A method for creating colored barley rich in anthocyanins

Abstract

This invention relates to the field of plant genetic engineering technology, specifically to a method for creating colored barley rich in anthocyanins. The invention provides different gene expression cassettes and corresponding multi-gene co-expression vectors that can significantly increase the anthocyanin content in barley seed endosperm. Transforming this vector into cultivated barley (Golden Promise) results in transgenic plants with significantly increased anthocyanin content in the seed endosperm. Based on this multi-gene co-expression vector, the invention provides a method for cultivating crops with anthocyanin-rich seed endosperm. This method can increase the anthocyanin content in the seed endosperm, resulting in higher nutritional value and providing an effective method for creating anthocyanin-rich plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for creating colored barley rich in anthocyanins. Background Technology

[0002] Anthocyanins are flavonoid compounds with the characteristic C6-C3-C6 carbon skeleton structure of this class of compounds, and mainly exist in plant vacuoles in the form of glycosides. The stability of anthocyanins is greatly affected under neutral and high pH conditions. Plants employ various methods to enhance anthocyanin stability, including modification with different functional groups, adjustment of vacuolar pH, and co-precipitation of pigments, while simultaneously regulating color. Delphinidin and its methylated derivatives—petunidin and malvidin—are sources of deep blue and purple anthocyanins, while cyanidin and pelargonidin are the main sources of bright red anthocyanins.

[0003] Anthocyanin synthesis is primarily controlled by the combined action of structural genes and regulatory genes. Structural genes are enzymes involved in anabolic metabolism, while regulatory genes are transcription factors. Studies in model plants have shown that metabolic structural genes and transcription factors play a decisive role in the overall flavonoid metabolism. The ternary complex MBW, composed of MYB, bHLH, and WD40, is considered to play a major regulatory role. MYB transcription factors typically contain 1-4 MYB repeat sequences and can be classified into families such as R1-MYB, R2R3-MYB, and R3-MYB based on the number of repeat sequences. In anthocyanin regulation, the R2R3-MYB transcription factor plays a dominant role. bHLH proteins are zinc finger proteins, mainly composed of two amphipathic α-helices connected by a loop of variable length. Members of the IIIf subgroup of the bHLH family regulate flavonoid biosynthesis by binding to MYB proteins. Members of the WD40 protein family all end in tryptophan-aspartic acid (Trp-Asp, WD), and their core region contains 40 amino acid residues, hence the name WD40. Members of the WD40 family often bind with members of the MYB and bHLH families to exert their effects. Given the important physiological functions of anthocyanins, increasing their content in plants is of great significance. However, there are currently no reports of increasing anthocyanin content in barley. Summary of the Invention

[0004] This invention provides a method for creating colored barley rich in anthocyanins.

[0005] This invention aims to create colored barley germplasm rich in anthocyanins. First, the expression of key genes for anthocyanin synthesis in barley seeds was analyzed. Further research was conducted on key genes for anthocyanin synthesis in other plants (such as purple sweet potatoes and corn), and candidate genes for barley transformation were preliminarily identified. After extensive screening, three gene combinations that significantly increase the anthocyanin content in barley seed endosperm were finally determined. Transforming barley with these three gene combinations significantly increases the anthocyanin content in barley seed endosperm and can be used to create barley germplasm rich in anthocyanins.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides the application of a gene combination in increasing the anthocyanin content of plants, said gene combination comprising anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes derived from purple sweet potato (Ipomoea batatas), and any one group of anthocyanin regulatory genes selected from the following (1)-(3):

[0008] (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas);

[0009] (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas);

[0010] (3) The ZmPL and ZmLC genes derived from maize (Zea mays L.);

[0011] The plants in question are those other than purple sweet potatoes and corn.

[0012] Compared with other gene combinations, the above three gene combinations are significantly more effective in increasing the anthocyanin content of barley and other plant seeds.

[0013] Preferably, the plant is a grass (Poaceae). The grass is preferably barley, wheat, or oats.

[0014] In some embodiments of the present invention, the plant is barley.

