CebHLH113 gene for regulating and controlling synthesis of colocasia esculenta anthocyanin and application thereof

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to a CebHLH113 gene for regulating and controlling synthesis of colocasia esculenta anthocyanin and application of the CebHLH113 gene. The amino acid sequence of the protein coded by the CebHLH113 gene is as shown in SEQ ID NO: 1. According to the invention, a key gene CebHLH113 for positively regulating and controlling anthocyanin synthesis is separated from red-pulp taro No. 3 of Ganxi taro for the first time. Overexpression of the CebHLH113 gene can significantly promote accumulation of anthocyanin in taro calluses, and silencing of the gene in stem tips significantly reduces the accumulation level of anthocyanin. The taros anthocyanin metabolism transcription regulation and control method fills the blank of a taros anthocyanin metabolism transcription regulation and control mechanism, is beneficial for providing important theoretical basis and technical support for improving the color and luster quality of taros by a molecular breeding technology and cultivating high-quality taros with high anthocyanin content, and has wide application value.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a method for regulating anthocyanin synthesis in taro. CebHLH113 Genes and their applications. Background Technology

[0002] taro( Colocasia esculenta Taro (L.) Schott, also known as taro root or taro vine, is a perennial monocotyledonous herbaceous plant belonging to the genus *Colocasia* of the family Araceae. As a traditional vegetable and grain crop in my country, its cultivation history spans over 9,000 years. Taro not only carries a rich historical and cultural significance but is also rich in nutrients such as starch, high-quality protein, polysaccharides, vitamins, and dietary fiber. In traditional Chinese medicine, it is believed to have nourishing, spleen-strengthening, stomach-nourishing, and detoxifying effects, making it highly valuable for human health. In recent years, thanks to its unique flavor and rich nutritional value, taro has increasingly gained popularity among consumers and attracted widespread attention from researchers.

[0003] Anthocyanins are important water-soluble secondary metabolites in plants, mainly stored in cell vacuoles. They not only impart diverse colors to flowers, fruits, tubers, and other organs, but also enhance plant resistance to stresses such as drought, salinity, and pests. Furthermore, their strong antioxidant activity makes them widely applicable in the food and medical fields, giving them significant development value. The white, yellow, red, and purple colors of taro corms are primarily determined by anthocyanins. Studies have detected components such as pelargonidin-3-glucoside and cyanidin-3-rhamnoside in the corms, and anthocyanin-3-rutinosyl has also been identified in the stems, confirming that anthocyanins are the key substances in the formation of taro's color.

[0004] Basic / helix-loop-helix (bHLH) transcription factors are the second largest family of transcription factors in plants, widely involved in numerous biological processes such as growth and development, signal transduction, metabolic regulation, and stress response. Members of this family have been identified in various plants, and their functions have been thoroughly investigated. Studies have shown that bHLH transcription factors play diverse regulatory roles in different plants. For example, approximately 10% of bHLH members in Arabidopsis thaliana are involved in maintaining iron homeostasis; in rice… OsbHLH107 By regulating the proliferation of ear husk cells, bHLH transcription factors influence grain size. In cotton, they are also closely related to the accumulation of secondary metabolites such as cottonseed oil, gossypol, and anthocyanins. Although the functions of bHLH transcription factors in plant growth and development are well understood, due to species-specific differences, the mechanisms by which this family regulates anthocyanin biosynthesis may be species-specific. Related research is still incomplete and requires further in-depth exploration.

[0005] It is evident that the bHLH gene family is characterized by a large number of members and significant functional differentiation, with different members playing distinct roles in biological processes. Whether bHLH family genes participate in and regulate the synthesis of anthocyanins in taro remains unclear. Therefore, identifying key genes in taro that regulate anthocyanin biosynthesis will provide important theoretical basis and technical support for improving the color and quality of taro through molecular breeding techniques and cultivating high-quality taro varieties with high anthocyanin content. Summary of the Invention

[0006] The purpose of this invention is to provide a method for regulating anthocyanin synthesis. CebHLH113 Genes and their applications. (The following is a separate section...) CebHLH113 Gene overexpression significantly promotes anthocyanin accumulation in taro callus tissue, while gene silencing in shoot tips significantly reduces anthocyanin accumulation. This finding provides important theoretical basis and technical support for improving taro color quality and cultivating high-anthocyanin-content taro varieties through molecular breeding techniques, and has broad application value.

[0007] This invention provides a method for regulating anthocyanin synthesis in taro. CebHLH113 Genes, the ones mentioned CebHLH113 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1.

[0008] As a preferred embodiment, the CebHLH113 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0009] The present invention also provides a method for regulating CebHLH113 Biological materials with high gene expression levels, including overexpression CebHLH113 Gene biomaterials, inhibition of the above CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression; the method of inhibition includes silencing.

[0010] As a preferred embodiment, the overexpression CebHLH113 Biological materials for gene overexpression include: CebHLH113 Gene amplification primers, overexpression... CebHLH113 Recombinant vectors of genes, overexpression of the aforementioned CebHLH113 Recombinant microorganisms of genes, overexpression of the aforementioned CebHLH113 Transgenic cell lines and overexpression of the gene CebHLH113 Any one or more genes in the tissues of transgenic plants; The basic framework of the recombinant vector includes the pSuper1300 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium.

[0011] As a preferred embodiment, the amplification primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:4.

[0012] As a preferred solution, suppress the CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression include: those containing the above. CebHLH113 Silencing vectors containing the gene silencing target, CebHLH113 A gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, a recombinant microorganism containing the knockout vector, and silencing. CebHLH113 Plant tissues and gene knockout CebHLH113 Any one or more genes in plant tissues; The basic framework of the silencing vector includes pTRV2.

