Method for creating colored barley rich in anthocyanin

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a method for creating colored barley rich in anthocyanin. The invention provides different gene expression cassettes capable of remarkably improving the anthocyanin content of barley seed endosperm and a corresponding multi-gene co-expression vector, and the anthocyanin content of the seed endosperm of an obtained transgenic plant is remarkably improved by transforming and cultivating the vector to barley Gloden promise. Based on the polygene co-expression vector, the invention provides a method for cultivating crops with seed endosperm rich in anthocyanin, the method can increase the anthocyanin content of the seed endosperm, the seed endosperm has high nutritional value, and an effective method is provided for creating plants rich in anthocyanin.

Description

Technical Field

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

[0002] Anthocyanin is a flavonoid compound with a unique C6-C3-C6 carbon skeleton structure, and is mainly present in the plant vacuole in the form of glycosides. The stability of anthocyanin is greatly affected under neutral and high pH conditions. In order to enhance the stability of anthocyanin, plants use different group modifications, adjust the vacuolar pH and co-pigmentation methods to adjust the color at the same time. Among them, delphinidin and its methylated derivatives, petunidin and malvidin, are the sources of dark blue and purple, and cyanidin and pelargonidin are the main sources of bright red.

[0003] Anthocyanins are mainly synthesized by structural genes and regulatory genes. Structural genes are enzymes in anabolism, while regulatory genes are transcription factors in anabolism. Studies in model plants have shown that metabolic structural genes and transcription factors in plants play a decisive role in the entire flavonoid metabolism, among which the ternary complex MBW composed of MYB, bHLH and WD40 is considered to play a major regulatory role. MYB transcription factors usually contain 1 to 4 MYB repeat sequences, and can be divided into R1-MYB, R2R3-MYB and R3-MYB families according to the number of repeat sequences. In the regulation of anthocyanins, R2R3-MYB transcription factors play a dominant role. bHLH proteins belong to zinc finger proteins, which are 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 with tryptophan-aspartic acid (Trp-Asp, WD), and their core region has 40 amino acid residues, so they are named WD40. WD40 family members often bind to MYB and bHLH family members to exert their effects. Based on the important physiological functions of anthocyanins, it is of great significance to increase the anthocyanin content of plants. However, there are no reports on increasing the anthocyanin content in barley. Summary of the invention

[0004] The invention provides a method for creating colorful barley rich in anthocyanin.

[0005] The present invention aims to create colored barley germplasm rich in anthocyanins. First, the expression of key genes for anthocyanin synthesis in barley seeds is analyzed, and further the key genes for anthocyanin synthesis in other plants (such as purple sweet potatoes, corn, etc.) are investigated to preliminarily determine the candidate genes for transforming barley. After a large number of screenings, three gene combinations that can significantly increase the anthocyanin content in the endosperm of barley seeds are finally determined. These three gene combinations are transformed into barley respectively, and can significantly increase the anthocyanin content in the endosperm of barley seeds, and can be used to create barley germplasm rich in anthocyanins.

[0006] Specifically, the present invention provides the technical solution described below.

[0007] In a first aspect, the present invention provides an application of a gene combination in increasing the anthocyanin content of a plant, wherein the gene combination comprises anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes derived from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); Wherein, the plants are plants other than purple sweet potatoes and corn.

[0008] Compared with other gene combinations, the above three gene combinations are significantly better in increasing the anthocyanin content in seeds of plants such as barley.

[0009] Preferably, the plant is a grass plant. The grass plant is preferably barley, wheat or oats.

[0010] In some embodiments of the invention, the plant is barley.

[0011] The present invention also provides an application of a gene combination in cultivating plants with increased anthocyanin content, wherein the gene combination comprises anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes derived from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); The plants are plants other than purple sweet potatoes and corn.

[0012] Preferably, the plant is a grass plant. The grass plant is preferably barley, wheat or oats.

[0013] In some embodiments of the invention, the plant is barley.

[0014] In the above application, the gene combination is transformed into the plant through transgenic technology to obtain positive transgenic plants.

[0015] In the present invention, the increased anthocyanin content is preferably an increased anthocyanin content in seeds, and more preferably an increased anthocyanin content in seed endosperm.

[0016] The present invention provides a method for creating a plant with increased anthocyanin content, the method comprising: modifying the plant so that the plant expresses a gene combination; The gene combination includes anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); The plants are plants other than purple sweet potatoes and corn.

[0017] The plant is preferably a grass plant. The grass plant can be barley, wheat or oats.

[0018] In some embodiments of the invention, the plant is barley.

[0019] Preferably, the method comprises: transforming a vector containing the gene combination into the plant.