[0015] The present invention also provides the application of gene combinations in cultivating plants with increased anthocyanin content, said gene combination comprising anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes derived from purple sweet potato (Ipomoea batatas), and any one group of anthocyanin regulatory genes selected from the following (1)-(3):

[0016] (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas);

[0017] (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas);

[0018] (3) The ZmPL and ZmLC genes derived from maize (Zea mays L.);

[0019] The plants mentioned are those other than purple sweet potatoes and corn.

[0020] Preferably, the plant is a grass (Poaceae). The grass is preferably barley, wheat, or oats.

[0021] In some embodiments of the present invention, the plant is barley.

[0022] In the above applications, the gene combination is transformed into the plant using transgenic technology to obtain positive transgenic plants.

[0023] In this invention, the increase in anthocyanin content is preferably an increase in the anthocyanin content of the seeds. More preferably, it is an increase in the anthocyanin content of the seed endosperm.

[0024] This invention provides a method for creating plants with increased anthocyanin content, the method comprising: modifying the plant to express a gene combination;

[0025] The gene combination includes the anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR from purple sweet potato (Ipomoea batatas), and any one of the following anthocyanin regulatory genes (1)-(3):

[0026] (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas);

[0027] (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas);

[0028] (3) The ZmPL and ZmLC genes derived from maize (Zea mays L.);

[0029] The plants mentioned are those other than purple sweet potatoes and corn.

[0030] The plant is preferably a grass (Poaceae). The grass may be barley, wheat, or oats.

[0031] In some embodiments of the present invention, the plant is barley.

[0032] Preferably, the method includes: transforming the plant with a vector containing the gene combination.

[0033] Preferably, in the vector, each gene in the gene combination exists as an independent expression cassette; the expression cassette includes a promoter, the gene in the gene combination, and a terminator.

[0034] This invention discovers that, compared to using different vectors to carry different genes in the gene combination or expressing multiple genes in the gene combination under a single promoter, constructing each gene in the gene combination with its own independent expression cassette structure (i.e., each gene uses its own promoter to control expression) into a single vector for plant transformation can better ensure the expression effect of each gene, which is beneficial to promoting the metabolic balance of the anthocyanin synthesis pathway in plants, promoting anthocyanin synthesis, and increasing its content.

[0035] Preferably, the increase in anthocyanin content refers to an increase in the anthocyanin content of the seeds. More preferably, it refers to an increase in the anthocyanin content of the seed endosperm.

[0036] To specifically increase the anthocyanin content in the endosperm of plant seeds, the promoter is preferably an endosperm-specific promoter.

[0037] Preferably, the promoter is selected from any one of the promoters TaWx-A, TaWx-B, TaWx-D, and 1Dx5. The nucleotide sequences of promoters 1Dx5, TaWx-A, TaWx-B, and TaWx-D are shown in SEQ ID NO.1, 2, 3, and 4, respectively.

[0038] Preferably, in the vector, the ANS, CHI, and DFR genes are transcribed using the TaWx-A promoter;

[0039] The CHS, F3'H, and F3H genes are transcribed starting with the TaWx-B promoter;

[0040] The 4CL gene is transcribed using the 1Dx5 promoter;

[0041] The transcription of IbMYB1 and ZmPL genes is initiated by the TaWx-D promoter;

[0042] The IbbHLH1, IbbHLH2, and ZmLC genes are transcribed starting with the 1Dx5 promoter.

[0043] For the three gene combinations mentioned above, promoters can be selected according to the promoter pairing method described above, based on the genes contained in each gene combination.

[0044] In each of the above expression cassettes, a spacer sequence is attached downstream of the terminator, which is used to enhance the effect of the terminator in terminating transcription.

[0045] This invention enhances the transcription termination effect by connecting the downstream spacer sequence of the terminator in each gene expression cassette, which helps ensure that each gene is successfully transcribed under the control of its corresponding promoter.

[0046] Preferably, the length of the interval sequence is 1000-2500 bp. More preferably, it is 2000-2500 bp.