[0013] The present invention also provides the aforementioned CebHLH113 The application of genes or the aforementioned biological materials in regulating anthocyanin synthesis and / or breeding high-anthocyanin taro varieties.

[0014] As a preferred embodiment, the regulation includes: overexpressing the CebHLH113 Genes that promote anthocyanin synthesis in taro; and genes that inhibit the synthesis of anthocyanins in taro. CebHLH113 Gene expression or knockout CebHLH113 Genes that inhibit the accumulation of anthocyanins in taro.

[0015] This invention also provides a method for promoting the synthesis of taro anthocyanins, comprising the following steps: overexpressing the aforementioned anthocyanins in a target taro plant. CebHLH113 Genes promote the synthesis of anthocyanins in taro.

[0016] This invention also provides a method for breeding high-anthocyanin taro varieties, comprising the following steps: overexpressing the aforementioned anthocyanin in the target taro. CebHLH113 Genes were used to obtain taro varieties with high anthocyanin content.

[0017] Beneficial effects: This invention provides a method for regulating anthocyanin synthesis. CebHLH113 Genes, the ones mentioned CebHLH113 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:1. This invention is the first to isolate a protein from the "Ganyu No. 3" red-fleshed taro variety that can promote anthocyanin accumulation in taro. CebHLH113 Genes. The results of this invention's embodiments show that genes obtained through genetic engineering... CebHLH113Transgenic taro callus tissue exhibited higher anthocyanin accumulation, while the anthocyanin content was reduced in the stem tips of the "Ganyu No. 3" red-fleshed taro variety after the gene was silenced. This demonstrates the advantages of the present invention. CebHLH113 Genes can regulate the synthesis of anthocyanins from taro. This invention fills the technical gap in the study of the functional analysis of bHLH family genes in the molecular regulation of anthocyanin biosynthesis in taro. CebHLH113 The application of gene technology has broad prospects, providing key technical support and theoretical basis for the molecular breeding of taro color and quality improvement and high-anthocyanin content high-quality varieties. After application, it can enhance the nutritional and economic value of taro, and has significant economic and social benefits. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 for CebHLH113 Image showing the results of gene amplification; Figure 2 Anthocyanin content at different developmental stages of the "Ganyu No. 3" red-fleshed taro and CebHLH113 The results of the relative gene expression analysis are shown in the figure; where A represents the anthocyanin content of "Ganyu No. 3" red-fleshed taro at different developmental stages; and B represents the anthocyanin content of "Ganyu No. 3" red-fleshed taro at different developmental stages. CebHLH113 Relative gene expression analysis; Figure 3 for CebHLH113 Figure showing the results of bioinformatics analysis of gene-encoded proteins; where A represents hydrophobicity prediction; B represents signal peptide prediction; C represents transmembrane peptide prediction; and D represents tertiary structure prediction. Figure 4 For 35S: CebHLH113 - Subcellular localization of the GFP fusion protein in tobacco leaves; from left to right: green fluorescence, nuclear labeling, bright field, and superimposed field; scale bar = 20 µm; Figure 5 for CebHLH113 The result of gene overexpression promoting anthocyanin synthesis in taro callus tissue; where A represents overexpression. CebHLH113 Color of taro callus tissue; B represents the anthocyanin content determination in wild-type WT and CebHLH113-0E taro callus tissue; among which... express P <0.005; C represents wild-type WT and CebHLH113-0E taro callus tissue. CebHLH113 Gene expression levels; among which, expressP <0.001; Figure 6 To remain silent in the stem tips of the "Ganyu No. 3" red-fleshed taro CebHLH113 Genetic results diagram; where A represents silencing in taro stem tips. CebHLH113 Gene-derived shoot tip tissue color; B represents the anthocyanin content determination in the VIGS control group CK and pTRV2-CebHLH113 of taro shoot tips; among which... express P <0.05; C represents the VIGS control group CK and pTRV2-CebHLH113 in taro stem tips. CebHLH113 Gene expression levels; among which, express P <0.01. Detailed Implementation

[0020] This invention provides a method for regulating anthocyanin synthesis in taro. CebHLH113 Genes, the ones mentioned CebHLH113The amino acid sequence of the protein encoded by the gene such as SEQ ID NO:1 shows: MAVGHQEEDMPRRRLRRQLAAAVHSIQWSYAILWSASSRQPGMLEWNDGYYNGDIKTRKTTQPMELKADQMALQRSEQLRQLYMSLSAGDGEQLARRPSALLSPEDLSDSEWYYLVCMSFTFHAGHGLVGRTLACGNHIWLFDAPSAESKIFTRSLLAKSASV QTVFCCPFMDGVLELGTTETVLEDPDLIKKVTTSFWDYPKTLSSGQSSSSNLKVEEDEDQMAPPPSRDHDMLNPASSENLHAGADRHTQSGDGVAGFAPTYHTHSSMKIAGVDEDRTEKLQADSSDELKTGSPDYCSNDCCANQHTDEFTMMQGPDGKSQARSWQFMDDE LSNGLHGSLNSSDDKCQSFSDLQVVSSPKEERIEYVNGLQICHNMKLTPLDPGADNAHYTRTLSAIFRNPAQADVMPFLPNGSHASSFEAWRKAQRLQIPMSQKLLKKILFNVALMHGGKTPSAPERNEAKSKTWKPEGDAVGLSHQLSERKRREKMNKKFLILTSLLPS ISKIDKASILGDTIDYLKELERRVGYLESYMGDLEAKGGKKHPDVAERTSDNYGRDEIACGKKSPLSKRKACDVDEQGFENHWILSKDGMADINVTIMGKEVLLQLSCPWRDSLLLEIVDAISNLHLDALSVQSSTVDGNLTLTIKSKFRGSVIASPGMIKCTLQRVISKC In one specific embodiment, the CebHLH113 protein has a molecular weight of 74990.75 Da and a theoretical isoelectric point (PI) of 6.02. Its instability coefficient is 53.8, classifying it as an unstable protein; its aliphatic coefficient is 74.72; and its average hydrophilicity is -0.529, classifying it as a hydrophilic protein. CebHLH113 lacks a signal peptide and is not a secretory protein, therefore it cannot be guided to extracellular or organelle membrane structures such as the endoplasmic reticulum or chloroplasts. CebHLH113 has zero transmembrane helices and lacks transmembrane domains, thus it is not a transmembrane protein. CebHLH113 contains a typical HLH (basic helix-loop-helix) domain and multiple α-helices and β-sheets, consistent with the characteristics of bHLH transcription factors. Subcellular localization shows that CebHLH113 is located in the cell nucleus.

