阅读说明：本技术 花青素合成相关蛋白AcMYB306及其编码基因与应用 (Anthocyanin synthesis related protein AcMYB306 and coding gene and application thereof ) 是由 李晓杰 梁毅 焦棒棒 曹琳娇 于 2020-12-15 设计创作，主要内容包括：本发明公开了花青素合成相关蛋白AcMYB306及其编码基因与应用。本发明所提供的花青素合成相关蛋白AcMYB306为氨基酸序列是SEQ ID NO:2所示的蛋白质。实验证明,与野生型拟南芥相比,转AcMYB306基因拟南芥幼苗中的花青素含量显著提高。花青素合成相关蛋白AcMYB306及其编码基因在调控植物花青素合成中具有重要的理论意义和实用价值。本发明在农业领域具有重要的应用和市场前景。(The invention discloses an anthocyanin synthesis related protein AcMYB306, and a coding gene and application thereof. The anthocyanin synthesis related protein AcMYB306 provided by the invention is a protein with an amino acid sequence shown in SEQ ID NO. 2. Experiments prove that compared with wild type arabidopsis, the anthocyanin content in the seedling of the AcMYB306 gene-transferred arabidopsis is obviously improved. The anthocyanin synthesis related protein AcMYB306 and the coding gene thereof have important theoretical significance and practical value in regulating and controlling plant anthocyanin synthesis. The invention has important application and market prospect in the agricultural field.)

An AcMYB306 protein that is 1) or 2) or 3) or 4) as follows:

1) the amino acid sequence is protein shown as SEQ ID NO. 2;

2) 2, the N end or/and the C end of the protein shown in SEQ ID NO. 2 is connected with a label to obtain fusion protein;

3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in 1) or 2), is derived from onion and is related to anthocyanin synthesis;

4) has 80 percent or more than 80 percent of homology with the amino acid sequence defined by SEQ ID NO. 2, is derived from onion and is related to anthocyanin synthesis.

2. A nucleic acid molecule encoding the AcMYB306 protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as (a1) or (a2) or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in 408 th to 1193 th positions from the 5' end of SEQ ID NO. 1;

(a2) the nucleotide sequence is a DNA molecule shown as SEQ ID NO. 1;

(a3) a DNA molecule derived from onion and encoding the AcMYB306 protein of claim 1 having 75% or greater homology to the nucleotide sequence defined in (a1) or (a 2);

(a4) a DNA molecule derived from onion and encoding the AcMYB306 protein of claim 1 that hybridizes under stringent conditions to the nucleotide sequence defined in (a1) or (a 2).

4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5. Use of an AcMYB306 protein of claim 1, or a nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector or recombinant microorganism comprising a nucleic acid molecule of claim 2 or 3, for modulating anthocyanin synthesis.

6. Use of an AcMYB306 protein of claim 1, or a nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector or recombinant microorganism comprising a nucleic acid molecule of claim 2 or 3, for the production of transgenic plants with altered anthocyanin levels.

7. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the AcMYB306 protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has an increased anthocyanin content as compared to the recipient plant.

8. The method of claim 7, wherein: said increasing the expression level and/or activity of an AcMYB306 protein of claim 1 in a recipient plant is effected by introducing into a recipient plant a nucleic acid molecule encoding said AcMYB306 protein.

9. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of an AcMYB306 protein of claim 1 in a plant, thereby increasing the anthocyanin content of the plant.

10. The use according to claim 6 or the method according to any of claims 7 to 9, wherein: the plant is any one of c1) to c 9): c1) a monocot plant; c2) a dicotyledonous plant; c3) a cruciferous plant; c4) arabidopsis thaliana; c5) wild type Arabidopsis thaliana Columbia-0 subtype; c6) a plant of the Liliaceae family; c7) onion; c8) red skin onion XiuQiu; c9) white skin onion Ring Master.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anthocyanin synthesis related protein AcMYB306, and a coding gene and application thereof.