[0047] In principle, there are no special restrictions on the selection of spacer sequences. Any segment in the barley genome that does not overlap with other genomic segments, has a uniform base distribution, and a moderate GC content can be selected.

[0048] This invention does not limit the terminators used for each gene expression cassette; any sequence that can terminate transcription can be used. As an example, the terminator for the carmine synthase gene (NOS) can be selected.

[0049] The present invention also provides a recombinant vector comprising an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, an F3'H gene expression cassette, an F3H gene expression cassette, a DFR gene expression cassette, an IbMYB1 gene expression cassette, and an IbbHLH1 gene expression cassette.

[0050] Alternatively, it may include the ANS gene expression cassette, CHS gene expression cassette, 4CL gene expression cassette, CHI gene expression cassette, F3'H gene expression cassette, F3H gene expression cassette, DFR gene expression cassette, IbMYB1 gene expression cassette, and IbbHLH2 gene expression cassette.

[0051] Alternatively, it may include the ANS gene expression cassette, CHS gene expression cassette, 4CL gene expression cassette, CHI gene expression cassette, F3'H gene expression cassette, F3H gene expression cassette, DFR gene expression cassette, ZmPL gene expression cassette, and ZmLC gene expression cassette.

[0052] Among them, the genes ANS, CHS, 4CL, CHI, F3'H, F3H, DFR, IbMYB1, IbbHLH1, and IbbHLH2 are derived from purple sweet potato (Ipomoea batatas).

[0053] The ZmPL and ZmLC genes are derived from maize (Zea mays L.).

[0054] Preferably, the expression cassette includes a promoter, a gene from the gene combination, and a terminator.

[0055] Preferably, the promoters for the ANS, CHI, and DFR gene expression cassettes are the TaWx-A promoters;

[0056] The promoters for the CHS, F3'H, and F3H gene expression cassettes are the TaWx-B promoters.

[0057] The promoter for the 4CL gene expression cassette is the 1Dx5 promoter;

[0058] The promoters for the IbMYB1 and ZmPL gene expression cassettes are the TaWx-D promoters;

[0059] The promoters for the IbbHLH1, IbbHLH2, and ZmLC gene expression cassettes are the 1Dx5 promoters.

[0060] In each of the above expression cassettes, a spacer sequence is attached downstream of the terminator, which is used to enhance the effect of the terminator in terminating transcription.

[0061] This invention enhances the transcription termination effect by connecting the downstream spacer sequence of the terminator in each gene expression cassette, which helps ensure that each gene is successfully transcribed under the control of its corresponding promoter.

[0062] Preferably, the length of the interval sequence is 1000-2500 bp. More preferably, it is 2000-2500 bp.

[0063] The present invention provides the application of the above-described recombinant vector or biological material containing the recombinant vector in increasing the anthocyanin content of plants or cultivating plants with increased anthocyanin content.

[0064] The biomaterials mentioned above include microbial cells or plant cells.

[0065] In the above applications, the plant is any plant other than purple sweet potato and corn. The plant is preferably a grass (Poaceae); the grass may be barley, wheat, or oats.

[0066] In some embodiments of the present invention, the plant is barley.

[0067] The present invention also provides a transgenic barley with increased anthocyanin content, wherein the transgenic barley expresses a gene combination;

[0068] The gene combination includes the ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes derived from purple sweet potato (Ipomoea batatas), and any one of the following (1)-(3):

[0069] (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas);

[0070] (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas);

[0071] (3) The ZmPL and ZmLC genes derived from maize (Zea mays L.).

[0072] Preferably, the genetically modified barley is prepared using the method described above for creating plants with increased anthocyanin content.

[0073] Preferably, the transgenic barley with increased anthocyanin content is transgenic barley with increased anthocyanin content in its seeds.

