0026] This invention also provides a visible selection marker gene, which generates a visible blue selection marker in plant tissues and organs by co-expressing the ThMYB1 or ThMYB2 gene with any bHLH transcription factor. Alternatively, it generates a visible blue selection marker in plant tissues and organs by co-expressing the ThR1 or ThR2 gene provided by this invention with any MYB-type transcription factor.

[0027] The screening marker genes disclosed in this invention can be used to distinguish between transgenic and non-transgenic materials.

[0028] Specifically, the screening marker gene provided by this invention can be used as a screening marker in the reproduction process of male sterile lines. After the fertility restoration gene, pollen inactivation gene and the screening marker gene provided by this invention are transferred into the male sterile line, the fertility restoration gene can restore the fertility of the male sterile line, and the pollen inactivation gene can inactivate pollen containing the transformed exogenous gene, that is, lose its fertilization ability. The screening marker gene provided by this invention can be used for sorting transgenic seeds or tissues and non-transgenic seeds or tissues. The sorted non-transgenic seeds are used to produce hybrids from sterile lines, and the transgenic seeds are used to maintain the continuous and stable production of sterile lines.

[0029] The screening marker gene provided by this invention can also be used as a screening marker in the reproduction process of female-sterile lines. The female fertility gene, pollen inactivation gene, and the screening marker gene provided by this invention can be transferred into female-sterile lines. Specifically, the female fertility gene can restore fertility to the female-sterile transformation recipient material, and the pollen inactivation gene can inactivate pollen containing the transformed exogenous construct, i.e., disable its fertilization ability. The screening marker gene provided by this invention can be used for sorting transgenic and non-transgenic seeds. The sorted non-transgenic seeds can be used to produce hybrids from female-sterile lines, while the transgenic seeds can be used as maintainer lines to continuously and stably produce female-sterile lines and female-sterile maintainer lines.

[0030] This invention also provides a promoter for the blue grain gene, which exhibits aleurone layer-specific expression characteristics, and its nucleotide sequence is shown in SEQ ID NO:13, 14, 15, or 16. SEQ ID NO:13, 14, 15, or 16 is linked to the reporter gene GUS to construct a vector that transforms rice and wheat. The expression activity and pattern of GUS in the transgenic plants are detected and analyzed. GUS staining analysis of the roots, stems, leaves, flowers, and seeds of the transgenic plants reveals that the promoter provided by this invention drives the expression of the GUS gene in the aleurone layer of plant seeds. This indicates that the blue grain gene promoter SEQ ID NO:13, 14, 15, or 16 provided by this invention is an aleurone layer-specific expression promoter.

[0031] The aleurone layer-specific expression promoter provided by this invention contains nucleotide sequences as shown in SEQ ID NO:13, 14, 15, or 16, or contains nucleotide sequences with more than 90% similarity to the nucleotide sequences listed in SEQ ID NO:13, 14, 15, or 16, or contains 500 or more consecutive nucleotide fragments derived from the sequences in SEQ ID NO:13, 14, 15, or 16, and can drive the expression of nucleotide sequences operatively linked to this promoter in the aleurone layer of plant seeds. Expression vectors, transgenic cell lines, and host bacteria containing the above sequences are all within the scope of protection of this invention. Primer pairs for amplifying any nucleotide fragment of the promoters in SEQ ID NO:13, 14, 15, or 16 disclosed in this invention are also within the scope of protection of this invention.

[0032] The term "promoter" as used in this invention refers to a DNA regulatory region that typically contains a TATA box that directs RNA polymerase II to initiate RNA synthesis at a suitable transcription start site for a specific coding sequence. Promoters may also contain other recognition sequences, typically located upstream or at the 5' end of the TATA box, often referred to as upstream promoter elements, which regulate transcription efficiency. Those skilled in the art will recognize that while nucleotide sequences for the promoter regions disclosed herein have been identified, the isolation and identification of other regulatory elements located upstream of the TATA box in the specific promoter regions identified herein are also within the scope of this invention. Therefore, the promoter regions disclosed herein are generally further defined as containing upstream regulatory elements, such as those elements that regulate the tissue and temporal expression of coding sequences, enhancers, etc. Similarly, promoter elements that enable expression in target tissues (e.g., male tissues) can be identified and isolated, and used in conjunction with other core promoters to verify male-preferred expression. A core promoter refers to the minimum sequence required to initiate transcription, such as a sequence called a TATA box, which is typically present in promoters of protein-coding genes. Therefore, optionally, the aleurone layer-specific expression promoter provided by the present invention can be used in association with its own or other core promoters. The core promoter can be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (US Patent No. 5,352,605), the ubiquitin promoter (US Patent No. 5,510,474), the IN2 core promoter (US Patent No. 5,364,780), or the Scrophularia mosaic virus promoter.

