Application of TaMYB6 gene in regulation and control of plant anthocyanin

Abstract

The invention provides application of a TaMYB6 gene in regulation and control of plant anthocyanin, and belongs to the technical field of plant genetic engineering. The invention provides application of a TaMYB6 gene in regulation and control of plant anthocyanin, and the TaMYB6 is highly homologous with OsC1 reported in rice and possibly participates in regulation and control of anthocyanin synthesis. According to the embodiment of the invention, the gene TaMYB6 is transformed into a wheat variety Fielder to obtain a transformed plant for overexpressing the TaMYB6, and in the overexpressed mutant, the anthocyanin content in stalks, stem nodes and leaf sheaths is obviously increased.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a TaMYB6 gene in regulating plant anthocyanins. BACKGROUND

[0002] Anthocyanins are important water-soluble natural pigments in plants, belong to flavonoid metabolism branch, and are widely distributed in flowers, fruits, leaves, stems and seeds of plants. As an important part of plant secondary metabolism network, anthocyanins play multiple physiological and ecological functions in the life cycle of plants. First, anthocyanins give plant tissues bright red, purple and blue colors, participate in attracting pollinators and seed dispersers, and have significant ecological adaptive value. Second, anthocyanins play a key role in plant stress resistance, can regulate redox balance by removing reactive oxygen species (ROS), and enhance the tolerance of plants to high light, low temperature, drought, salt stress and other adverse environments. In addition, under the stress of low phosphorus or low nitrogen and other soil low-nutrient environments, plants can accumulate anthocyanins, regulate nutrient allocation and metabolic pathways, and enhance plant resilience. Anthocyanins not only improve the adaptability of plants to abiotic stress, but also have certain biochemical barrier function against pathogenic bacteria and insects. With the rapid development of plant metabolomics, transcriptomics and functional genomics, more and more researches on the regulation mechanism of anthocyanin biosynthesis, tissue-specific distribution pattern and environmental response regulation are continuously deepened, which provides a new perspective for understanding the molecular basis of plant adaptation to environmental changes, and also provides important metabolic engineering targets for plant breeding.

[0003] A variety of transcription factors involved in regulating anthocyanin synthesis in Arabidopsis, rice, corn and other plants have been reported. Different transcription factors in these reports regulate different genes in the anthocyanin synthesis pathway, and have different effects on anthocyanin synthesis. Therefore, it is particularly important to explore the transcription factors involved in regulating anthocyanin synthesis in wheat for targeted improvement of anthocyanin content in wheat. SUMMARY

[0004] The application provides application of a TaMYB6 gene in regulating plant anthocyanins, and the TaMYB6 gene positively regulates the content of plant anthocyanins.

[0005] The application provides application of a TaMYB6 gene in regulating plant anthocyanins.

[0006] In a preferred mode of the application, the protein encoded by the TaMYB6 gene has the amino acid sequence shown in any one of the following:

[0007] (1) the amino acid sequence shown in SEQ ID No. 3;

[0008] (2) an amino acid sequence obtained by substitution, addition, deletion, or mutation of one or more amino acids in the amino acid sequence shown in SEQ ID No. 3;

[0009] (3) an amino acid sequence having 90% or more identity with the amino acid sequence described in (1) or (2).

[0010] In a preferred mode of the present application, the TaMYB6 gene comprises the full-length nucleotide sequence shown in SEQ ID No. 2.

[0011] In a preferred mode of the present application, the TaMYB6 gene comprises the CDS sequence shown in SEQ ID No. 1.

[0012] In a preferred mode of the present application, the regulation comprises that the anthocyanin content in the plant tissue is increased after overexpression of the TaMYB6 gene.

[0013] In a preferred mode of the present application, when the plant is wheat, the plant tissue comprises stem, stem node, and leaf sheath.

[0014] The present application also provides an overexpression vector of the TaMYB6 gene.

[0015] In a preferred mode of the present application, the TaMYB6 gene in the overexpression vector is obtained by PCR amplification, wherein the primer pair used comprises TaMYB6-F shown in SEQ ID No. 4 and TaMYB6-R shown in SEQ ID No. 5.

[0016] The present application also provides the use of the above-mentioned overexpression vector in the construction of plants with high anthocyanin content.

[0017] The present application also provides a method for increasing the anthocyanin content in plants, comprising overexpression of the TaMYB6 gene in the genome of the plant.

