CN110857316B

CN110857316B - Anthocyanin synthesis related protein and application thereof in regulating and controlling plant anthocyanin content

Info

Publication number: CN110857316B
Application number: CN201810920439.4A
Authority: CN
Inventors: 林浩; 王崇楠; 季文楷; 牛丽芳
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2018-08-14
Filing date: 2018-08-14
Publication date: 2021-02-26
Anticipated expiration: 2038-08-14
Also published as: CN110857316A

Abstract

The invention discloses an anthocyanin synthesis related protein and application thereof in regulating and controlling plant anthocyanin content. The anthocyanin synthesis related protein disclosed by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 1; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). The anthocyanin synthesis related protein can regulate and control the content of plant anthocyanin, can be used for cultivating plants with increased or reduced anthocyanin content, and can also be used for regulating and controlling the color of plant plants.

Description

Anthocyanin synthesis related protein and application thereof in regulating and controlling plant anthocyanin content

Technical Field

The invention relates to an anthocyanin synthesis related protein and application thereof in regulating and controlling plant anthocyanin content in the technical field of biology.

Background

Anthocyanins are water-soluble flavonoids widely present in plants, are one of the main pigments constituting the color of petals and fruits, and play an important role in plant physiology; meanwhile, the anthocyanin is also a natural plant antioxidant, can protect plants and human bodies from being damaged by harmful substances such as free radicals and the like, and has a plurality of benefits on the health of human beings.

The medicago truncatula is a new leguminous model plant and has good collinearity with important leguminous crops and pasture such as soybean, alfalfa and the like. With the deep development of the functional genomics of medicago truncatula, some genes related to anthocyanin synthesis pathways and family transcription factors such as MYB and WD40 are cloned in the medicago truncatula, and researches find that the genes play an important role in the biosynthesis and metabolic regulation of anthocyanin in the medicago truncatula, but due to the special physiological structural characteristics of leguminous plants, the expression patterns and biological functions of the genes are different from those reported in researches in a dicotyledonous model plant Arabidopsis thaliana, which indicates that genetic diversity exists in the synthesis and metabolic regulation pathways of anthocyanin among different species.

Disclosure of Invention

The invention aims to provide an anthocyanin synthesis related protein, and a coding gene and application thereof.

The invention firstly provides any one of the following applications of an anthocyanin synthesis related protein (RH1 protein) or a substance for regulating the activity or content of the anthocyanin synthesis related protein:

D1) regulating and controlling the content of plant anthocyanin;

D2) preparing a product for regulating and controlling the content of plant anthocyanin;

D3) cultivating plants with increased anthocyanin content;

D4) preparing and cultivating plant products with increased anthocyanin content;

D5) cultivating plants with reduced anthocyanin content;

D6) preparing and cultivating plant products with reduced anthocyanin content;

D7) regulating and controlling the plant color;

D8) preparing a product for regulating and controlling the plant color;

D9) cultivating plants with changed colors;

D10) preparing a product for regulating and controlling the plant color;

the anthocyanin synthesis related protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

The RH1 protein in A2) is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The RH1 protein in A2) above may be synthesized artificially, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the RH1 protein in A2) described above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID NO. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence of the tag shown in the above table. Wherein, the DNA molecule shown in the sequence 2 encodes RH1 protein shown in the sequence 1.

The invention also provides any one of the following applications of the biological material related to the anthocyanin synthesis related protein:

D1) regulating and controlling the content of plant anthocyanin;

D3) cultivating plants with increased anthocyanin content;

D5) cultivating plants with reduced anthocyanin content;

D6) preparing and cultivating plant products with reduced anthocyanin content;

D7) regulating and controlling the plant color;

D8) preparing a product for regulating and controlling the plant color;

D9) cultivating plants with changed colors;

D10) preparing a product for regulating and controlling the plant color;

the biomaterial is any one of the following B1) to B9):

B1) nucleic acid molecules encoding the anthocyanin synthesis-associated protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule for reducing the expression level of the protein related to anthocyanin synthesis;

B9) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B8).