[0033] The function of the gene promoter described in this invention can be analyzed by the following methods: operably linking the promoter sequence with a reporter gene to form a transformable vector, then transferring the vector into a plant, and in obtaining transgenic offspring, confirming its expression characteristics by observing the expression of the reporter gene in various tissues and organs of the plant; or subcloning the above vector into an expression vector for transient expression experiments, and detecting the function of the promoter or its regulatory region through transient expression experiments.

[0034] The choice of an appropriate expression vector for testing promoter or regulatory region function will depend on the host and the method of introducing the expression vector into the host, methods well known to those skilled in the art. For eukaryotes, the regions in the vector include those controlling transcription initiation and processing. These regions are operatively linked to reporter genes, including YFP, UidA, GUS genes, or luciferases. Expression vectors containing putative regulatory regions located within genomic fragments can be introduced into intact tissues, such as staged pollen, or into callus tissue for functional validation.

[0035] Furthermore, the nucleotide sequence or fragments or variants of the aleurone layer-specific expression promoter provided by this invention can be assembled together with a heterologous nucleotide sequence into an expression cassette for expression in a target plant, more specifically, in the seeds of that plant. The expression cassette has suitable restriction enzyme sites for inserting the promoter and the heterologous nucleotide sequence. These expression cassettes can be used to genetically manipulate any plant to obtain the desired phenotype.

[0036] The aleurone layer-specific expression promoter disclosed in this invention can be used to drive the expression of the following genes so that the transformed plants acquire the corresponding phenotypes. These genes include, but are not limited to, genes related to yield increase, genes that improve seed nutritional value, genes that increase anthocyanin content, and fluorescent protein genes.

[0037] The present invention also provides an expression cassette, vector, or engineered strain, wherein the expression cassette, vector, or engineered strain contains the aleurone layer-specific expression promoter SEQ ID NO:13, 14, 15, or 16 provided by the present invention, or contains 500 or more consecutive nucleotide fragments derived from the sequence SEQ ID NO:13, 14, 15, or 16.

[0038] The aleurone layer-specific expression promoter provided by this invention can be used for the specific expression of exogenous genes in seeds, thereby avoiding the adverse effects of continuous expression of the exogenous gene in other plant tissues, and has important application value in plant genetic engineering research.

[0039] The nucleotide sequence and promoter sequence or expression cassette of the blue grain gene provided by this invention can be inserted into vectors, plasmids, yeast artificial chromosomes, bacterial artificial chromosomes, or any other vector suitable for transformation into host cells. Preferred host cells are bacterial cells, especially those used for cloning or storing polynucleotides or for transforming plant cells, such as *Escherichia coli*, *Agrobacterium rhizogenes*, and *Agrobacterium capillaris*. When the host cell is a plant cell, the expression cassette or vector can be inserted into the genome of the transformed plant cell. Insertion can be targeted or random.

[0040] The transfer, introduction, or transformation of nucleotide sequences, vectors, or expression cassettes into or into plants, as described in this invention, refers to the transfer of nucleotide sequences, vectors, or expression cassettes into recipient cells or recipient plants using conventional transgenic methods. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform recombinant expression vectors into plant cells to produce the transgenic plants of this invention. Transformation methods can include direct and indirect methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, gene gun introduction, electroporation, and microinjection. The transformation methods also include Agrobacterium-mediated plant transformation methods, etc.

[0041] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a blue grain gene and its promoter, which can increase the anthocyanin content in plants. Since anthocyanins have antioxidant functions, in an era of increasing environmental pollution and people's growing pursuit of healthy living, the discovery of this anthocyanin synthesis-related gene undoubtedly increases the nutritional and medical value of edible plant parts. Simultaneously, while increasing the anthocyanin content of plants, this blue grain gene can also be used as a selection marker, eliminating the need for marker removal in transgenic processes, saving time and steps in transgenic bioengineering, and has significant application value in practical production.

[0042] References

[0043] Zhou K,Wang S,Feng Y,Liu Z,Wang G.The 4E-system of producing hybridwheat.Crop Sci.2006;46(1):250-255.