[0021] As one specific implementation method, the CebHLH113

[0022] As one specific implementation method, the CebHLH113 The gene originates from the red-fleshed taro variety "Ganyu No. 3". The red-fleshed taro variety "Ganyu No. 3" was disclosed in "Chen Yuewen, Zeng Chenghong, Cheng Qi, et al. Identification and expression analysis of R2R3-MYB gene family related to anthocyanin synthesis in taro[J]. Journal of Southern Agriculture, 2025, 56(08):2421-2438.", with variety right number: CNA20211008747.

[0023] The present invention also provides a method for regulating CebHLH113 Biological materials with high gene expression levels, including overexpression CebHLH113 Gene biomaterials, inhibition of the above CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression; the method of inhibition includes silencing.

[0024] The overexpression described in this invention CebHLH113 Biological materials for gene overexpression include: CebHLH113 Gene amplification primers, overexpression... CebHLH113 Recombinant vectors of genes, overexpression of the aforementioned CebHLH113 Recombinant microorganisms of genes, overexpression of the aforementioned CebHLH113 Transgenic cell lines and overexpression of the gene CebHLH113 The recombinant vector can be any one or more of the following: a transgenic plant tissue containing the gene. As one specific embodiment, the basic framework of the recombinant vector includes the pSuper1300 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium. As one specific embodiment, the amplification primers include a forward primer and a reverse primer.

[0025] The present invention overexpressed the above CebHLH113 The primers for gene amplification are shown below: Forward primer (SEQ ID NO:3): 5'-ATGGCTGTTGGGCATCAGGA-3'; Reverse primer (SEQ ID NO:4): 5'-TCAGCACTTGCTTATGACTCTCT-3'.

[0026] In a specific embodiment of the present invention, the overexpression described CebHLH113 The recombinant vector for the gene was pSuper1300-35S-CebHLH113, and the gene was overexpressed. CebHLH113 The recombinant microorganism containing the pSuper1300-35S-CebHLH113 vector was Agrobacterium GV3101, which overexpressed the gene. CebHLH113 The transgenic plant tissues of the gene are overexpressed CebHLH113The "Ganyu No. 2" callus tissue.

[0027] As one specific implementation method, suppressing the CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression include: those containing the above. CebHLH113 Silencing vectors containing the gene silencing target, CebHLH113 A gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, a recombinant microorganism containing the knockout vector, and silencing. CebHLH113 Plant tissues and gene knockout CebHLH113 Any one or more genes in plant tissues; the basic framework of the silencing vector includes pTRV2.

[0028] In a specific embodiment of the present invention, the following is included: CebHLH113 The silencing vector for the gene silencing target includes the pTRV2-CebHLH113 vector, and the recombinant microorganism containing the silencing vector is Agrobacterium GV3101 containing the pTRV2-CebHLH113 vector, which silences the target gene. CebHLH113 Plant tissues are silent in terms of gene expression. CebHLH113 The "Ganyu No. 3" gene callus.

[0029] In a specific embodiment of the present invention, the CebHLH113 Sequence of gene silencing target such as SEQ ID Shown in NO:11: 5'-ATGGCTGTTGGGCATCAGGAAGAAGACATGCCTAGGAGGCGCCTTAGGAGACAGCTTGCCGCTGCCGTCCACAGCATACAGTGGAGTTACGCAATCTTGTGGTCCGCTTCAAGCAGGCAACCAGGTATGTTAGAATGGAATGATGG GTACTACAATGGTGATATAAAGACAAGAAAGACAACCCAGCCCATGGAACTAAAAGCTGATCAGATGGCTCTGCAAAGAAGTGAGCAACTGAGACAGCTCTATATGTCACTCTCAGCTGGTGACGGTGAGCAGCTGGCTAGAAGGCCTTCTGCC-3'.

[0030] The present invention also provides the aforementioned CebHLH113 The application of the gene or the described biological material in regulating anthocyanin synthesis and / or breeding high-anthocyanin taro varieties. As one specific embodiment, the regulation includes: overexpressing the described gene... CebHLH113 Genes that promote anthocyanin synthesis in taro; and genes that inhibit the synthesis of anthocyanins in taro.CebHLH113 Gene expression or knockout CebHLH113 Genes that inhibit the accumulation of anthocyanins in taro.