[0020] Preferably, in the vector, each gene in the gene combination exists as an independent expression cassette; the expression cassette comprises a promoter, the gene in the gene combination and a terminator.

[0021] The present invention finds that, compared with using different vectors to carry different genes in the gene combination or expressing multiple genes in the gene combination under one promoter, constructing each gene in the gene combination into one vector with its own independent expression cassette structure (i.e., each gene uses its own promoter to control expression) to transform plants 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.

[0022] Preferably, the increased anthocyanin content is an increased anthocyanin content in seeds, more preferably an increased anthocyanin content in seed endosperm.

[0023] In order to specifically increase the anthocyanin content in the endosperm of plant seeds, the promoter is preferably an endosperm-specific promoter.

[0024] Preferably, the promoter is any one selected from TaWx-A, TaWx-B, TaWx-D, and 1Dx5 promoters. The nucleotide sequences of promoters 1Dx5, TaWx-A, TaWx-B, and TaWx-D are shown in SEQ ID NOs. 1, 2, 3, and 4, respectively.

[0025] Preferably, in the vector, the ANS, CHI, and DFR genes initiate transcription with the TaWx-A promoter; The CHS, F3'H, and F3H genes initiate transcription from the TaWx-B promoter; The 4CL gene initiates transcription from the 1Dx5 promoter; IbMYB1 and ZmPL genes initiate transcription from the TaWx-D promoter; The transcription of IbbHLH1, IbbHLH2, and ZmLC genes is initiated by the 1Dx5 promoter.

[0026] For the above three gene combinations, the promoter can be selected according to the genes contained in each gene combination according to the above promoter matching method.

[0027] In each of the above expression cassettes, a spacer sequence is further connected downstream of the terminator, and the spacer sequence is used to enhance the effect of the terminator in terminating transcription.

[0028] The present invention strengthens the function of terminating transcription by connecting a spacer sequence downstream of the terminator in each gene expression cassette, which is beneficial to ensure that each gene is successfully transcribed under the control of its corresponding promoter.

[0029] Preferably, the length of the spacer sequence is 1000-2500 bp, more preferably 2000-2500 bp.

[0030] In principle, the present invention has no particular limitation on the selection of the spacer sequence, and any segment in the barley genome that does not overlap with other genome segments and has a uniform base distribution and a moderate GC content can be selected.

[0031] The present invention has no limitation on the terminator used in each gene expression cassette, and any sequence that can terminate transcription can be used. As an example, the nopaline synthase gene (NOS) terminator can be selected.

[0032] The present invention also provides a recombinant vector, which comprises an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, an IbMYB1 gene expression cassette, and an IbbHLH1 gene expression cassette; Alternatively, comprising an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, an IbMYB1 gene expression cassette, and an IbbHLH2 gene expression cassette; Alternatively, it comprises an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, a ZmPL gene expression cassette, and a ZmLC gene expression cassette; Among them, ANS, CHS, 4CL, CHI, F3'H, F3H, DFR, IbMYB1, IbbHLH1, and IbbHLH2 genes were derived from purple sweet potato ( Ipomoea batatas ); The ZmPL and ZmLC genes are derived from maize (Zea mays L.).

[0033] Preferably, the expression cassette comprises a promoter, a gene in the gene combination and a terminator.

[0034] Preferably, the promoter of the ANS, CHI, DFR gene expression cassette is TaWx-A promoter; The promoter of the CHS, F3'H, and F3H gene expression cassettes is the TaWx-B promoter; The promoter of the 4CL gene expression cassette is the 1Dx5 promoter; The promoter of the IbMYB1 and ZmPL gene expression cassettes is the TaWx-D promoter; The promoter of IbbHLH1, IbbHLH2, and ZmLC gene expression cassettes is the 1Dx5 promoter.

[0035] In each of the above expression cassettes, a spacer sequence is further connected downstream of the terminator, and the spacer sequence is used to enhance the effect of the terminator in terminating transcription.

[0036] The present invention strengthens the function of terminating transcription by connecting a spacer sequence downstream of the terminator in each gene expression cassette, which is beneficial to ensure that each gene is successfully transcribed under the control of its corresponding promoter.

[0037] Preferably, the length of the spacer sequence is 1000-2500 bp, more preferably 2000-2500 bp.

[0038] The present invention provides the use of the above-mentioned recombinant vector or a biological material containing the recombinant vector in increasing the anthocyanin content of plants or cultivating plants with increased anthocyanin content.

[0039] The biological materials mentioned above include microbial cells or plant cells.