[0044] Li Zhensheng, Mu Sumei. Study on blue-grained monotypic wheat (Part 1) [J]. Acta Genetica Sinica, 1982(6): 15.

[0045] Zheng Q.,Li B.,Mu S.,Zhou H.,Li Z.(2006).Physical mapping of the blue-grained gene(s) from Thinopyrum ponticum by GISH and FISH in a set oftranslocation lines with different seed colors in wheat.Genome 49,1109-1114.

[0046] Dubcovsky,J.,Luo,MC,Zhong,GY,Bransteitter,R.,Desai,A.,Kilian,A.,et al.(1996).Genetic map of diploid wheat,Triticum monococcum L.,and its comparison with maps of Hordeum vulgare L.Genetics 143,983-999.

[0047] Himi,E.,and Noda,K.(2005).Red grain color gene(R)of wheat is a Myb-type transcription factor.Euphytica 143,239-242.

[0048] Khlestkina,EKGenes determining coloration of different organs in wheat.Russ.J.Genet.Appl.Res.2013,3,54-65.

[0049] Shoeva, OY, Gordeeva, EL, and Khlestkina, EK (2014). The regulation of anthocyanin synthesis in the wheat pericarp. Molecules 19, 20266-20279.

[0050] Gong Xia, Xue Jing, Zhang Xiaodong. 2011. Research progress on regulatory genes in the anthocyanin synthesis pathway in plants. Progress in Biotechnology 1(6):381-390

[0051] Ahmed N, Maekawa M, Utsugi S, Himi E, Ablet H, Rikiishi K, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize C1 and B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1):29-34.

[0052] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. Attached Figure Description

[0053] Figure 1 These are the grain colors of blue wheat and white wheat. The left image shows the dark blue grains of blue wheat 3114BB, while the right image shows the white grains of its parent, white wheat 3114.

[0054] Figure 2 The origin of the four blue-grained wheat genes ThMYB1, ThMYB2, ThR1, and ThR2 was verified using genomic PCR. The left image shows the agarose gel electrophoresis of the PCR products of the ThMYB1 and ThMYB2 genes, and the right image shows the agarose gel electrophoresis of the PCR products of the ThR1 and ThR2 genes. Expression of the four genes was not detected in the genome of white-grained wheat 3114, but it was detected in the genomes of blue-grained wheat 3114BB and long-spike wheatgrass.

[0055] Figure 3 The origin of the four blue-grained wheat genes was verified using semi-quantitative RT-PCR. Agarose gel electrophoresis of the PCR products showed that the expression of the four genes was not detected in the cDNA of white-grained wheat 3114, but it was detected in the cDNA of blue-grained wheat 3114BB, among which ACTIN is a housekeeping protein gene.

[0056] Figure 4 Semi-quantitative RT-PCR was used to verify the expression patterns of four blue grain genes in blue-grained wheat 3114BB. The left image shows agarose gel electrophoresis of PCR products of blue grain genes in different plant organs or tissues, and the right image shows agarose gel electrophoresis of PCR products of blue grain genes at various developmental stages in the aleurone layer, where ACTIN is the housekeeping protein gene. Expression of the four genes was not detected in cDNA from roots, stems, leaves, embryos, and endosperm, but high levels of expression of all four genes were detected in the aleurone layer. Furthermore, the expression levels of different blue grain genes were not entirely consistent at different developmental days in the aleurone layer. DPA refers to days post anthesis, i.e., "days after flowering".

[0057] Figure 5This study used a gene gun transient transformation experiment to verify that four blue-grain genes could induce the production of red anthocyanin spots in wheat coleoptiles. The blue-grain genes were combined in pairs and co-transformed into wheat coleoptiles. After culturing in a light incubator for 16 hours, microscopic observation of the wheat coleoptiles revealed that the ThMYB1+ThR1 and ThMYB2+ThR1 combinations induced the production of red anthocyanin spots in most cells, the ThMYB1+ThR2 combination only induced anthocyanin production in a very small number of cells, and the ThMYB2+ThR2 combination did not induce anthocyanin production at all.

[0058] Figure 6 This is a schematic diagram of the construction of a plant transformation vector used in wheat transgenic experiments. LB and RB represent the left and right boundaries of the T-DNA, respectively; the expression of the Bar resistance gene is driven by Ubip (the promoter of the Ubi gene) and terminated by the Nos terminator; the expression of the ThMYB1 and ThR1 genes is regulated by their respective promoters and terminators.