Background

Anthocyanins belong to the class of bioflavonoids, and the most important physiological active functions of flavonoids are free radical scavenging and antioxidant capacity. Researches prove that the anthocyanin is the most effective antioxidant found by people at present and is also the most effective free radical scavenger, and the antioxidant performance of the anthocyanin is 50 times higher than that of VE and 20 times higher than that of VC. The anthocyanin not only makes the plant show colorful colors, but also has the health care function. The extract with certain anthocyanin concentration can effectively prevent canceration at different stages, but the individual effect of the anthocyanin is not determined. In addition, with the development of science and technology, people pay more and more attention to the safety of food additives, and the development and utilization of natural additives become the general trend of the development and use of additives. The anthocyanin can be used as a nutrition enhancer in food, can also be used as a food preservative to replace synthetic preservatives such as benzoic acid and the like, can be used as a food colorant to be applied to common beverages and food, and meets the general requirements of people on the nature, safety and health of food additives. Therefore, the method has important application value in mining related genes for anthocyanin synthesis.

Disclosure of Invention

The invention aims to improve the anthocyanin content of plants.

The invention firstly protects AcMYB306 protein, which can be 1) or 2) or 3) or 4) as follows:

1) the amino acid sequence is protein shown as SEQ ID NO. 2;

2) 2, the N end or/and the C end of the protein shown in SEQ ID NO. 2 is connected with a label to obtain fusion protein;

3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in 1) or 2), is derived from onion and is related to anthocyanin synthesis;

4) has 80 percent or more than 80 percent of homology with the amino acid sequence defined by SEQ ID NO. 2, is derived from onion and is related to anthocyanin synthesis.

Wherein SEQ ID NO 2 consists of 261 amino acid residues.

In order to facilitate the purification of the protein of 1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein shown in SEQ ID NO: 2.

TABLE 1 sequence of tags

The protein according to 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in 3) above can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of 3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in positions 408 to 1193 from the 5 ' end of SEQ ID NO. 1, and/or performing missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 to the 5 ' end and/or 3 ' end thereof.

The invention also protects a nucleic acid molecule encoding the AcMYB306 protein.

The nucleic acid molecule encoding the AcMYB306 protein can be a DNA molecule shown in the following (a1), or (a2), or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in 408 th to 1193 th positions from the 5' end of SEQ ID NO. 1;

(a2) the nucleotide sequence is a DNA molecule shown as SEQ ID NO. 1;

(a3) a DNA molecule which has 75% or more homology with the nucleotide sequence defined in (a1) or (a2), is derived from onion and encodes the AcMYB306 protein;

(a4) a DNA molecule which is derived from onion and encodes the AcMYB306 protein and hybridizes with the nucleotide sequence defined in (a1) or (a2) under strict conditions.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO. 1 consists of 1490 nucleotides, and the nucleotides shown at positions 408 to 1193 from the 5' end of SEQ ID NO. 1 encode the amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence encoding the AcMYB306 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that are artificially modified to have 75% or greater identity to the nucleotide sequence of the AcMYB306 protein isolated according to the present invention, provided that they encode the AcMYB306 protein, are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that is 75% or greater, or 80% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to the nucleotide sequence of the present invention encoding the AcMYB306 protein consisting of the amino acid sequence set forth in SEQ ID NO 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The invention also protects an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the nucleic acid molecules described above.

The recombinant vector containing any one of the nucleic acid molecules can be a recombinant plasmid obtained by inserting the DNA molecules shown in the positions 408 to 1193 from the 5' end of SEQ ID NO. 1 into the multiple cloning sites of the expression vector.

The recombinant vector can be specifically a recombinant plasmid SUP1300-AcMYB 306. The recombinant plasmid SUP1300-AcMYB306 can be a recombinant plasmid obtained by replacing a small DNA fragment between restriction enzyme SalI and KpnI restriction recognition sites of the super-expression vector SUP1300 with a DNA molecule shown in the positions 408 to 1193 from the 5' end of SEQ ID NO. 1.

The recombinant microorganism containing any of the above-described nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-described nucleic acid molecules into a starting microorganism.

The starting microorganism can be agrobacterium or escherichia coli. The agrobacterium may specifically be agrobacterium tumefaciens. The agrobacterium tumefaciens can be specifically agrobacterium tumefaciens GV 3101.