[0040] In the above application, the plant is a plant other than purple sweet potato and corn. The plant is preferably a grass plant; the grass plant can be barley, wheat or oats.

[0041] In some embodiments of the invention, the plant is barley.

[0042] The present invention also provides a transgenic barley with increased anthocyanin content, wherein the transgenic barley expresses a gene combination; The gene combination includes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes from purple sweet potato (Ipomoea batatas), and any one of the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.).

[0043] Preferably, the transgenic barley is prepared by the above-mentioned method for creating a plant with increased anthocyanin content.

[0044] Preferably, the transgenic barley with increased anthocyanin content is transgenic barley with increased anthocyanin content in seeds.

[0045] The beneficial effects of the present invention include at least: the present invention provides a gene combination that can significantly increase the anthocyanin content of barley seed endosperm, an expression cassette of each gene, and a multi-gene expression vector containing each gene expression cassette, and the multi-gene expression vector corresponding to each gene combination is transformed into barley, and the anthocyanin content of the seed endosperm of the obtained transgenic plant is significantly increased. Based on the above-mentioned gene combinations and their corresponding multi-gene expression vectors, the present invention provides a method for cultivating plants with increased anthocyanin content in seed endosperm, which can increase the anthocyanin content in seed endosperm, improve nutritional value, and have a high breeding efficiency, providing an effective method for creating plants rich in anthocyanins. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 The results of the analysis of the expression levels of anthocyanin synthesis-related genes in different organs of barley in Example 1 of the present invention are shown.

[0048] Figure 2 This is the result of detecting the expression of the key genes for anthocyanin synthesis in barley seeds in Example 1 of the present invention.

[0049] Figure 3 Schematic diagram of the framework structure of each gene expression cassette in the four intermediate vectors in Example 3 of the present invention and the upstream and downstream restriction sites.

[0050] Figure 4 and Figure 5 This is a schematic diagram of the expression cassette structure and its upstream and downstream restriction sites after each gene in Example 3 of the present invention is connected to the intermediate vector.

[0051] Figure 6 Schematic diagram of the structure of three multi-gene expression vectors in Example 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.

[0052] Figure 7 These are the expression level detection results of anthocyanin synthesis-related genes in different transgenic strains in Example 5 of the present invention.

[0053] Figure 8 This is the result of detecting the grain phenotype of the transgenic barley plant in Example 5 of the present invention.

[0054] Figure 7 and Figure 8 In the table, GP is barley Gloden promise (control), and ZM313, IB313-1, and IB313-2 are all transgenic barley plants. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] Example 1 Detection of the expression of key genes for anthocyanin synthesis in barley

[0057] First, the published barley transcriptome database (https: / / bar.utoronto.ca / eplant_barley / ) was used to search and analyze the expression levels of anthocyanin synthesis-related genes in different organs of barley. Figure 1 The results showed that only ANS, B1, Ant1, and Ant2 genes were not expressed in barley.

[0058] Furthermore, the expression of key genes for anthocyanin synthesis in barley seeds was detected. RNA was extracted from barley seeds, and the expression of key genes for anthocyanin synthesis in barley seeds, such as PAL, ANS, 4CL, C4H, bHLH, CHS, CHI, MYB, F3'H, F3H, WD40, and DFR, was detected by RT-PCR. The results are as follows: Figure 2 The results showed that ANS, CHS, and MYB genes were not detected in barley seeds, and although 4CL and F3'H genes were expressed, the expression levels were very low.

[0059] Example 2 Screening and determination of gene combinations

[0060] Based on the expression results of the key genes for anthocyanin synthesis in barley seeds in Example 1, a preliminary screening of multiple anthocyanin synthesis-related genes from plants of different origins was performed to determine candidate genes. The expression of some candidate genes and their source plants, gene accession numbers, and homologous genes of the candidate genes in barley are shown in Table 1.

[0061] Table 1

[0062] After combined screening, the following three gene combinations were finally determined to increase the anthocyanin content of barley seeds: (1)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH1, F3H, DFR; (2)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH2, F3H, DFR; (3)ANS, CHS, 4CL, ZmPL, CHI, F3'H, ZmLC, F3H, DFR.

[0063] The plant sources and gene accession numbers of the above genes are shown in Table 1.

[0064] Example 3 Construction of multi-gene expression vector

[0065] The three gene combinations determined in Example 2 were respectively connected to plant expression vectors to construct a multi-gene expression vector. The specific method is as follows: Using the plant expression vector pTRIDT313 as the starting vector, the pTRIDT313 vector and all DNA sequences that need to be connected to the vector are analyzed for restriction sites to determine the endonucleases that are not contained in the vector and the connected sequences. pTRIDT313 is modified to add the above-determined restriction sites, including: synthesizing the restriction site sequence, and then connecting it to the multi-cloning site of the vector by homologous recombination.