[0031] The embodiments of this invention show that, in the overexpression experiment, the relative anthocyanin content of the OE-CebHLH113 overexpression material was significantly higher than that of the WT control ( P <0.005), indicating CebHLH113 Gene overexpression promotes anthocyanin synthesis and accumulation in taro callus tissue. Compared with the WT control, CebHLH113 Overexpression material CebHLH113 The relative expression level of the gene was significantly increased ( P <0.001), indicating that the target gene has been successfully overexpressed, and the significant increase in its expression level is consistent with the significant accumulation of anthocyanin content, indicating that... CebHLH113 Genes positively regulate anthocyanin synthesis in taro. In the silencing experiment, the relative anthocyanin content in the taro stem tips of pTRV2-CebHLH113 was significantly lower than that in the control CK. P <0.05). Compared to the control group, the pTRV2-CebHLH113 treatment group showed significantly higher concentrations. CebHLH113 Gene expression levels were significantly downregulated. P The expression level was <0.01, indicating that the gene had been successfully transiently silenced. Furthermore, the decrease in expression level was consistent with the significant decrease in anthocyanin content, suggesting... CebHLH113 Genes are involved in regulating the synthesis of anthocyanins.

[0032] This invention also provides a method for promoting the synthesis of taro anthocyanins, comprising the following steps: overexpressing the aforementioned anthocyanins in a target taro plant. CebHLH113 Genes promote the synthesis of anthocyanins in taro.

[0033] In a specific embodiment of the present invention, referring to existing technology (a patent application numbered CN202411731651.8 entitled "A Method for Genetic Transformation of Taro"), the pSuper1300-35S-CebHLH113 vector was introduced into the callus tissue of the taro variety "Ganyu No. 2" (variety rights number: CNA20201006425). After steps such as pre-culture, infection, co-culture, and Kanamycin resistance screening, a transgenic taro was obtained. CebHLH113 The callus tissue (named OE-CebHLH113) showed a significantly increased accumulation of anthocyanins.

[0034] This invention also provides a method for breeding high-anthocyanin taro varieties, comprising the following steps: overexpressing the aforementioned anthocyanin in the target taro. CebHLH113 Genes were used to obtain taro varieties with high anthocyanin content.

[0035] To further illustrate the present invention, the following examples demonstrate a method for regulating anthocyanin synthesis provided by the present invention. CebHLH113 The genes and their applications are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0036] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0037] Example 1 Figure 1 Acquisition of genes taro CebHLH113 taro CebHLH113 The gene encodes a protein containing 674 amino acids, with the sequence shown in SEQ ID NO:1: MAVGHQEEDMPRRRLRRQLAAAVHSIQWSYAILWSASSRQPGMLEWNDGYYNGDIKTRKTTQPMELKADQMALQRSEQLRQLYMSLSAGDGEQLARRPSALLSPEDLSDSEWYYLVCMSFTFHAGHGLVGRTLACGNHIWLFDAPSAESKIFTRSLLAKSASVQTVFCCPFMDGVLELGTTETVLEDPDLIKKVTTSFWDYPKTLSSGQSLSSNLKVEEDEDQMAPPPSRDHDMLNPASSENLHAGADRHTQSGDGVAGFAPTYHTHSSMKIAGVDEDRTEKLQADSSDELKTGSPDYCSNDCCANQHTDEFTMMQGPDGKSQARSWQFMDDE LSNGLHGSLNSSDDKCQSFSDLQVVSSPKEERIEYVNGLQICHNMKLTPLDPGADNAHYTRTLSAIFRNPAQADVMPFLPNGSHASSFEAWRKAQRLQIPMSQKLLKKILFNVALMHGGKTPSAPERNEAKSKTWKPEGDAVGLSHQLSERKRREKMNKKFLILTSLLPS ISKIDKASILGDTIDYLKELERRVGYLESYMGDLEAKGGKKHPDVAERTSDNYGRDEIACGKKSPLSKRKACDVDEQGFENHWILSKDGMADINVTIMGKEVLLQLSCPWRDSLLLEIVDAISNLHLDALSVQSSTVDGNLTLTIKSKFRGSVIASPGMIKCTLQRVISKC .

[0038] In this embodiment, the gene is used for cloning. Figure 2 The primer pairs are as follows: the forward primer sequence is shown in SEQ ID NO:3: 5'-ATGGCTGTTGGGCATCAGGA-3'; the reverse primer sequence is shown in SEQ ID NO:4: 5'-TCAGCACTTGCTTATGACTCTCT-3'.

[0039] CebHLH113 The specific process of gene amplification is as follows: (1) The corms, roots, leaf stems and leaves of the red-fleshed taro “Ganyu No. 3” (“Ganyu No. 3”: Chen Yuewen, Zeng Chenghong, Cheng Qi, et al. Identification and expression analysis of R2R3-MYB gene family related to anthocyanin synthesis of taro [J]. Journal of Southern Agriculture, 2025, 56(08):2421-2438.) were ground in liquid nitrogen and mixed evenly. RNA was extracted using the instructions of the polysaccharide and polyphenol plant RNA extraction kit provided by Nanjing Novizan Biotechnology Co., Ltd.

[0040] (2) cDNA synthesis method: Using the RNA from step (1) as a template, the first-strand cDNA was synthesized by reverse transcription according to the instructions of the Hifair Ⅲ 1st Strand cDNA Synthesis Kit (gDNA digester plus) from Shanghai Yisheng Biotechnology Co., Ltd.

[0041] (3) Using the cDNA obtained in step (2) as a template, PCR amplification was performed using forward and reverse primers. The forward primer was 5'-ATGGCTGTTGGGCATCAGGA-3' (SEQ ID NO:3); the reverse primer was 5'-TCAGCACTTGCTTATGACTCTCT-3' (SEQ ID NO:4). The amplification system was as follows: 2 μL forward primer (primer concentration 10 μM), 2 μL reverse primer (primer concentration 10 μM), 5 μL template (“Ganyu No. 3” red taro cDNA, template concentration 500 ng / μL), 25 μL 2×Hieff canace Plus PCR Master Mix (With Dye) (Shanghai Yisheng Biotechnology Co., Ltd.), and 16 µL sterile ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 60 s, 72℃ extension for 1 min, for a total of 34 cycles, and finally 72℃ final extension for 5 min.