[0074] The beneficial effects of this invention include at least the following: This invention provides gene combinations that significantly increase the anthocyanin content in barley seed endosperm, expression cassettes for each gene, and multi-gene expression vectors containing each gene expression cassette. Transforming barley with the multi-gene expression vectors corresponding to each gene combination results in transgenic plants with significantly increased anthocyanin content in the seed endosperm. Based on the above gene combinations and their corresponding multi-gene expression vectors, this invention provides a method for cultivating plants with increased anthocyanin content in seed endosperm. This method can increase the anthocyanin content in seed endosperm, improve nutritional value, and has high breeding efficiency, providing an effective method for creating anthocyanin-rich plants. Attached Figure Description

[0075] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0076] Figure 1 The results show the expression levels of anthocyanin synthesis-related genes in different organs of barley in Example 1 of this invention.

[0077] Figure 2 This is the result of detecting the expression of key genes for anthocyanin synthesis in barley seeds in Example 1 of the present invention.

[0078] Figure 3 This is a schematic diagram of the framework structure of each gene expression cassette in the four intermediate vectors of Example 3 of the present invention and its upstream and downstream restriction enzyme sites.

[0079] Figure 4 and Figure 5 This is a schematic diagram of the expression cassette structure and upstream and downstream restriction sites of each gene after it is ligated into the intermediate vector in Example 3 of the present invention.

[0080] Figure 6This is a schematic diagram of the structure of three multi-gene expression vectors in Embodiment 3 of the present invention; wherein, A, B, D5, and D represent promoters TaWx-A, TaWx-B, 1Dx5, and TaWx-D, respectively; DNA-1 and DNA-2 represent invalid DNA sequence 1 and invalid DNA sequence 2, respectively.

[0081] Figure 7 The results show the expression levels of anthocyanin synthesis-related genes in different transgenic lines in Example 5 of this invention.

[0082] Figure 8 The results of grain phenotypic detection of the barley transgenic plant in Example 5 of this invention are shown.

[0083] Figure 7 and Figure 8 In the study, GP represents the barley Golden Promise (control), while ZM313, IB313-1, and IB313-2 are all transgenic barley plants. Detailed Implementation

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0085] Example 1: Detection of expression of key genes in anthocyanin synthesis in barley

[0086] First, the expression levels of anthocyanin synthesis-related genes in different organs of barley were retrieved and analyzed using the published barley transcriptome database (https: / / bar.utoronto.ca / eplant_barley / ). The results are as follows: Figure 1 As shown in the figure. The results showed that only the ANS, B1, Ant1, and Ant2 genes were not expressed in barley.

[0087] Furthermore, the expression of key genes involved in anthocyanin synthesis in barley seeds was investigated. RNA was extracted from barley seeds, and the expression levels of key genes involved in anthocyanin synthesis, including PAL, ANS, 4CL, C4H, bHLH, CHS, CHI, MYB, F3'H, F3H, WD40, and DFR, were detected using RT-PCR. The results are shown below. Figure 2 As shown in the figure. The results showed that the ANS, CHS, and MYB genes were not detected to be expressed in barley seeds, and although the 4CL and F3'H genes were expressed, the expression levels were very low.

[0088] Example 2: Screening and Determination of Gene Combinations

[0089] Based on the expression results of key genes for anthocyanin synthesis in barley seeds in Example 1, a preliminary screening of multiple anthocyanin synthesis-related genes from different plant sources was conducted to identify candidate genes. Table 1 shows some candidate genes, their source plants, gene accession numbers, and the expression of homologous genes of the candidate genes in barley.

[0090] Table 1

[0091]

[0092] Through combination screening, the following three gene combinations were finally identified for increasing the anthocyanin content of barley seeds:

[0093] (1)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH1, F3H, DFR;

[0094] (2)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH2, F3H, DFR;

[0095] (3)ANS, CHS, 4CL, ZmPL, CHI, F3'H, ZmLC, F3H, DFR.

[0096] The plant sources and gene accession numbers of the above genes are shown in Table 1.