[0018] Beneficial effects: the application utilizes HPLC-MS / MS technology to detect anthocyanin content of a natural population of wheat, and locates a SNP site (AX-07839221) on chromosome 5A to be significantly associated with peonidin-3-glucoside content in wheat leaves, the gene annotation is Myb transcription factor, the gene number is TraesCS5A01G495500, and the gene is named as TaMYB6. The TaMYB6 in the application is highly homologous to OsC1 reported in rice, and may be involved in the regulation of anthocyanin synthesis. In the embodiment of the application, the gene TaMYB6 is transformed into wheat variety Fielder to obtain a transformed plant overexpressing TaMYB6, and the anthocyanin content in the stem, stem node and leaf sheath of the overexpression mutant is significantly increased. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure for the result of locating TaMYB6 by genome-wide association analysis of anthocyanin peonidin-3-glucoside content in wheat;

[0020] Figure 2 Sequence alignment of TaMYB6 and homologous genes;

[0021] Figure 3 Phylogenetic tree of homologous sequence analysis of TaMYB6 protein;

[0022] Figure 4 Schematic diagram of genetic transformation vector structure for overexpressing TaMYB6 in wheat;

[0023] Figure 5 Difference in TaMYB6 gene expression and anthocyanin content in wheat leaves and grains between wild-type wheat and transgenic wheat overexpressing TaMYB6; wherein, *** represents P<0.001;

[0024] Figure 6 Heat map of expression amount of anthocyanin synthesis pathway related genes in transgenic wheat. DETAILED DESCRIPTION

[0025] The application provides application of the TaMYB6 gene in regulating plant anthocyanin.

[0026] The TaMYB6 gene in the application is detected in a natural population of wheat and can be located on chromosome 5A, and the protein encoded by the TaMYB6 gene has any one of the following amino acid sequences:

[0027] (1) the amino acid sequence shown in SEQ ID No. 3;

[0028] (2) based on the amino acid sequence shown in SEQ ID No. 3, an amino acid sequence obtained after substitution, addition, reduction or mutation of one or more amino acids;

[0029] (3) an amino acid sequence having 90% or more identity with the amino acid sequence described in (1) or (2).

[0030] The TaMYB6 gene described in the application includes the full-length nucleotide sequence shown in SEQ ID No. 2, and includes the CDS sequence shown in SEQ ID No. 1.

[0031] In an embodiment of the application, the anthocyanin content in plant tissues is increased after overexpression of the TaMYB6 gene in a wheat variety by genetic transformation technology. The plants described in the application include wheat, and in wheat, the anthocyanin content in stems, stem nodes and leaf sheaths is significantly increased after overexpression of the TaMYB6 gene.

[0032] The application also provides an overexpression vector of the TaMYB6 gene.

[0033] The TaMYB6 gene in the overexpression vector described in the application is obtained by PCR amplification, wherein the primer pair used includes TaMYB6-F shown in SEQ ID No. 4 and TaMYB6-R shown in SEQ ID No. 5.

[0034] The application also provides the use of the above-mentioned overexpression vector in the construction of plants with high anthocyanin content.

[0035] The TaMYB6 gene or the overexpression vector described in the application can be used to construct transgenic plants with high expression of anthocyanin by genetic transformation methods.

[0036] The application also provides a method for increasing the anthocyanin content in plants, comprising overexpression of the TaMYB6 gene in the genome of the plant.

[0037] The method for overexpression is not particularly limited in the application, and conventional genetic transformation methods in the art can be used.

[0038] In order to further illustrate the application, the application of the TaMYB6 gene provided in the application in the regulation of anthocyanin in plants is described in detail below with reference to the examples, but they should not be understood as limiting the scope of protection of the application.

[0039] Example 1

[0040] Obtaining of the TaMYB6 gene

[0041] 1. Determination and structural analysis of the TaMYB6 gene

[0042] The anthocyanin content of the natural population material of wheat was detected by HPLC-MS / MS technology, and a SNP site (AX-07839221) was located on chromosome 5A, which was significantly associated with the content of peonidin-3-glucoside in wheat leaves ( Figure 1 ), the gene annotation is Myb transcription factor, the gene number is TraesCS5A01G495500, and the gene is named as TaMYB6. The full-length genomic sequence of TaMYB6 is 811 bp (SEQ ID No. 1), the coding sequence is 729 bp (SEQ ID No. 2), and it encodes 242 amino acids (SEQ ID No. 3). The gene has one exon. Sequence alignment analysis ( Figure 2 ) and phylogenetic tree analysis ( Figure 3 ) were carried out on the reported MYB family genes in rice, corn and Arabidopsis and TaMYB6, and the results showed that TaMYB6 was highly homologous to the reported OsC1 in rice, and may be involved in the regulation of anthocyanin synthesis.