The recombinant microorganism containing any one of the nucleic acid molecules can be specifically a recombinant agrobacterium obtained by introducing a recombinant plasmid SUP1300-AcMYB306 into agrobacterium tumefaciens GV 3101.

The invention also provides application of any AcMYB306 protein, any nucleic acid molecule or an expression cassette, a recombinant vector or a recombinant microorganism containing any nucleic acid molecule in regulation and control of anthocyanin synthesis.

In the application, the regulation of anthocyanin synthesis can be used for improving anthocyanin synthesis or reducing anthocyanin synthesis.

The anthocyanin synthesis can be plant anthocyanin synthesis.

The invention also protects the application of any one of the AcMYB306 proteins, any one of the nucleic acid molecules or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the nucleic acid molecules in cultivation of transgenic plants with changed anthocyanin content.

In the application, the cultivation of the transgenic plant with the changed anthocyanin content can be the cultivation of a transgenic plant with increased anthocyanin content or the cultivation of a transgenic plant with reduced anthocyanin content.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: increasing the expression level and/or activity of any AcMYB306 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has an increased anthocyanin content as compared to the recipient plant.

In the above method, the "increasing the expression level and/or activity of any one of the AcMYB306 proteins in the recipient plant" may be achieved by a method known in the art, such as transgenosis, multicopy, promoter change, regulatory factor change, and the like, to increase the expression level and/or activity of any one of the AcMYB306 proteins in the recipient plant.

In the above method, said "increasing the expression level and/or activity of any of said AcMYB306 proteins in a recipient plant" may specifically be achieved by introducing into a recipient plant a nucleic acid molecule encoding any of said AcMYB306 proteins.

The nucleic acid molecule encoding any one of the AcMYB306 proteins can be a DNA molecule shown in the following (a1), or (a2), or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in 408 th to 1193 th positions from the 5' end of SEQ ID NO. 1;

(a2) the nucleotide sequence is a DNA molecule shown as SEQ ID NO. 1;

(a3) a DNA molecule which has 75% or more homology with the nucleotide sequence defined in (a1) or (a2), is derived from onion and encodes the AcMYB306 protein;

(a4) a DNA molecule which is derived from onion and encodes the AcMYB306 protein and hybridizes with the nucleotide sequence defined in (a1) or (a2) under strict conditions.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO. 1 consists of 1490 nucleotides, and the nucleotides shown at positions 408 to 1193 from the 5' end of SEQ ID NO. 1 encode the amino acid sequence shown in SEQ ID NO. 2.

In the above method, said introducing into a recipient plant a nucleic acid molecule encoding any of the above AcMYB306 proteins may be specifically effected by introducing into a recipient plant a recombinant vector comprising any of the above nucleic acid molecules.

The recombinant vector containing any one of the nucleic acid molecules can be specifically a recombinant plasmid SUP1300-AcMYB 306. The recombinant plasmid SUP1300-AcMYB306 can be a recombinant plasmid obtained by replacing a small DNA fragment between restriction enzyme SalI and KpnI restriction recognition sites of the super-expression vector SUP1300 with a DNA molecule shown in the positions 408 to 1193 from the 5' end of SEQ ID NO. 1.

The invention also provides a plant breeding method, which comprises the following steps: increasing the expression level and/or activity of any AcMYB306 protein in the plant, thereby increasing the anthocyanin content of the plant.

Any of the plants described above may be any of c1) to c 9): c1) a monocot plant; c2) a dicotyledonous plant; c3) a cruciferous plant; c4) arabidopsis thaliana; c5) wild type Arabidopsis thaliana Columbia-0 subtype; c6) a plant of the Liliaceae family; c7) onion; c8) red skin onion XiuQiu; c9) white skin onion Ring Master.

AcMYB306 gene is introduced into wild type Arabidopsis thaliana to obtain AcMYB306 gene-transferred Arabidopsis thaliana. Experiments prove that compared with wild type arabidopsis, the anthocyanin content in the seedling of the AcMYB306 gene-transferred arabidopsis is obviously improved. Therefore, the protein AcMYB306 related to anthocyanin synthesis can regulate and control plant anthocyanin synthesis. The invention has important application and market prospect in the agricultural field.