[0042] (4) The PCR product obtained in step (3) totaled 2025 bp. The specific results are as follows: CeActin As shown. Based on its classification within the taro bHLH gene family, it is named... CebHLH113 .

[0043] Example 2 CebHLH113 Gene expression analysis at different developmental stages of the "Ganyu No. 3" red-fleshed taro (1) “Ganyu No. 3” red-fleshed taro is a taro rich in anthocyanins, and the anthocyanin content of its corms varies at different developmental stages. The fleshy part (F) and cortical tissue (C) of the corms of “Ganyu No. 3” red-fleshed taro were collected at 8-10 days (S1), 50-60 days (S2) and 90-100 days (S3) after corm formation. The samples were placed in a pre-cooled mortar and ground into powder with liquid nitrogen for later use.

[0044] (2) Anthocyanin determination: Take 0.1 g of the sample powder from step (1) above, add 1 mL of pre-cooled methanol containing 1% hydrochloric acid, vortex mix, and incubate at 4℃ in the dark for 12 h; then centrifuge at 4℃ and 12000 g for 20 min, and carefully aspirate the supernatant, which is the anthocyanin extract. Use a UV-Vis spectrophotometer to measure the optical density (A value) of the extract at wavelengths of 530 nm (characteristic absorption peak of anthocyanins), 620 nm (impurity absorption correction), and 650 nm (chlorophyll absorption correction). Calculate the relative anthocyanin content according to the formula: [(A530-A620)-0.1×(A650-A620)] / fresh weight of sample (g). The results are as follows: CeActin As shown in Table A and Table 1, the anthocyanin content of the "Ganyu No. 3" red-fleshed taro was highest in the S1 period and lower in the S2 and S3 periods; the anthocyanin content of the cortex tissue was higher than that of the corm flesh in the same period.

[0045] (3) To further test Figure 2 The expression differences in the corm flesh and cortical tissue of the "Ganyu No. 3" red-fleshed taro at different developmental stages were investigated. Total RNA was extracted from the samples obtained in step (1) above, and the first-strand cDNA of the corm was synthesized by reverse transcription. Using this cDNA as a template, the expression differences were investigated. CebHLH113 The gene was used as an internal control, and the detection was performed by real-time quantitative PCR. CebHLH113 The level of expression.

[0046] (4) Primer design: For quantitative real-time PCR detection, a primer pair consisting of qpCebHLH113-F and primer qpCebHLH113-R was used. CebHLH113 Gene expression was detected using a primer pair consisting of primers CeActin-F and CeActin-R. CebHLH113 Gene expression.

[0047] qpCebHLH113-F (SEQ ID NO:5): 5'-ATCCTGGTGCTGACAATGCA-3'; qpCebHLH113-R (SEQ ID NO:6): 5'-GGAGCACTTGGGGTCTTACC-3'; CeActin-F (SEQ ID NO:7): 5'-CTAGTGGTCGCACAACAGGT-3'; CeActin-R (SEQ ID NO:8): 5'-TTCACGCTCAGCAGTGGTAG-3'.

[0048] (5) The preferred quantitative PCR amplification system is 20 µL, using the quantitative PCR reagent (Hieff UNICON Universal Blue qPCR SYBR Green Master Mix) provided by Shanghai Yisheng Biotechnology Co., Ltd., China. The system includes: 10 µL of Hieff UNICON Universal Blue qPCR SYBR Green Master Mix, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 7.2 µL of RNase-free ddH2O, and 2 µL of cDNA template. The quantitative PCR reaction program includes: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 39 cycles. The melting curve is between 65℃ and 95℃, with an increase of 0.5℃ every 5 s.

[0049] (6) Using 2 -ΔΔCt The relative expression levels of genes were calculated using this method, and the results are as follows: Figure 3 As shown in B and Table 2, Figure 3 The gene expression level was relatively high in the early stage of taro enlargement (S1), and lower in the S2 and S3 stages. Moreover, the expression level in the cortical tissue was higher than that in the fleshy part of the corm. Figure 3 The trend of expression level changes is consistent with the trend of anthocyanin content changes.

[0050] Table 1. Anthocyanin content (mg / g FW) of "Ganyu No. 3" red-fleshed taro at different developmental stages

[0051] Table 2. Different developmental stages of "Ganyu No. 3" red-fleshed taro Figure 3 Relative expression analysis of genes

[0052] Example 3 CebHLH113 Bioinformatics analysis of encoded proteins The physicochemical properties of the CebHLH113 gene protein were predicted using the online ExPAsy tool (https: / / web.expasy.org / protparam). The results showed that the CebHLH113 protein has a molecular weight of 74990.75 Da and a theoretical isoelectric point (PI) of 6.02. The protein instability coefficient was 53.8, classifying it as an unstable protein; the aliphatic coefficient was 74.72; and the average hydrophilicity was -0.529, classifying it as a hydrophilic protein.​ A represents the predicted hydrophilicity / hydrophobicity of the CebHLH113 protein.

[0053] The signal peptide of the CebHLH113 protein was predicted using the SignalP4.1Server online website (https: / / services.healthtech.dtu.dk / services / SignalP-4.1 / ). The results are as follows: ​ As shown in Figure B, the CebHLH113 protein does not have a signal peptide, indicating that it is not a secretory protein and will not be guided to extracellular or organelle membrane structures such as the endoplasmic reticulum and chloroplasts.

[0054] The transmembrane structure of the CebHLH113 protein was predicted using the TMHMMv.Server 2.0 online website (http: / / www.cbs.dtu.dk / services / TMHMM / ). The results are as follows: ​ As shown in Figure C, the predicted number of transmembrane helices in the CebHLH113 protein is 0, indicating that the protein has no transmembrane domain and is not a transmembrane protein.