[0059] Figure 7 This study used Agrobacterium-mediated transformation to obtain the grain color of T1 generation plants of ThMYB1+ThR1 transgenic wheat in the fielder wheat variety background. The left image shows white grains of the non-transgenic wheat variety fielder, the middle image shows a line of T1 generation transgenic plants with dark blue grains, and the right image shows a line of T1 generation transgenic plants with light blue grains. Detailed Implementation

[0060] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0061] Example 1: Cloning of the Blue Grain Gene

[0062] To clone the blue-grain gene from chromosome 4Ag of *Thinopyrum longicornis*, this invention performed differential expression analysis on blue-grained and white-grained wheat (see...). Figure 1Theoretically, the differentially expressed genes between blue and white wheat include two types: 1) genes expressed by the 4Ag chromosome of *Thinopyrum elongatum*, which includes the blue-grain gene that this invention aims to clone; 2) downstream gene changes caused by the expression of the 4Ag chromosome of *Thinopyrum elongatum*, which are derived from the wheat genome. This invention aims to analyze and obtain the blue-grain gene described in 1). The materials selected were blue wheat 3114BB and its parent, white wheat 3114. Because the blue color of the aleurone layer of wheat grains begins to appear about 20 days after flowering, and the entire aleurone layer turns blue after about 25 days, this invention extracted aleurone layer samples 25 days after flowering. Two samples each of blue and white grains were taken, designated as blue 1, blue 2, white 1, and white 2, respectively. RNA was extracted and high-throughput sequencing (PE125) was performed, with each sample yielding 9G of data.

[0063] Because the published wheat reference genome only covers 61% of the entire wheat genome, and the gene annotations are incomplete and the scaffolds are fragmented, direct sequence alignment and differential gene expression analysis cannot identify target genes. This invention employs a three-step elimination method: first, excluding paired-end sequences that accurately align to the wheat reference genome; second, excluding genes highly expressed in white-grained wheat from the remaining paired-end short sequence reassembled genes; and finally, excluding genes unrelated to anthocyanin metabolism from the differentially expressed genes that meet the criteria.

[0064] Based on the above analysis, this invention identified 139 differentially expressed genes on non-wheat reference genomes, of which 35 were highly expressed in blue-grained wheat and almost not expressed in white-grained wheat. After gene functional annotation analysis, excluding genes unrelated to anthocyanin metabolism, only two target genes remained, encoding MYB and bHLH proteins, respectively. These were named ThMYB1 and ThR1. Based on the known cDNA sequences, this invention obtained the coding region genomic sequences of these two genes through PCR amplification. During this process, this invention discovered that both ThMYB1 and ThR1 yielded another highly homologous sequence during PCR amplification. Therefore, this invention cloned these two homologous sequences and named them ThMYB2 and ThR2, respectively. Using chromosome walking technology, this invention obtained the promoter and terminator sequences of these four genes.

[0065] The genomic nucleotide sequence of the ThMYB1 gene from start codon to stop codon is shown in SEQ ID NO:1, the nucleotide sequence of its coding region (CDS) is shown in SEQ ID NO:2, the amino acid sequence encoded by its CDS is shown in SEQ ID NO:3, its promoter sequence is shown in SEQ ID NO:13, and its terminator sequence is shown in SEQ ID NO:17. Similarly, the genomic nucleotide sequence of the ThMYB2 gene from start codon to stop codon is shown in SEQ ID NO:4, the nucleotide sequence of its coding region (CDS) is shown in SEQ ID NO:5, the amino acid sequence encoded by its CDS is shown in SEQ ID NO:6, its promoter sequence is shown in SEQ ID NO:14, and its terminator sequence is shown in SEQ ID NO:18. The genomic nucleotide sequence of the ThR1 gene from start codon to stop codon is shown in SEQ ID NO:7, its coding region (CDS) nucleotide sequence is shown in SEQ ID NO:8, the amino acid sequence encoded by its CDS is shown in SEQ ID NO:9, its promoter sequence is shown in SEQ ID NO:15, and its terminator sequence is shown in SEQ ID NO:19. The genomic nucleotide sequence of the ThR2 gene from start codon to stop codon is shown in SEQ ID NO:10, its coding region (CDS) nucleotide sequence is shown in SEQ ID NO:11, the amino acid sequence encoded by its CDS is shown in SEQ ID NO:12, its promoter sequence is shown in SEQ ID NO:16, and its terminator sequence is shown in SEQ ID NO:20.