Drawings

FIG. 1 shows that relative expression of AcMYB306 gene in red-skin onion XiuQiu and white-skin onion Ring Master is detected by real-time fluorescent quantitative PCR.

FIG. 2 shows the expression level of AcMYB306 gene in AcMYB306 gene-transferred Arabidopsis thaliana by semi-quantitative PCR detection.

FIG. 3 shows phenotypic characterization of AcMYB306 transgenic Arabidopsis.

FIG. 4 is the determination of total anthocyanin content in AcMYB306 transgenic Arabidopsis.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The experimental procedures used in the examples described below are, unless otherwise specified, generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (second edition, written by J. SammBruk et al, Huang Petang et al, scientific Press, 2002), or according to the manufacturer's recommendations.

Red skin onion XiuQiu and white skin onion Ring Master are described in the following documents: chunsha Zhang, Xiaojie Li, Zongxiang Zhang, Aisong Zeng, Guojun Chang, Yi Liang. Transcriptome Sequencing and Metabolim Analysis improvements the roll of Cyanidin Metabolim in Dark-red Onion (Allium cepa L.) Bulb, Scientific Reports, 2018, 8(1): 14109.

wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004,36: 167-.

The super expression vector SUP1300 is described in the following documents: DEXH box RNA helicase-mediated heterologous reaction specific oxygen species production in Arabidopsis thaliana proteins cross talk between beta antisense and axin signaling [ J ] Plant Cell, 2012, 24 (5): 1815-1833 in this document the name pCAMBIA1300 recombinant overexpression promoter super.

Example 1 obtaining of AcMYB306 Gene

The inventor of the invention obtains the AcMYB306 gene by separating from onion through a large amount of experiments. The nucleotide sequence of the AcMYB306 gene is shown in SEQ ID NO. 1.

The AcMYB306 gene codes the AcMYB306 protein shown in SEQ ID NO. 2; specifically, the DNA molecule shown in the 408 th to 1193 rd positions from the 5' end of SEQ ID NO. 1 codes for the AcMYB306 protein shown in SEQ ID NO. 2.

Real-time fluorescent quantitative PCR detects the relative expression of AcMYB306 genes in the red skin onion XiuQiu and white skin onion Ring Master. The method comprises the following specific steps:

1. RNA of the red skin onion XiuQiu and the white skin onion Ring Master is respectively extracted and then reverse transcription is carried out to obtain corresponding cDNA.

2. The cDNA of red skin onion XiuQiu and white skin onion Ring Master are respectively used as templates, and the relative expression quantity of the AcMYB306 gene is detected by real-time fluorescent quantitative PCR (with the AcActin gene as an internal reference).

Primers for detecting the AcMYB306 gene are 5'-GGAATACTCATCTGAAACGAAAGC-3' and 5'-AGCAACCAAGTCTCCAGCATC-3'.

Primers for detecting AcActin gene were 5'-GCCGAGCGTGAGATAGTCCG-3' and 5'-CCTATCAGCAATGCCAGGAAAC-3'.

The relative expression level of AcMYB306 gene in the red-skin onion XiuQiu is obtained by taking the relative expression level of AcMYB306 gene in the white-skin onion Ring Master as 1.

The results are shown in FIG. 1(XiuQiu is red skin onion XiuQiu, Ring Master is white skin onion Ring Master). The result shows that the relative expression level of the AcMYB306 gene in the red-skin onion XiuQiu is obviously higher than that of the white-skin onion Ring Master.

It is described in the literature (Transcriptome Sequencing and Metabolism Analysis results the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulb, Scientific Reports, 2018, 8(1):14109) that the content of anthocyanins in red Onion XiuQiu is about 25 times that in white Onion Ring Master. It is speculated that the AcMYB306 gene may be involved in anthocyanin synthesis.

Example 2 obtaining of AcMYB306 Gene-transferred Arabidopsis thaliana and determination of anthocyanin content

Construction of recombinant plasmid SUP1300-AcMYB306 and acquisition of recombinant agrobacterium

1. RNA of the red skin onion XiuQiu is extracted and then reverse transcription is carried out to obtain cDNA of the red skin onion XiuQiu.