[0043] SEQ ID No. 1: 5'-CTAACACAATACACACCGGAGATGAGGTGAGATAG AGAGGTTAGTTCAGAGAAAGAGGAAGAGGAAGATAGAGAGGGAGAGAATGAGGAGGGCGTGCAGTGCGAAGGAAGGGGTGAAGAGAGGGGCATGGACGAGCAAGGAGGACGAAACCTTGGCCTCCTACATCAAGGCGCATGGCGAAGGCAGATGGAAGGAAGTCCCCTTGAAAGCTGGCCTGCGGCGGTGCGGCAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGAGCATCAAGCGGGGCAACATCTCCGACGAGGAGGAGCTCATCGTCAGGCTCCACCGCCTCCTCGGCAACAGGTGGTCCCTCATCGCGGGCAGGCTGCCCGGCCGAACAGACAACGAAATCAAGAACTACTGGAACAGCACCCTCGGGCGGAAGGCGCTCCCGGAGCGAGCTGCCACCAGGGTGGTCGCCACGCCCTGCGCCTCCGCCAGCTCCGGCTCCTCCACAGGGGCGTCGACAGCAGGGCTGTCCTCTAACTGCGGGGCTGGTACAAGTGCCAAGGCTGCGGGGCTGCTGTCGTCGGCCTCGGTGTGGGTGCCCAAGCCCGTGAGGTGCACAGGCGGTCTCTTCCTCAGCGGGGATGCGCCGCTGCCCGCGCCGGTCACGCAGACGCGGGCCGTGGGTGGAGACGGAGATGAGTGCAGCGGCAGCAGCTTGGCGGCATCGTCGGGCGGCGGCGACTGGATGGACGACGTGAGAGCCTTGGCGTCGTTCCTCGAGTCCGACGAGGACTGGGTCAACTCCCTGCACATGGCGGATTAA-3';

[0044] SEQ ID No. 2: ATGAGGAGGGCGTGCAGTGCGAAGGAAGGGGTGAAG AGAGGGGCATGGACGAGCAAGGAGGACGAAACCTTGGCCTCCTACATCAAGGCGCATGGCGAAGGCAGATGGAAGGAAGTCCCCTTGAAAGCTGGCCTGCGGCGGTGCGGCAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGAGCATCAAGCGGGGCAACATCTCCGACGACGAGGAGGAGCTCATCGTCAGGCTCCACCGCCTCCTCGGCAACAGGTGGTCCCTCATCGCGGGCAGGCTGCCCGGCCGAACAGACAACGAAATCAAGAACTACTGGAACAGCACCCTCGGGCGGAAGGCGCTCCCGGAGCGAGCTGCCACCAGGGTGGTCGCCACGCCCTGCGCCTCCGCCAGCTCCGGCTCCTCCACAGGGGCGTCGACAGCAGGGCTGTCCTCTAACTGCGGGGCTGGTACAAGTGCCAAGGCTGCGGGGCTGCTGTCGTCGGCCTCGGTGTGGGTGCCCAAGCCCGTGAGGTGCACAGGCGGTCTCTTCCTCAGCGGGGATGCGCCGCTGCCCGCGCCGGTCACGCAGACGCGGGCCGTGGGTGGAGACGGAGATGAGTGCAGCGGCAGCAGCTTGGCGGCATCGTCGGGCGGCGGCGACTGGATGGACGACGTGAGAGCCTTGGCGTCGTTCCTCGAGTCCGACGAGGACTGGGTCAACTCCCTGCACATGGCGGATTAA.

[0045] SEQ ID No. 3: MRRACSAKEGVKRGAWTSKEDETLASYIKAHGEGRWKE VPLKAGLRRCGKSCRLRWLNYLRPSIKRGNISDDEEELIVRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNSTLGRKALPERAATRVVATPCASASSGSSTGASTAGLSSNCGAGTSAKAAGLLSSASVWVPKPVRCTGGLFLSGDAPLPAPVTQTRAVGGDGDECSGSSLAASSGGGDWMDDVRALASFLESDEDWVNSLHMAD*.

[0046] 2. Amplification of TaMYB6 gene

[0047] The total RNA of the wheat variety Zhonghuanchun was extracted, and reverse-transcribed into cDNA, and then amplified by using specific primers. The amplification system was 15 μL, including: DNA (20 ng / μL) 2 μL, forward primer (5 μM) 1 μL, reverse primer (5 uM) 1 μL, 2x PCR mixture 7.5 μL, and ddH2O 3.5 μL. After the PCR amplification system was configured, PCR amplification was performed, and the PCR amplification program included 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 1 min 30 s, 34 cycles; 72°C for 5 min, 25°C for 1 min.