[0097] Example 3 Construction of a multi-gene expression vector

[0098] The three gene combinations determined in Example 2 were respectively ligated into plant expression vectors to construct multi-gene expression vectors. The specific methods are as follows:

[0099] Using the plant expression vector pTRIDT313 as the starting vector, restriction enzyme site analysis was performed on the pTRIDT313 vector and all DNA sequences to be ligated into it to identify restriction enzymes not present in the vector or the ligation sequences. pTRIDT313 was then modified to add the identified restriction enzyme sites, including synthesizing restriction enzyme site sequences, and then ligated into the multiple cloning site of the vector via homologous recombination.

[0100] The pBI121 vector was modified to serve as an intermediate vector, facilitating the sequential ligation of gene expression cassettes into the modified expression vector. The modification of the pBI121 vector mainly included:

[0101] (1) Replace the 35S promoter with 1Dx5, TaWx-A, TaWx-B and TaWx-D promoters respectively;

[0102] (2) Introduce the same tail enzyme and the required restriction site upstream of the promoter and downstream of the terminator, respectively;

[0103] (3) Add an invalid DNA sequence (about 2000 bp) downstream of the NOS terminator element to enhance the termination effect of the NOS terminator and ensure that each gene is successfully transcribed under the control of its corresponding promoter.

[0104] (4) Synthesize the Pme I, Pac I, and Asc I enzyme cleavage sites and insert them downstream of the invalid DNA sequence.

[0105] The invalid DNA sequence was selected from a 5286 bp segment located downstream of the Wx-D gene sequence on barley chromosome 7D, at a distance of 37201524 bp–37196239 bp. NCBI alignment showed no overlap with any other gene in this region, and the sequence exhibited a uniform base distribution and a GC content of 48.7%. This segment was divided into two parts, which were used as invalid DNA sequences inserted downstream of the NOS terminator. The nucleotide sequences of invalid DNA sequence 1 and invalid DNA sequence 2 are shown in SEQ ID NO. 5 and 6, respectively.

[0106] The aforementioned carrier modification can be achieved using the homologous recombination method.

[0107] The framework structure of each gene expression cassette and its upstream and downstream restriction enzyme sites in the four intermediate vectors obtained by the above modifications are as follows: Figure 3 As shown.

[0108] Each gene from the three gene combinations in Example 2 was cloned from purple sweet potato and corn, respectively.

[0109] Each gene in the gene combination was ligated into the different intermediate vectors mentioned above, according to its corresponding promoter and the order in which it was ligated.

[0110] The promoter information for each gene is as follows:

[0111] ANS, CHI, and DFR initiate transcription with the TaWx-A promoter;

[0112] The CHS, F3'H, and F3H genes are transcribed starting with the TaWx-B promoter;

[0113] The 4CL gene is transcribed using the 1Dx5 promoter;

[0114] The transcription of IbMYB1 and ZmPL genes is initiated by the TaWx-D promoter;

[0115] The IbbHLH1, IbbHLH2, and ZmLC genes are transcribed starting with the 1Dx5 promoter.

[0116] After each gene is ligated into the intermediate vector, its expression cassette structure and its upstream and downstream restriction enzyme sites are as follows: Figure 4 and Figure 5 As shown.

[0117] Using the aforementioned intermediate vectors, the expression cassettes of each gene were sequentially ligated into the modified pTRIDT313 expression vector, resulting in three multi-gene expression vectors, the structural diagrams of which are shown below. Figure 6 As shown.

[0118] Example 4: Construction of transgenic barley

[0119] The three multi-gene expression vectors constructed in Example 3 were transformed into barley to construct transgenic barley plants with multiple gene expression vectors. The specific methods are as follows:

[0120] Using the previously established barley genetic transformation system, the three multi-gene expression vectors constructed above were transformed into highly infectious Agrobacterium strains. AGL1 The embryos of the barley variety Golden Promise were infected, and after transformation culture processes including co-culture, screening, differentiation, and rooting, regenerated plants were obtained through resistance screening. Transgenic positive plants were then screened using specific marker PCR. Specific procedures were described in reference (…). Transgenic Research 2019, 28: 225-235). After screening, transgenic positive plants IB313-1, IB313-2, and ZM313 were obtained. These three transgenic plants correspond to the following gene combinations (1), (2), and (3) respectively to construct multi-gene expression vectors:

[0121] (1)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH1, F3H, DFR;

[0122] (2)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH2, F3H, DFR;

[0123] (3)ANS, CHS, 4CL, ZmPL, CHI, F3'H, ZmLC, F3H, DFR.