2. Taking cDNA of the onion XiuQiu with red skin as a template, adopting a primer MYB 306-SalI-F: 5' -CGCGTCGACATGGTGAGATCGCCTTGCTGCGA-3' (underlined recognition site for restriction enzyme SalI) and MYB 306-KpnI-TAA-R: 5' -CGGGGTACCTCAAAAGAAAACAGAATTATCAT-3' (recognition site of restriction enzyme KpnI is underlined) was subjected to PCR amplification to obtainTo about 804bp PCR amplification product.

3. And (3) carrying out enzyme digestion on the PCR amplification product obtained in the step (2) by using restriction enzymes SalI and KpnI, and recovering the enzyme digestion product.

4. The overexpression vector SUP1300 was digested with restriction enzymes SalI and KpnI, and the vector backbone of about 10070kb was recovered.

5. And connecting the enzyme digestion product with a vector framework to obtain the recombinant plasmid SUP1300-AcMYB 306.

The recombinant plasmid SUP1300-AcMYB306 was sequenced. The sequencing result shows that the recombinant plasmid SUP1300-AcMYB306 is a recombinant plasmid obtained by replacing a small DNA fragment between restriction enzyme SalI and KpnI restriction recognition sites of the super-expression vector SUP1300 with a DNA molecule shown in the 408 th to 1193 th positions from the 5' end of SEQ ID NO. 1, and the rest is unchanged.

6. The recombinant plasmid SUP1300-AcMYB306 is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium.

Second, AcMYB306 transgenic Arabidopsis

1. Transformation of recombinant Agrobacterium into wild-type Arabidopsis thaliana by the floral dip transformation method of Arabidopsis thaliana (Clough, S.J., and Bent, A.F.; Floraldip: expressed method for Agrobacterium-mediated transformation of Arabidopsis thaliana plant J. (1998)16, 735-₁The seed is transferred to AcMYB306 gene Arabidopsis thaliana.

2. The T obtained in the step 1₁The seed of the transgenic AcMYB306 arabidopsis is sown on an MS solid culture medium containing 30mg/L hygromycin, and the arabidopsis (resistant seedling) capable of growing normally is T₁Transformation of AcMYB306 Gene-Positive seedlings, T₁The seeds received by the positive seedlings of the transfer AcMYB306 gene are T₂The seed is transferred to AcMYB306 gene Arabidopsis thaliana.

3. The T of different strains screened in the step 2₂The seeds of the arabidopsis thaliana with the AcMYB306 gene transferred are sown on an MS solid medium containing 30mg/L hygromycin for screening, and if the ratio of the number of arabidopsis thaliana (resistant seedlings) capable of normally growing in a certain strain to the number of arabidopsis thaliana (non-resistant seedlings) incapable of normally growing in the strain is 3: 1, then theThe strain line is a strain line with one copy of AcMYB306 gene inserted therein, and the seeds received by the resistant seedlings in the strain line are T₃The seed is transferred to AcMYB306 gene Arabidopsis thaliana.

4. The T screened out in the step 3₃The seeds of the transgenic AcMYB306 arabidopsis are sown on an MS solid culture medium containing 30mg/L hygromycin again for screening, and the seeds which are all resistant seedlings are T₃The generation is homozygous AcMYB306 gene Arabidopsis thaliana. 1 of them is T₃The generation-homozygous AcMYB306 gene-transferred Arabidopsis strain was named OEAcMYB 306.

Three, half quantitative PCR detection

The Arabidopsis seed to be tested is T of OEAcMYB306₃Generation seed or wild type arabidopsis seed.

1. Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then spread on MS solid medium and vernalize for 2 days at 4 ℃.

2. And (3) after the step 1 is finished, taking the arabidopsis thaliana seeds to be detected, and carrying out light-dark alternate culture (16h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 7 days to obtain the arabidopsis thaliana seedlings to be detected.

3. And extracting RNA of the arabidopsis seedling to be detected, and then carrying out reverse transcription to obtain cDNA of the arabidopsis seedling to be detected.