[0048] TaMYB6-F (SEQ ID No. 4): 5'-ATGAGGAGGGCGTGCAGT-3';

[0049] TaMYB6-R (SEQ ID No. 5): 5'-TTAATCCGCCATGTGCAGG-3';

[0050] The PCR amplified target fragment was connected with a blunt-ended vector, and the reaction product was transformed into E. coli DH5α, 1 mL of LB was recovered for 1 h, 150 μL of the recovered bacterial liquid was coated on a LA medium plate containing resistance AMP, and incubated at 37°C for 12 h. The single colony was subjected to PCR detection, and the plasmid was extracted and sequenced. The PCR product was subjected to sequencing analysis, and the cDNA sequence of the TaMYB6 gene was obtained. The 729 bp TaMYB6 gene sequence amplified from Zhonghuanchun was compared with the sequence, and then homologous recombination was performed with the target vector.

[0051] Example 2

[0052] Regulation of TaMYB6 genetic material on the content of anthocyanin in wheat

[0053] 1. Construction of TaMYB6 genetic transformation vector

[0054] The 729 bp TaMYB6 gene fragment amplified from Chinese Spring in Example 1 was subjected to homologous recombination with the target vector pLGY, and the enzyme digestion site was BamHI, to obtain the recombinant vector. The vector map is shown in Figure 4 . The vector carries the Agrobacterium-mediated genetic transformation vector with the characteristics of constitutive and overexpression of the maize ubiquitin gene promoter.

[0055] 2. TaMYB6 genetic transformation

[0056] According to the existing technology (Hayta S, Smedley M A, Demir S U, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.), the plasmid correctly cloned in step 1 was introduced into the wheat variety Fielder through Agrobacterium EHA105 mediation and wheat genetic transformation system. After pre-culture, infection, co-culture, selection of glufosinate-resistant calli, differentiation, rooting, seedling culture and transplantation, etc., transgenic plants were obtained.

[0057] 4. Analysis of TaMYB6 overexpression transgenic material gene expression and wheat leaf and grain anthocyanin content

[0058] The DNA level of T1 generation transgenic wheat plants was detected to confirm the positive plants of TaMYB6 construction. The RNA samples and metabolic samples of the leaf at the stage of emergence and the seed of 3 weeks of filling were taken from the T1 generation positive transgenic wheat plants overexpressing TaMYB6 and wild type wheat plants, and the expression amount of TaMYB6 and the content of anthocyanin were analyzed. The results showed that compared with the control wild type Fielder, the expression amount of TaMYB6 gene in the positive transformation plants overexpressing TaMYB6 was greatly improved ( Figure 4 A), and the anthocyanin content in the stem, stem node and leaf sheath was significantly increased ( Figure 5 ).

[0059] The transcriptome sequencing of the leaf sheath of T1 generation transgenic wheat and wild type plants shows that the expression of chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid-3'-hydroxylase (F3'H), dihydroflavonol-4-reductase (DFR), anthocyanin synthase (ANS) and their modification genes glucosyltransferase (UGT), acyltransferase (MAT, ACT), methyltransferase (OMT) in the anthocyanin synthesis pathway of transgenic wheat are significantly up-regulated. Figure 6

[0060] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.​

Claims

1. Application of TaMYB6 gene in regulating plant anthocyanin.

2. Use according to claim 1, characterized in that, The protein encoded by the TaMYB6 gene has an amino acid sequence as shown in any one of the following: (1) the amino acid sequence shown in SEQ ID No. 3; (2) an amino acid sequence obtained by substitution, addition, deletion or mutation of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 3; (3) an amino acid sequence having more than 90% identity with the amino acid sequence of (1) or (2).

3. Use according to claim 2, characterized in that, The TaMYB6 gene comprises the full-length nucleotide sequence shown in SEQ ID No.

2.

4. Use according to claim 2 or 3, characterized in that, The TaMYB6 gene comprises the CDS sequence shown in SEQ ID No.

1.

5. The use according to claim 1, characterized in that, The regulation comprises increasing the anthocyanin content in plant tissues after overexpression of the TaMYB6 gene.

6. Use according to claim 5, characterized in that, When the plant is wheat, the plant tissues comprise stems, stem nodes and leaf sheaths.

7. A TaMYB6 gene overexpression vector.

8. The overexpression vector according to claim 7, wherein, The TaMYB6 gene in the overexpression vector is obtained by PCR amplification, wherein the primer pair used comprises TaMYB6-F shown in SEQ ID No. 4 and TaMYB6-R shown in SEQ ID No.

5.

9. Application of the overexpression vector of claim 7 or 8 in constructing a plant with high anthocyanin content.

10. A method for increasing anthocyanin content in a plant, characterized by, The TaMYB6 gene is overexpressed in the genome of the plant.

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