[0055] The tertiary structure of the CebHLH113 protein was predicted using the online tool SWISS MODEL (https: / / swissmodel.expasy.org), and the results are as follows: ​ As shown in Figure D, the CebHLH113 protein contains a typical HLH (basic helix-loop-helix) domain and multiple α-helices and β-sheets, consistent with the characteristics of bHLH transcription factors.

[0056] Example 4 Subcellular localization analysis of CebHLH113 protein To clarify the subcellular localization of the CebHLH113 protein and verify its transcription factor functional characteristics, this invention used pSuper1300 as a vector (Chen X, Sun J, Shan N, et al. DaMYB75 and DaMYB56 antagonistically regulate anthocyanin biosynthesis by binding to the DaANS promoter in Dioscorea alata[J].The Crop Journal[2026-03-11].DOI:10.1016 / j.cj.2025.03.009) to construct the pSuper1300-35S-CebHLH113 fusion expression vector. This vector was then transformed into *Nicotiana benthamiana* via Agrobacterium-mediated transformation. The fluorescence distribution was observed using a laser scanning confocal microscope in conjunction with the Nuclear-RFP nuclear marker. The specific steps are as follows: (1) Primer design: Based on the HindIII and SpeI restriction sites of the pSuper1300 vector, primers were designed to be used in combination with... ​ Homologous recombination primers were designed based on the gene nucleotide sequence (SEQ ID NO:2, excluding the stop codon). The primer sequences are as follows: pSuper1300-CebHLH113-F (SEQ ID NO:9): 5'-GACTCTAGAAAGCTTCTGCAGATGGCTGTTGGGCATCAGGA-3'; pSuper1300-CebHLH113-R (SEQ ID NO:10): 5'-CGCCCTTGCTCACCATGGTACCGCACTTGCTTATGACTCTCT-3'.

[0057] (2) Amplification of the target fragment: obtained by cloning in Example 1 CebHLH113 Using the nucleotide PCR gel recovery product as a template, PCR amplification was performed using the primer pairs from step (1) above. The amplification system was the same as in Example 1, yielding a product containing homologous recombination arms. CebHLH113 Target segment.

[0058] (3) Vector linearization by double enzyme digestion: A 50 µL double enzyme digestion reaction system was prepared, consisting of 5 µL rCutSmart buffer, 1 µL HindIII restriction endonuclease, 1 µL SpeI, 4 µL pSuper1300 vector plasmid (plasmid concentration 500 ng / μL), and 39 µL ddH2O. The reaction conditions were: digestion at 37℃ for 2 h, followed by enzyme inactivation at 65℃ for 20 min to obtain the linearized pSuper1300 vector. The linearization effect was verified by agarose gel electrophoresis before use.

[0059] (4) Homologous recombination: According to the instructions of the homologous recombination kit of Yisheng Biotechnology (Shanghai) Co., Ltd., the amplification product obtained in step (2) and the linearized vector product obtained in step (3) above are subjected to homologous recombination. The reaction product can be directly converted or stored in a -20℃ freezer.

[0060] (5) Escherichia coli transformation: The ligation product obtained in step (4) above was transformed into competent Escherichia coli cells (according to the instructions for transformation of competent Escherichia coli cells from Qingke Biotechnology Co., Ltd.), and cultured overnight at 37°C for 14 h on LB solid medium (containing 50 mg / L Kan). Five single colonies were picked and cultured on LB liquid medium (containing 50 mg / L Kan) at 37°C and 200 rpm for 6 h with shaking. After the bacterial culture became turbid, it was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0061] (6) Agrobacterium transformation: The single colony that was successfully sequenced was preserved and amplified to extract plasmid (extraction was performed according to the high purity plasmid DNA mini-extraction kit of Qingke Biotechnology). The successfully extracted plasmid was introduced into GV3101 Agrobacterium competent cells by freeze-thaw method to obtain recombinant Agrobacterium GV3101 / pSuper1300-35S-CebHLH113.

[0062] (7) Preparation of Agrobacterium culture: The three Agrobacterium species corresponding to pSuper1300-35S-CebHLH113, pSuper1300 empty vector, and Nuclear-RFP nuclear marker vector were inoculated into LB liquid medium containing 50 mg / L Kan and 25 mg / L rifampin (Rif), and cultured at 28°C and 200 rpm until OD. 600 =0.8. Then, centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in infection buffer (containing 10 mM MES, 200 µM acetylsalicylic acid, and 10 mM MgCl2). Mix "pSuper1300-35S-CebHLH113 Agrobacterium + Nuclear-RFP Agrobacterium" and "pSuper1300 empty vector Agrobacterium + Nuclear-RFP Agrobacterium" at a 1:1 volume ratio, and adjust the OD of the mixed bacterial solutions. 600 =1.0, and after standing in the dark for 2 h, the leaves of Tobacco Benedict were infected.

[0063] (8) Cultivation and sample preparation: The tobacco plants infected in step (7) above were cultured for 2 days at 25°C under 16 h light / 8 h darkness. The leaves at the injection site were cut off or the lower epidermis was torn off to prepare temporary slides for later use.

[0064] (9) Confocal microscopy observation: Observation was performed using an FV3000 laser scanning confocal microscope (Olympus, Tokyo, Japan). A 488 nm laser channel was used to detect GFP green fluorescence (GFP fusion protein), and a 546 nm laser channel was used to detect red fluorescence (Nuclear-RFP nuclear marker). Results are as follows: Figure 4 As shown, the green fluorescence of CebHLH113-GFP completely overlaps with the red fluorescence of the nuclear label, and the specific signal is detected only in the cell nucleus, indicating that the CebHLH113 protein is localized in the cell nucleus.