[0066] This invention verified the origin of these four genes using genomic PCR and semi-quantitative RT-PCR. Genomic PCR showed that ThMYB1, ThMYB2, ThR1, and ThR2 were not amplified in white wheat 3114, but these four genes were amplified in the genomes of blue wheat 3114BB and *Thymus chinensis*, indicating that these four genes indeed originated from chromosome 4Ag of *Thymus chinensis* and not from common wheat (see...). Figure 2 Semi-quantitative RT-PCR results of cDNA in aleurone layer tissue 25 days after flowering also showed that ThMYB1, ThMYB2, ThR1, and ThR2 were not expressed in the aleurone layer of white wheat but were highly expressed only in the aleurone layer of blue wheat (see...). Figure 3 The above results indicate that the four genes ThMYB1, ThMYB2, ThR1, and ThR2 all originate from chromosome 4Ag of *Thymus chinensis* and are highly expressed in the aleurone layer of *Wheatgrass spp.*, and are likely the blue grain genes sought in this invention.

[0067] Example 2: Expression pattern of the blue grain gene

[0068] This invention used semi-quantitative RT-PCR to verify the expression patterns of these four genes. The results are as follows: Figure 4 As shown. First, the expression of the four genes was not detected in the roots, stems, and leaves of the blue wheat 3114BB vegetative organs. In different tissues of the seed 20 days after flowering (20 DPA), the four genes were specifically expressed in the aleurone layer tissue, but were not detected in the embryo and endosperm tissues. This indicates that the four genes are aleurone layer-specific genes, and their promoters are promoters that drive the specific expression of the genes in the aleurone layer. Furthermore, this invention analyzed the expression patterns of ThMYB1, ThMYB2, ThR1, and ThR2 in the aleurone layer of blue wheat seeds at different post-flowering days. It was found that the expression patterns of the two MYB genes and the two bHLH genes were not entirely the same: ThMYB1 and ThMYB2 were expressed at very low levels in the aleurone layer at 10 and 15 DPA, suddenly showing high expression at 20 DPA, and then gradually decreasing at 25 and 30 DPA; ThR1 and ThR2 were undetectable in the aleurone layer at 10 and 15 DPA, but showed sustained high expression from 20 to 30 DPA. These results indicate that ThMYB1, ThMYB2, ThR1, and ThR2 are all wheat aleurone layer-specific genes, and their expression exhibits spatiotemporal specificity.

[0069] The promoter sequences SEQ ID NO:13, 14, 15 and 16 of the above four genes were used to drive the GUS gene in rice, maize and other plants for functional verification. It was found that the above promoters all drive GUS to be specifically expressed in the aleurone layer, indicating that the promoters provided by the present invention are all aleurone layer-specific expression promoters.

[0070] Example 3: Experiment on transient transformation of wheat coleoptiles using a gene gun

[0071] In previous studies, the gene gun bombardment method was used to transiently transform wheat coleoptiles with maize MYB family transcription factor C1 and bHLH family transcription factor B1, which induced red anthocyanin spots in the wheat coleoptiles (Ahmed N, Maekawa M, Utsugi S, Himi E, Ablet H, Rikiishi K, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize C1 and B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1):29-34). To verify whether the two MYB genes and two bHLH genes obtained in this invention have the same function, this invention constructed a gene gun transient transformation vector using these four genes. First, the NOS terminator was cloned into the pEASY-T1 simple vector (TransGen). Then, the coding frames of the four genes ThMYB1, ThMYB2, ThR1, and ThR2 were inserted before the NOS terminator. Finally, the Ubi (Ubiquitin) promoter from maize was inserted before the gene coding frames using in-fusion to drive gene expression, resulting in four vectors: Ubi::ThMYB1, Ubi::ThMYB2, Ubi::ThR1, and Ubi::ThR2. The experimental method followed (Ahmed et al., 2003). The transformation vectors of the MYB and bHLH genes were combined in pairs: ThMYB1+ThR1, ThMYB1+ThR2, ThMYB2+ThR1, and ThMYB2+ThR2 were co-transformed into wheat coleoptiles. After culturing in a light incubator for 16 hours, the results were observed and photographed under a microscope. Figure 5 As shown, the combination of ThMYB1+ThR1 and ThMYB2+ThR1 can induce the production of red anthocyanins, and ThMYB1+ThR2 can also induce cells to produce anthocyanins.