4. The cDNA of arabidopsis thaliana to be detected is used as a template, and the expression quantity of the AcMYB306 gene is detected by semi-quantitative PCR (AtActin 2 gene is used as an internal reference).

The primer for detecting the AcMYB306 gene is MYB 306-F: 5'-GGAATACTCATCTGAAACGAAAGC-3' and MYB 306-R: 5'-AGCAACCAAGTCTCCAGCATC-3' are provided.

Primers for detecting AtActin2 gene are Primer-TF: 5'-AGCACTTGCACCAAGCAGCATG-3' and Primer-TR: 5'-ACGATTCCTGGACCTGCCTCATCTGCTCATACGGTCAGCGATA-3' are provided.

The results of the detection are shown in FIG. 2(M is DNA Marker, Col is wild type Arabidopsis). The results show that T of OEAcMYB306 is comparable to wild type Arabidopsis seedlings₃The expression level of AcMYB306 gene in seedlings obtained from the seeds of the generation is obviously increased.

Thus, T of OEAcMYB306 is shown₃The seedlings obtained by the generation of seeds are all positive seedlings.

Phenotypic identification of AcMYB306 transgenic Arabidopsis

The Arabidopsis seed to be tested is T of OEAcMYB306₃Generation seed or wild type arabidopsis seed.

1. Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then spread on MS solid medium and vernalize for 2 days at 4 ℃.

2. And (3) after the step 1 is finished, taking the arabidopsis thaliana seeds to be detected, and carrying out light-dark alternate culture (16h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 7 days to obtain the arabidopsis thaliana seedlings to be detected.

3. The color of the Arabidopsis seedlings was observed under a microscope.

The results are shown in FIG. 3(Col is wild type Arabidopsis). The results showed that the roots and leaves of the wild type Arabidopsis seedlings were green, and that the T of OEAcMYB306₃The roots and partial leaves of seedlings obtained by seed generation are purple red.

Fifthly, determination of total anthocyanin content of AcMYB306 transgenic arabidopsis

The Arabidopsis seed to be tested is T of OEAcMYB306₃Generation seed or wild type arabidopsis seed.

1. Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then spread on MS solid medium and vernalize for 2 days at 4 ℃.

2. And (3) after the step (1) is finished, taking the arabidopsis thaliana seeds to be detected, and carrying out light-dark alternate culture (16h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 14 days to obtain the arabidopsis thaliana seedlings to be detected.

3. And detecting the anthocyanin content of the arabidopsis seedlings to be detected. The method for measuring the anthocyanin content is described in the following documents: a radius Basic Helix-Loop-Helix transformation Factor, RsTT8 Acts a Positive Regulator for Anthocaryin biosyntheses, front Plant Sci 8: 1917.

the results of the detection are shown in FIG. 4(Col is wild type Arabidopsis thaliana). The results show that T of OEAcMYB306 is comparable to wild type Arabidopsis seedlings₃The anthocyanin content in the seedlings obtained by the generation of seeds is obviously improved.

The result shows that the anthocyanin content of wild arabidopsis thaliana can be obviously improved by over-expressing the AcMYB306 gene, namely the AcMYB306 protein can promote the synthesis of anthocyanin.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> agriculture and forestry academy of sciences of Beijing City

<120> anthocyanin synthesis related protein AcMYB306, and coding gene and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1490

<212> DNA

<213> Allium cepa L.