[0066] The pBI121 vector was modified and used as an intermediate vector to facilitate the sequential insertion of each gene expression cassette into the modified expression vector. The modification of the pBI121 vector mainly includes: (1) Replace the 35S promoter with 1Dx5, TaWx-A, TaWx-B, and TaWx-D promoters respectively; (2) Introducing the homotyping enzyme and the required restriction site upstream of the promoter and downstream of the terminator, respectively; (3) Add an invalid DNA sequence (about 2000 bp) downstream of the NOS terminator element to strengthen the termination effect of the NOS terminator and ensure that each gene is successfully transcribed under the control of its corresponding promoter; (4) Synthesize the restriction enzyme cleavage sites Pme I, Pac I, and Asc I sequences and connect them downstream of the invalid DNA sequence.

[0067] Among them, the invalid DNA sequence was selected from the 5286bp sequence at 37201524bp–37196239bp downstream of the Wx-D gene sequence of barley chromosome 7D. After comparison with NCBI, it was found that this segment did not overlap with any gene, and the base distribution of the sequence was uniform, with a GC content of 48.7%. It was divided into two segments, 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.

[0068] The above-mentioned vector transformation can be carried out by homologous recombination method.

[0069] The framework structure of each gene expression cassette in the four intermediate vectors obtained by the above transformation and the upstream and downstream restriction sites are as follows: Figure 3 shown.

[0070] Each gene in the three gene combinations in Example 2 was cloned from purple sweet potato and corn, respectively.

[0071] Each gene in the gene combination is connected to the above-mentioned different intermediate vectors according to the corresponding promoter used and the order of connection.

[0072] The promoter information corresponding to each gene is as follows: ANS, CHI, and DFR initiate transcription with the TaWx-A promoter; The CHS, F3'H, and F3H genes initiate transcription from the TaWx-B promoter; The 4CL gene initiates transcription from the 1Dx5 promoter; IbMYB1 and ZmPL genes initiate transcription from the TaWx-D promoter; The transcription of IbbHLH1, IbbHLH2, and ZmLC genes is initiated by the 1Dx5 promoter.

[0073] After each gene is connected to the intermediate vector, its expression cassette structure and its upstream and downstream restriction sites are as follows Figure 4 and Figure 5 shown.

[0074] The expression cassettes of each gene were sequentially connected to the modified pTRIDT313 expression vector using the above intermediate vectors to construct three multi-gene expression vectors, the structural schematic diagram of which is shown in FIG. Figure 6 shown.

[0075] Example 4 Construction of transgenic barley

[0076] The three multi-gene expression vectors constructed in Example 3 were transformed into barley respectively to construct transgenic plants of multi-gene transformed barley. The specific method is as follows: Using the barley genetic transformation system established earlier, the three multi-gene expression vectors constructed above were respectively transferred into the highly infective Agrobacterium strains AGL1 , infected young embryos of barley variety Golden Promise, and obtained regenerated plants through transformation culture processes such as co-culture, screening, differentiation, and rooting, combined with resistance screening, and selected transgenic positive plants using specific marker PCR. The specific operation is referred to the literature ( Transgenic Research 2019, 28: 225-235). After screening, transgenic positive plants IB313-1, IB313-2, and ZM313 were obtained. These three transgenic plants corresponded to the multi-gene expression vectors constructed by the following gene combinations (1), (2), and (3), respectively: (1)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH1, F3H, DFR; (2)ANS, CHS, 4CL, IbMYB1, CHI, F3'H, IbbHLH2, F3H, DFR; (3)ANS, CHS, 4CL, ZmPL, CHI, F3'H, ZmLC, F3H, DFR.

[0077] Example 5 Detection of gene expression, phenotype and anthocyanin content in transgenic plants

[0078] Real-time fluorescence quantitative PCR was used to detect the expression levels of anthocyanin synthesis-related genes in the seed endosperm of the barley transgenic plants constructed in Example 4. The results are as follows: Figure 7 The results showed that compared with the control plants (GP), the expression of genes related to the anthocyanin synthesis pathway in the seed endosperm of the transgenic plants was basically upregulated.

[0079] The content of anthocyanin components in the seed endosperm of the transgenic barley plants was detected by high performance liquid chromatography, 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 the transgenic plants IB313-1 and IB313-2 were significantly increased, and the contents of cyanidin and malvidin in ZM313 were significantly increased, but there was no significant difference in peonidin compared with the wild type. The results showed that the three multi-gene expression vectors constructed in Example 3 were able to effectively increase the anthocyanin content in the seed endosperm of barley after being introduced into the barley.