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b12) a DNA molecule shown in 3179-position 6011 of a sequence 3 in a sequence table;

b13) a cDNA molecule or a genome DNA molecule which has 75 percent or more identity with the nucleotide sequence defined by b11) or b12) and codes the protein related to the anthocyanin synthesis;

b14) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by b11) or b12) or b13) under strict conditions and codes the anthocyanin synthesis related protein;

B2) the expression cassette may be b11) or b12) or b13) as follows:

b21) DNA molecule shown in sequence 3 in the sequence table;

b22) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b21) and has the same function;

b23) a DNA molecule which is hybridized with the nucleotide sequence defined by b21) or b22) under strict conditions and has the same function.

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.

The nucleotide sequence of the invention encoding the RH1 protein can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the RH1 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode RH1 protein and have the function of RH1 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. 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.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding RH1 protein (RH1 gene expression cassette) described in B2) refers to a DNA capable of expressing RH1 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of RH1 gene but also a terminator for terminating transcription of RH1 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); fromChemically inducible promoter of tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beach et al (1985) EMBO J. 4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant vector containing the RH1 gene expression cassette can be constructed by using an existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be pBI121 vector or pCAMBIA2300 vector.

B3) The recombinant vector can be pCAMBIA2300-RH1pro:: RH 1. RH1 is a recombinant vector obtained by replacing a DNA fragment between EcoRI and PstI recognition sequences of a pCAMBIA2300 vector with a DNA fragment shown in a sequence 3.

B8) The recombinant vector can be a recombinant vector which can reduce the content of RH1 and is prepared by utilizing a crisper/cas9 system. The recombinant vector can express a sgRNA targeted to B1) the nucleic acid molecule. The target sequence of the sgRNA can be 400-419 th position of the sequence 2 or 175-193 th position of the sequence 2 in the sequence table.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes AGL 1.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides any one of the following methods:

x1) cultivating the plant with increased anthocyanin content, which comprises the steps of enabling the receptor plant to express the protein related to anthocyanin synthesis, or increasing the content of the protein related to anthocyanin synthesis in the receptor plant, or increasing the activity of the protein related to anthocyanin synthesis in the receptor plant, so as to obtain the target plant with increased anthocyanin content;

x2) cultivating the plant with reduced anthocyanin content, which comprises reducing the content of the protein related to anthocyanin synthesis in the receptor plant, or reducing the activity of the protein related to anthocyanin synthesis in the receptor plant, so as to obtain the target plant with reduced anthocyanin content;

x3) to obtain plant with changed plant color, which comprises changing the content of the protein related to anthocyanin synthesis in the receptor plant or changing the activity of the protein related to anthocyanin synthesis in the receptor plant.

In the above method, X1) may be carried out by introducing a gene encoding the protein involved in anthocyanin synthesis into the recipient plant and expressing the gene;

x2) the method can be effected by inhibiting expression of a gene encoding the protein involved in anthocyanin synthesis in the recipient plant;

x3) can be carried out by introducing a gene encoding the protein involved in anthocyanin synthesis into the recipient plant and allowing the gene to be expressed, or by inhibiting the expression of a gene encoding the protein involved in anthocyanin synthesis in the recipient plant.

In the above method, the encoding gene may be B1).

In the above method, the encoding gene of RH1 may be modified as follows, and then introduced into a recipient plant, so as to achieve a better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding RH1 according to the present invention may be changed to conform to the preference of a recipient plant while maintaining the amino acid sequence thereof; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding RH1 can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding RH 1. The recombinant expression vector can be specifically pCAMBIA2300-RH1pro:: RH 1.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

The plant of interest is understood to comprise not only the first generation plant in which the RH1 protein or the gene encoding it has been altered, but also its progeny. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

The present invention also provides a product having the following function of D1) or D2), which contains the anthocyanin synthesis-related protein or the biomaterial:

D1) regulating and controlling the content of plant anthocyanin;

D2) regulating and controlling the plant color.

The product can use the protein related to anthocyanin synthesis or the biological material as an active ingredient, and can also use the protein related to anthocyanin synthesis or the biological material together with other substances with the same function as the active ingredient.

In the present invention, the plant may be M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) leguminous plants;

m3) Medicago truncatula.

In the present invention, the plant color may be leaf color.

The anthocyanin synthesis related protein or the biological material also belongs to the protection scope of the invention.