<400> 1

gtccttggaa atctaatagg ttttgatttg taaatcttaa acatacagct actataaaca 60

gtattcatta actactctta taatagccca tgccttgttt tttctttcct ttttcccggt 120

gcattctcaa taaatacaca cggttctgcg cagcagcagt gctagcttta gatagaaaca 180

agttactttg taaatagaga tatttagttg acttttatgg tattatgagc aaaataattt 240

attgctcctt gctgtcatca tgcatgttga ctcttcgctc ctcttcaatt gtagtcaatg 300

cattcttttt cctataaatc tcactgcaaa ccatagcttt gtacatatac tgctacttca 360

atattcagtt acatatacac caaatataca taagtataca taagaaaatg gtgagatcgc 420

cttgctgcga taaagtaggt gtgaaaaaag gaccatggac tcctgaagaa gacatcactt 480

tggtttctta cattcaagaa catggtcctg gaaactggaa ggctgtccct gctaatactg 540

ggttattgag atgtagcaag agctgtaggc tcagatggac aaattattta aggccaggga 600

taaagcgtgg aaactttact gatcaagaag agaagcttat tattcatctt caagctcttt 660

tgggcaacag atgggctgca attgcttcat atcttccaga aagaacagac aacgacatca 720

agaactactg gaatactcat ctgaaacgaa agcttgagaa aaacaacaaa ggcagtaaca 780

acagcaacac aaactctaaa ggaagatggg aaaaaagatt acaaactgat atccacatgg 840

caaaacaagc tcttgaagac gctttatctt tagataacaa acaaatcact tgcgataaca 900

atgacaactt tgagataaag ccatccacta catacgcttc gagcgcagag aatatttcaa 960

ggcttttgga aggatggaag aagactagtc cgatgaactc gaagtatact gaatctagcg 1020

agagcaccag tcaaagtgtg aatgtggcat cagaggtttc tgagagtagc gagagcaagc 1080

catgttttga ggtccaaatg ccaatttcga tgctggagac ttggttgctt gacgatagtt 1140

ttggaggagg tatgtttgat atggcaatgg atgataattc tgttttcttt tgaggtggtg 1200

attttgtatt cgtgcaggtt ttaaatgcct gttttgatta gcaaaattgt acattaagaa 1260

gatagagatt agtattgtac aagtgttgct ggcttaatgt aaaaggctaa aatgctgttc 1320

gatttcatgt tttttcttta aatattcaag tgaggaagta tcttattaac ttgattgttg 1380

taaagatgta ggctgtgtta attgaaatag taagtcatgg atgctacaag aacaatatta 1440

acatggaatt atcagaagtg taaatcaaat actcagcatt tgcaactttt 1490

<210> 2

<211> 261

<212> PRT

<213> Allium cepa L.

<400> 2

Met Val Arg Ser Pro Cys Cys Asp Lys Val Gly Val Lys Lys Gly Pro

1 5 10 15

Trp Thr Pro Glu Glu Asp Ile Thr Leu Val Ser Tyr Ile Gln Glu His

20 25 30

Gly Pro Gly Asn Trp Lys Ala Val Pro Ala Asn Thr Gly Leu Leu Arg

35 40 45

Cys Ser Lys Ser Cys Arg Leu Arg Trp Thr Asn Tyr Leu Arg Pro Gly

50 55 60

Ile Lys Arg Gly Asn Phe Thr Asp Gln Glu Glu Lys Leu Ile Ile His

65 70 75 80

Leu Gln Ala Leu Leu Gly Asn Arg Trp Ala Ala Ile Ala Ser Tyr Leu

85 90 95

Pro Glu Arg Thr Asp Asn Asp Ile Lys Asn Tyr Trp Asn Thr His Leu

100 105 110

Lys Arg Lys Leu Glu Lys Asn Asn Lys Gly Ser Asn Asn Ser Asn Thr

115 120 125

Asn Ser Lys Gly Arg Trp Glu Lys Arg Leu Gln Thr Asp Ile His Met

130 135 140

Ala Lys Gln Ala Leu Glu Asp Ala Leu Ser Leu Asp Asn Lys Gln Ile

145 150 155 160

Thr Cys Asp Asn Asn Asp Asn Phe Glu Ile Lys Pro Ser Thr Thr Tyr

165 170 175

Ala Ser Ser Ala Glu Asn Ile Ser Arg Leu Leu Glu Gly Trp Lys Lys

180 185 190

Thr Ser Pro Met Asn Ser Lys Tyr Thr Glu Ser Ser Glu Ser Thr Ser

195 200 205

Gln Ser Val Asn Val Ala Ser Glu Val Ser Glu Ser Ser Glu Ser Lys

210 215 220

Pro Cys Phe Glu Val Gln Met Pro Ile Ser Met Leu Glu Thr Trp Leu

225 230 235 240

Leu Asp Asp Ser Phe Gly Gly Gly Met Phe Asp Met Ala Met Asp Asp

245 250 255

Asn Ser Val Phe Phe

260