[0124] Example 5: Detection of gene expression, phenotype, and anthocyanin content in transgenic plants.

[0125] The expression levels of anthocyanin synthesis-related genes in the seed endosperm of the barley transgenic plants constructed in Example 4 were detected using real-time quantitative PCR. The results are as follows: Figure 7As shown in the figure. The results showed that, compared with the control plant (GP), the expression of genes related to the anthocyanin synthesis pathway in the seed endosperm of the transgenic plant was generally upregulated.

[0126] The anthocyanin content in the seed endosperm of transgenic barley plants was detected by high-performance liquid chromatography (HPLC), and the results are shown in Table 2. The results showed that compared with the control plant (GP), the contents of cyanidin, peonidin, and malvidin in transgenic plants IB313-1 and IB313-2 were significantly increased. In ZM313, the contents of cyanidin and malvidin were significantly increased, but the peonidin content was not significantly different from that of the wild type. These results indicate that the three multi-gene expression vectors constructed in Example 3 can effectively increase the anthocyanin content in the seed endosperm of barley.

[0127] Table 2

[0128]

[0129] The grain phenotype of the barley transgenic plants constructed in Example 4 was detected, and the results are as follows: Figure 8 As shown in the figure. The results showed that the anthocyanin content in the seed endosperm of IB313-1 and IB313-2 was significantly increased, and the seed color was significantly different from that of the wild type. Although the anthocyanin content in the seed endosperm of ZM313 was also significantly increased, the seed endosperm color of ZM313 was not significantly different from that of the wild type.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of gene combination in increasing anthocyanin content in barley endosperm, characterized in that, The gene combination includes the anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR from purple sweet potato (Ipomoea batatas), and any one of the following anthocyanin regulatory genes (1)-(2): (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas); The application is accomplished by introducing a vector into barley, in which the ANS, CHI, and DFR genes are transcribed using the TaWx-A promoter. The CHS, F3'H, and F3H genes are transcribed starting with the TaWx-B promoter; The 4CL gene is transcribed using the 1Dx5 promoter; The IbMYB1 gene is transcribed starting with the TaWx-D promoter; The IbbHLH1 and IbbHLH2 genes are transcribed starting with the 1Dx5 promoter; The nucleotide sequences of the promoters 1Dx5, TaWx-A, TaWx-B, and TaWx-D are shown in SEQ ID NO. 1, 2, 3, and 4, respectively.

2. The application of gene combination in cultivating barley with increased anthocyanin content in the endosperm, characterized in that, The gene combination includes the anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR from purple sweet potato (Ipomoea batatas), and any one of the following anthocyanin regulatory genes (1)-(2): (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas); The application is accomplished by introducing a vector into barley, wherein the combination of genes and promoters in the vector and the promoter sequence are as described in claim 1.

3. A method for producing barley with increased anthocyanin content in the endosperm, characterized in that, The method includes: modifying barley to express a combination of genes; The gene combination includes the anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR from purple sweet potato (Ipomoea batatas), and any one of the following anthocyanin regulatory genes (1)-(2): (1) IbMYB1 and IbbHLH1 genes derived from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes derived from purple sweet potato (Ipomoea batatas); The application is accomplished by introducing a vector into barley, wherein the combination of genes and promoters in the vector and the promoter sequence are as described in claim 1.

4. The method according to claim 3, characterized in that, In the vector, each gene in the gene combination exists as an independent expression cassette; The expression cassette includes a promoter, a gene from the gene combination, and a terminator.

5. The method according to claims 3 and 4, characterized in that, Downstream of the terminator is a spacer sequence, which enhances the terminator's effect in terminating transcription.

6. The method according to claim 5, characterized in that, The length of the interval sequence is 1000-2500 bp.

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