[0065] Example 5: Regulation of anthocyanin content in taro using CebHLH113 genetic material To determine taro CebHLH113To determine whether the gene is involved in anthocyanin synthesis in taro, the overexpression vector of (OE) CebHLH113 was transformed into taro callus tissue using Agrobacterium-mediated transformation. Phenotypic observation, anthocyanin content determination, and gene expression analysis were then conducted to verify the gene's function. The specific steps are as follows: 1. CebHLH113 Genetic transformation Referring to existing technology (a patent application titled "A Method for Genetic Transformation of Taro," application number CN202411731651.8), the recombinant Agrobacterium GV3101 / pSuper1300-35S-CebHLH113 obtained in Example 4 was introduced into the callus tissue of the taro variety "Ganyu No. 2" (variety rights number: CNA20201006425). After steps such as pre-culture, infection, co-culture, and Kanamycin resistance screening, transgenic taro was obtained. CebHLH113 Callus tissue (named OE-CebHLH113); untransformed "Ganyu No. 2" callus tissue was used as wild-type control (WT). Figure 5 A in the middle represents overexpression. CebHLH113 Color of gene-modified taro callus tissue.

[0066] 2. CebHLH113 Analysis of anthocyanin content in overexpression materials The anthocyanin content of WT and OE-CebHLH113 callus tissues was determined using the anthocyanin content determination method described in Example 2. The results are as follows: Figure 5 As shown in Table B and Table 3, the relative anthocyanin content of the OE-CebHLH113 overexpression material was significantly higher than that of the WT control. P <0.005), indicating CebHLH113 Gene overexpression can promote the synthesis and accumulation of anthocyanins in taro callus tissue.

[0067] 3. CebHLH113 Analysis of relative gene expression levels in overexpression materials The fluorescence quantitative method described in Example 2 was used to analyze WT and OE-CebHLH113 callus tissues. CebHLH113 The results of the analysis of relative gene expression levels are as follows: Figure 5 As shown in Table C and Table 4, compared with the WT control, CebHLH113 Overexpression material CebHLH113 The relative expression level of the gene was significantly increased ( P <0.001), indicating that the target gene has been successfully overexpressed, and the significant increase in its expression level is consistent with the significant accumulation of anthocyanin content, indicating that... CebHLH113 Genes positively regulate the synthesis of taro anthocyanins.

[0068] Table 3. Determination of anthocyanin content in taro callus (mg / g FW)

[0069] Note: express P <0.005.

[0070] Table 4. Taro callus tissue CebHLH113 Gene expression level

[0071] Note: express P <0.001.

[0072] Example 6 CebHLH113 VIGS silencing of genes To further determine CebHLH113 The gene plays an important role in regulating anthocyanin synthesis in taro. This was demonstrated by transiently silencing the gene in the shoot tips of the "Ganyu No. 3" red-fleshed taro variety using VIGS virus silencing technology. CebHLH113 The specific steps for observing shoot tip phenotype, measuring anthocyanin content, and analyzing gene expression levels are as follows: 1. Construction of pTRV2-CebHLH113 silencing expression vector (1) This embodiment provides silence CebHLH113 The target sequence of the gene, totaling 300 bp, is shown in SEQ ID NO:11 as follows: 5'-ATGGCTGTTGGGCATCAGGAAGAAGACATGCCTAGGAGGCGCCTTAGGAGACAGCTTGCCGCTGCCGTCCACAGCATACAGTGGAGTTACGCAATCTTGTGGTCCGCTTCAAGCAGGCAACCAGGTATGTTAGAATGGAATGATGGGTACTACAATGGTGATATAAAGACAAGAAAGACAACCCAGCCCATGGAACTAAAAGCTGATCAGATGGCTCTGCAAAGAAGTGAGCAACTGAGACAGCTCTATATGTCACTCTCAGCTGGTGACGGTGAGCAGCTGGCTAGAAGGCCTTCTGCC-3'.

[0073] (2) Using the mixed cDNA from step (2) in Example 1 as a template, PCR amplification was performed using a primer pair composed of pTRV2-CebHLH113-F and pTRV2-CebHLH113-R. The forward primer was 5'-GAAGTTCCCAGGCGTCACATGGCTGTTGGGCATCAG-3' (SEQ ID NO:12); the reverse primer was 5'-CGTGAGCTCGGTACCGGATCCGGCAGAAGGCCTTCTAGC-3' (SEQ ID NO:13). The amplification system consisted of 2 μL of forward primer (primer concentration 10 M), 2 μL of reverse primer (primer concentration 10 μM), 5 μL of template (“Ganyu No. 3” red taro cDNA, template concentration 500 ng / μL), 25 μL of 2×Hieff canace Plus PCR Master Mix (With Dye) (Shanghai Yisheng Biotechnology Co., Ltd.), and 16 µL of sterile ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 20 s, 72℃ extension for 30 s, for a total of 34 cycles, followed by a final extension at 72℃ for 5 min, yielding a product containing... CebHLH113 PCR amplification products of gene-specific fragments.

[0074] (3) The vector pTRV2 was double-digested using the restriction endonucleases EcoRI-HF and BamHI-HF. The double digestion reaction system consisted of 5 µL rCutSmart buffer, 1 µL EcoRI-HF, 1 µL BamHI-HF, 3 µL pTRV2 vector plasmid (plasmid concentration 500 ng / μL), and 40 µL ddH2O. The double digestion reaction conditions were: digestion at 37℃ for 3 h, followed by enzyme inactivation at 65℃ for 20 min to obtain a linearized vector. The amplified product and the linearized vector were homologously recombined to obtain the recombinant vector pTRV2-CebHLH113.