[0080] Table 2

[0081] The barley transgenic plants constructed in Example 4 were subjected to grain phenotype detection, and the results were as follows: Figure 8 The results showed that the anthocyanin content in the endosperm of IB313-1 and IB313-2 seeds increased significantly, and the seed color changed significantly compared with the wild type. Although the anthocyanin content in the endosperm of ZM313 seeds also increased significantly, the endosperm color of ZM313 seeds did not change significantly compared with the wild type.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 the anthocyanin content of plants, characterized in that: The gene combination includes anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); The plants are plants other than purple sweet potatoes and corn; Preferably, the plant is a grass plant; more preferably barley, wheat or oats.

2. Use of a gene combination in cultivating plants with increased anthocyanin content, characterized in that: The gene combination includes anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); The plants are plants other than purple sweet potatoes and corn; Preferably, the plant is a grass plant; more preferably barley, wheat or oats.

3. A method for creating a plant with increased anthocyanin content, characterized in that: The method comprises: modifying a plant so that it expresses a gene combination; The gene combination includes anthocyanin structural genes ANS, CHS, 4CL, CHI, F3'H, F3H and DFR genes from purple sweet potato (Ipomoea batatas), and any group of anthocyanin regulatory genes selected from the following (1)-(3): (1) IbMYB1 and IbbHLH1 genes from purple sweet potato (Ipomoea batatas); (2) IbMYB1 and IbbHLH2 genes from purple sweet potato (Ipomoea batatas); (3) ZmPL and ZmLC genes from maize (Zea mays L.); The plants are plants other than purple sweet potatoes and corn; Preferably, the plant is a grass plant; More preferably, the plant is barley, wheat or oats.

4. The method according to claim 3, characterized in that: The method comprises: transforming a vector containing the gene combination into the plant.

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

6. The method according to claim 5, characterized in that The promoter is an endosperm-specific promoter; Preferably, the promoter is any one selected from TaWx-A, TaWx-B, TaWx-D, and 1Dx5 promoters.

7. The method according to claim 5 or 6, characterized in that: In the vector, the ANS, CHI, and DFR genes initiate transcription with the TaWx-A promoter; The CHS, F3'H, and F3H genes initiate transcription from the TaWx-B promoter; The 4CL gene initiates transcription from the 1Dx5 promoter; IbMYB1 and ZmPL genes initiate transcription from the TaWx-D promoter; The transcription of IbbHLH1, IbbHLH2, and ZmLC genes is initiated by the 1Dx5 promoter.

8. The method according to any one of claims 5 to 7, characterized in that: A spacer sequence is also connected downstream of the terminator, and the spacer sequence is used to enhance the effect of the terminator in terminating transcription; Preferably, the length of the spacer sequence is 1000-2500 bp.

9. A recombinant vector, characterized in that The vector comprises an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, an IbMYB1 gene expression cassette, and an IbbHLH1 gene expression cassette; Alternatively, comprising an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, an IbMYB1 gene expression cassette, and an IbbHLH2 gene expression cassette; Alternatively, it comprises an ANS gene expression cassette, a CHS gene expression cassette, a 4CL gene expression cassette, a CHI gene expression cassette, a F3'H gene expression cassette, a F3H gene expression cassette, a DFR gene expression cassette, a ZmPL gene expression cassette, and a ZmLC gene expression cassette; Among them, ANS, CHS, 4CL, CHI, F3'H, F3H, DFR, IbMYB1, IbbHLH1, and IbbHLH2 genes were derived from purple sweet potato ( Ipomoea batatas ); The ZmPL and ZmLC genes are derived from maize ( Zea mays L.); Preferably, the expression cassette comprises a promoter, a gene in the gene combination and a terminator; Preferably, the promoter of the ANS, CHI, DFR gene expression cassette is TaWx-A promoter; The promoter of the CHS, F3'H, and F3H gene expression cassettes is the TaWx-B promoter; The promoter of the 4CL gene expression cassette is the 1Dx5 promoter; The promoter of the IbMYB1 and ZmPL gene expression cassettes is the TaWx-D promoter; The promoter of IbbHLH1, IbbHLH2, and ZmLC gene expression cassettes is the 1Dx5 promoter.

10. Use of the recombinant vector according to claim 9 or a biological material containing the recombinant vector in increasing the anthocyanin content of plants or cultivating plants with increased anthocyanin content; The plants are plants other than purple sweet potatoes and corn; Preferably, the plant is a grass plant; More preferably, the plant is barley, wheat or oats.

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