[0072] Example 4: Construction of plant expression vectors

[0073] To further verify that ThMYB1, ThMYB2, ThR1, and ThR2 are indeed blue grain genes, this invention selected ThMYB1 and ThR1 genes to construct stable transformation vectors for wheat transgenic experiments. Using the binary expression vector pCAMBIA1300 as the backbone, the hygromycin resistance expression frame driven by the 35S promoter of the plant resistance selection gene on pCAMBIA1300 was first replaced with the Bar resistance gene expression frame driven by the Ubi promoter on the pAHC20 vector. Based on this, this invention inserted a 3215bp ThMYB1 genome sequence (containing a 1952bp promoter sequence, an 822bp genome sequence, and a 441bp terminator sequence) and a 4422bp ThR1 genome sequence (containing a 2084bp promoter sequence, a 1720bp CDS sequence, and a 618bp terminator sequence) into the multiple cloning site, thereby forming a plant expression vector (see...). Figure 6 ).

[0074] Example 5: Obtaining Transgenic Blue Wheat

[0075] The plant expression vector constructed in Example 4 was transformed into Agrobacterium strain C58C1 using electroporation. The vector was then transferred into the wheat variety Fielder using an Agrobacterium-mediated wheat transformation system, yielding 96 transgenic positive T0 generation plants. At harvest, the grain color of the T0 generation plants was observed, revealing that some lines showed dark blue grain segregation, while others (approximately 34%) showed light blue grain segregation. Detailed results are shown in [link to details]. Figure 7 As shown. The results of this transgenic study indicate that the co-expression of the ThMYB1 and ThR1 genes in the plant can increase the anthocyanin content in the plant. The ThMYB1, ThMYB2, ThR1, and ThR2 genes provided by this invention are blue grain genes in wheat.

[0076] Example 6: Functional verification of the blue grain gene in rice, Arabidopsis thaliana, and maize plants.

[0077] The ThMYB1 or ThMYB2 gene provided by this invention was combined with ThR1, ThR2, ZmR and ZmB genes in pairs, with one MYB gene plus one HLH gene, and then co-expressed in rice, Arabidopsis thaliana, maize and other plants. It was found that they also had the same function of increasing the anthocyanin content in the plants.

Claims

1. A blue grain gene for increasing anthocyanin content in wheat, characterized in that, The nucleotide sequence of the gene is selected from one of the following groups: (a) The nucleotide sequence shown in SEQ ID NO: 4; (b) The encoded amino acid sequence is the nucleotide sequence shown in SEQ ID NO:

6.

2. The blue grain gene according to claim 1, characterized in that, The CDS sequence of the blue grain gene is shown in SEQ ID NO:

5.

3. An expression box, characterized in that... The expression cassette contains a blue grain gene, the nucleotide sequence of which is selected from one of the following groups: (a) The nucleotide sequence shown in SEQ ID NO: 4; (b) The encoded amino acid sequence is the nucleotide sequence shown in SEQ ID NO:

6.

4. The expression box according to claim 3, characterized in that... The nucleotide sequence of the blue grain gene is shown in SEQ ID NO:

5.

5. The expression box according to claim 3, characterized in that... The blue grain gene can also be operatively linked to a promoter that can drive its expression, wherein the promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a spatiotemporally specific expression promoter.

6. The application of the blue grain gene as described in claim 1 or 2 or the expression cassette as described in claim 3 or 4 in increasing the anthocyanin content of wheat.

7. A method for increasing the anthocyanin content of wheat tissues or organs, the method comprising the step of co-expressing MYB-type transcription factors and bHLH transcription factors in plant tissues or organs, characterized in that, The MYB class transcription factor is the ThMYB2 gene, and its nucleotide sequence is selected from one of the following groups: (a) The nucleotide sequence shown in SEQ ID NO: 4; (b) The encoded amino acid sequence is the nucleotide sequence shown in SEQ ID NO: 6; The bHLH transcription factor is the ThR1 gene, and its nucleotide sequence is selected from one of the following groups: (a) The nucleotide sequence shown in SEQ ID NO: 7; (b) The encoded amino acid sequence is the nucleotide sequence shown in SEQ ID NO:

9.

8. The method according to claim 7, characterized in that The nucleotide sequence of the ThMYB2 gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the ThR1 gene is shown in SEQ ID NO:

8.

9. The application of the method of claim 7 in increasing the anthocyanin content of wheat tissues or organs.

Images