The invention clones RH1 gene from medicago truncatula, successfully constructs plant CRISPR knockout vector, and eliminates erythema of plant leaves with reduced anthocyanin content after RH1 gene is knocked out in agrobacterium-mediated leaf disc method transformed plants. When the RH1 gene is introduced into wild plants, the anthocyanin content is increased. The RH1 gene is related to the content of anthocyanin, and can be used for regulating the content of anthocyanin.

Drawings

FIG. 1 is a phenotypic picture of wild type leaves of Medicago truncatula R108 and red heart mutant leaves of Medicago truncatula rh 1.

FIG. 2 shows the result of determination of anthocyanin content in R108 leaf and RH1 leaf and the expression of RH1 gene therein.

FIG. 3 shows the leaf phenotype of the rh1 mutant obtained by genome editing and the molecular detection of the R108 and rh1 mutant genome edited plants. MtRH1-1/rh1 is a CRISPR-1 transgenic plant, and MtRH2-3/rh1 and MtRH2-2/rh1 are CRISPR-2 transgenic plants.

FIG. 4 shows the phenotype of RH1 transgenic plants and the expression level of RH1 gene. pRH1, RH1/R108-1 and pRH1, RH1/R108-2 are two positive trans-RH 1 gene plants respectively, ox-1 is pRH1, RH1/R108-1, ox-2 is pRH1, RH 1/R108-2.

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 experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments 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. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

Medicago truncatula R108 (wild type): supplied by The Nobel Foundation.

rh1 mutant: screening Medicago truncatula Tnt1 insertion mutant library in The Nobel Foundation, and isolating a mutant red heart1(rh1) with abnormal anthocyanin synthesis in Tnt1database (r)https:// medicago-mutant.noble.org/mutant/index.php) The mutant is numbered as NF7599, and mainly shows that anthocyanin is specifically accumulated on leaves and shows a phenotype of big red heart.

The pFGC5941-Cas9 vector is described in (Meng et al, 2017, Targeted mutagenesis by CRISPR/Cas9system in the model legume medical guide trunculata, Plant Cell Rep (2017)36: 371-374), which is publicly available from the applicant, is only used for repeating the experiments related to the present invention, and is not used for other purposes.

YEP solid medium: 10g of peptone, 10g of yeast extract, 5g of sodium chloride, 10g of agar powder and distilled water to a constant volume of 1L.

YEP liquid medium: 10g of peptone, 10g of yeast extract, 5g of sodium chloride and distilled water to a constant volume of 1L.

Callus induction liquid medium (PH 5.8): 100mL of macroelement mother liquor, 1mL of microelement mother liquor, 1mL of organic element mother liquor, 20mL of iron salt mother liquor, 100mg of inositol, 30g of sucrose, 4mg of auxin, 0.5mg of cytokinin, 200mg of cephalosporins and 250mg of timentin, and the constant volume is 1L. Callus induction solid medium (PH 5.8): 100mL of macroelement mother liquor, 1mL of microelement mother liquor, 1mL of organic element mother liquor, 20mL of iron salt mother liquor, 100mg of inositol, 30g of sucrose, 4mg of auxin, 0.5mg of cytokinin, 200mg of cephalosporin, 250mg of timentin and 3.2g of Phytagel, and the volume is up to 1L.

Differentiation medium (PH 5.8): 100mL of macroelement mother liquor, 1mL of trace element mother liquor, 1mL of organic element mother liquor, 20mL of ferric salt mother liquor, 100mg of inositol, 20g of sucrose, 200mg of cephalosporin, 250mg of timentin and 3.2g of Phytagel, and the volume is up to 1L.

Rooting medium (PH 5.8): murashige & Skoog basic Medium with Vitamins 2.215g/L (company: Phyto Technology Laboratories, cat. No.: 16B0519138A), agar powder 7 g/L.

Mother liquor of iron salt: 37.3mg of disodium ethylene diamine tetraacetate and 27.8mg of ferrous sulfate heptahydrate, and the volume is determined to be 1L.

Macroelement mother liquor: magnesium sulfate heptahydrate 1.85g, potassium nitrate 28.3g, ammonium sulfate 4.63g, calcium chloride dihydrate 1.66g, potassium dihydrogen phosphate 4g, distilled water to constant volume of 1L.