[0075] 2. Recombinant Agrobacterium The recombinant vector pTRV2-CebHLH113 obtained in step 1 above was introduced into Agrobacterium GV3101 using the freeze-thaw method to obtain recombinant Agrobacterium GV3101 / pTRV2-CebHLH113.

[0076] 3. Agrobacterium-mediated VIGS silencing in taro shoot tips Referring to existing technology (invention patent titled: Taro CePDS Gene and its VIGS Viral Vector and Silenting Method, application number: CN202411203373.9), VIGS control group infection solution and infection solution containing the above-mentioned GV3101 / pTRV2-CebHLH113 were obtained. Stem tips of the "Ganyu No. 3" red-fleshed taro were taken, washed with ddH2O and disinfected with 15% sodium hypochlorite, and then placed in the two infection solutions respectively. Vacuum permeation treatment was then performed, i.e., vacuuming for 15 min and standing at normal pressure for 30 min. The remaining bacterial solution was removed and aspirated, with aseptic operation throughout. The treated stem tips were inoculated onto MS solid medium, cultured for 2 days in the dark at 25℃, and then transferred to normal light conditions for another 10 days. Silencing was achieved in the taro stem tips. CebHLH113 Gene morphology diagram as follows Figure 6 As shown in Figure A.

[0077] 4. Anthocyanin content analysis of VIGS silent material in taro stem tips The anthocyanin content in taro stem tips was determined using the anthocyanin content determination method described in Example 2. The results are as follows: Figure 6 As shown in Table B and Table 5, the relative anthocyanin content in the taro stem tips of pTRV2-CebHLH113 was significantly lower than that in the control CK. P <0.05).

[0078] 5. CebHLH113 Analysis of relative gene expression levels in overexpression materials Using the same quantitative real-time PCR method as in Example 2, the levels of [unclear - possibly "pTRV2-CebHLH113"] in the taro stem tips of the control group (CK) and the experimental group (pTRV2-CebHLH113) were detected. CebHLH113 The relative expression levels of genes. Results are as follows: Figure 6 As shown in Table C and Table 6, compared with the control group, the pTRV2-CebHLH113 treatment group had [a certain percentage of positive results]. CebHLH113 Gene expression levels were significantly downregulated. P The expression level was <0.01, indicating that the gene had been successfully transiently silenced. Furthermore, the decrease in expression level was consistent with the significant decrease in anthocyanin content, suggesting... CebHLH113 Genes are involved in regulating the synthesis of anthocyanins.

[0079] Table 5. Determination of anthocyanin content in taro stem tips (mg / g FW)

[0080] Note: express P <0.05.

[0081] Table 6 Taro stem tip CebHLH113 Gene expression level

[0082] Note: express P <0.01.

[0083] Therefore, this invention is the first to isolate a key gene positively regulating anthocyanin synthesis from the "Ganyu No. 3" red-fleshed taro. CebHLH113 The aforementioned CebHLH113 Gene overexpression significantly promotes anthocyanin accumulation in taro callus tissue, while silencing the gene in shoot tips significantly reduces anthocyanin accumulation. This invention fills a gap in the transcriptional regulation mechanism of anthocyanin metabolism in taro, and is helpful in breeding high-anthocyanin, high-stress-resistant, and high-quality taro varieties, with broad application value.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for regulating anthocyanin synthesis in taro CebHLH113 Genes, characterized by, The CebHLH113 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

1.

2. As described in claim 1 CebHLH113 Genes, characterized by, The CebHLH113 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. For regulating the function described in claim 1 or 2 CebHLH113 Biomaterials with high gene expression levels are characterized by, Including overexpression CebHLH113 Gene biomaterials, inhibition of the above CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression; The suppression methods include silencing.

4. The biomaterial according to claim 3, characterized in that, The overexpression CebHLH113 Biological materials for gene overexpression include: CebHLH113 Gene amplification primers, overexpression... CebHLH113 Recombinant vectors of genes, overexpression of the aforementioned CebHLH113 Recombinant microorganisms of genes, overexpression of the aforementioned CebHLH113 Transgenic cell lines and overexpression of the gene CebHLH113 Any one or more genes in the tissues of transgenic plants; The basic framework of the recombinant vector includes the pSuper1300 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium.

5. The biomaterial according to claim 4, characterized in that, The amplification primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

4.

6. The biomaterial according to claim 3, characterized in that, Suppress the CebHLH113 Gene expression or knockout CebHLH113 Biological materials for gene expression include: those containing the above. CebHLH113 Silencing vectors containing the gene silencing target, CebHLH113 A gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, a recombinant microorganism containing the knockout vector, and silencing. CebHLH113 Plant tissues and gene knockout CebHLH113 Any one or more genes in plant tissues; The basic framework of the silencing vector includes pTRV2.

7. The claim 1 or 2 CebHLH113 The application of genes or the biological materials described in any one of claims 3 to 6 in regulating anthocyanin synthesis and / or cultivating high-anthocyanin taro varieties.

8. The application according to claim 7, characterized in that, The regulation includes: overexpressing the... CebHLH113 Genes that promote anthocyanin synthesis in taro; and genes that inhibit the synthesis of anthocyanins in taro. CebHLH113 Gene expression or knockout CebHLH113 Genes that inhibit the accumulation of anthocyanins in taro.

9. A method for promoting anthocyanin synthesis, characterized in that, Includes the following steps: Overexpression of the substance as described in claim 1 or 2 in the target taro CebHLH113 Genes promote the synthesis of anthocyanins in taro.

10. A method for cultivating high-anthocyanin taro varieties, characterized in that, Includes the following steps: Overexpression of the substance as described in claim 1 or 2 in the target taro CebHLH113 Genes were used to obtain taro varieties with high anthocyanin content.

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