And (3) a microelement mother solution: 1g of manganese sulfate monohydrate, 500mg of boric acid, 100mg of zinc sulfate heptahydrate, 100mg of potassium iodide, 10mg of sodium molybdate dihydrate, 20mg of copper sulfate pentahydrate and 10mg of cobalt chloride hexahydrate, and distilled water is added to a constant volume of 1L.

Organic element mother liquor: 500mg of nicotinic acid, 500mg of thiamine hydrochloride, 500mg of pyridoxine hydrochloride and distilled water to a constant volume of 1L.

Example 1, discovery of RH1 and Gene encoding RH1

Mutant acquisition and phenotypic analysis

A mutant with abnormal anthocyanin synthesis is obtained in The Nobel Foundation by screening a Zygophyllum alfalfa Tnt1 insertion mutant library, The mutant is numbered NF7599 in Tnt1database, mainly shows that anthocyanin is specifically accumulated on leaves and shows a red heart phenotype, and The mutant is named red heart1(rh 1). Hybridizing the mutant with wild type medicago truncatula R108 to obtain F₁Intermediate type with lighter red heart, F₂The population isolates three phenotypes of wild type, red heart light intermediate type and red heart phenotype, and meets the separation ratio of 1:2: 1(p is 0.754)>0.05). Indicating that the red heart character of the rh1 mutant is controlled by a semi-dominant single gene for the wild type R108.

The observation of leaf phenotype of the rh1 mutant and Medicago truncatula R108 (wild type) is shown in FIG. 1. In FIG. 1, the left panel shows the R108 leaf of wild-type Medicago truncatula with small red circles at the base of the leaflets; the middle diagram is the hybrid F of R108 and rh1 mutant₁Leaf, small red circle enlarged into virtual red heart; the right panel shows the rh1 mutant leaf blade, and the small red circle is enlarged to form a solid red heart.

And (3) determining the relative content of anthocyanin: the materials to be tested are leaves of medicago truncatula R108 (wild type) and rh1 mutant. Analyzing the anthocyanin content in the leaves of the plant to be detected, and the steps are as follows:

(1) material taking: selecting plants of medicago truncatula R108 (wild type) and rh1 mutant with consistent growth state, taking just developed mature compound leaves, respectively weighing about 100mg, recording actual weighing result (FW), freezing the taken leaves with liquid nitrogen for 20min, grinding, placing in 1mL of 1% (v/v) hydrochloric acid methanol solution (solution consisting of 990 mu L of methanol and 10 mu L of hydrochloric acid), shaking and uniformly mixing, and standing in dark at 4 ℃ for 1 day to obtain an extracting solution.

(2) Centrifuging: centrifuging the extract obtained in step (1) at 13,000rpm for 15min, and collecting the supernatant.

(3) Respectively measuring A on the supernatant obtained in the step (2) on an ultraviolet-visible spectrophotometer₅₃₀And A₆₅₇。

(4) Calculating the relative content of anthocyanin (Canthocyanine) ([ A ]₅₃₀-(1/4×A₆₅₇)]/FW。

The result of the measurement of the relative anthocyanin content in the leaves of the medicago truncatula R108 and rh1 mutant is shown in the left picture in FIG. 2, and the relative anthocyanin content in the leaves of the rh1 mutant is remarkably higher than that of the medicago truncatula R108.

Second, map-based cloning of candidate genes

F hybridizing with A17 (sequenced genomic information variety) and rh1 according to the molecular marker of medical go truncatula InDel developed by the applicant₂Map-based cloning is carried out, the gene is positioned on a chromosome 7, and a candidate gene RH1 gene is found by combining RNA-seq data. The expression level of the gene in the RH1 mutant and the medicago truncatula R108 is detected, the trend of the relative content of the anthocyanin in leaves is consistent, the RH1 gene expression in the RH1 mutant is obviously improved and is 7 times higher than that of the wild medicago truncatula R108, and the right graph in figure 2 is shown.

The gene is presumed to be a candidate gene causing the anthocyanin content in the RH1 mutant and controlling the leaf red heart phenotype, and is marked as an anthocyanin synthesis related gene, namely RH1 gene for short. The primers used for detecting the expression level of the RH1 gene are q40-F and q40-R, and the sequences are as follows:

q40-F:5'-cctctgctGTACAACCAAGTCT-3'；

q40-R:5'-ACTGCTCCCCATGTCCCATA-3'。

the sequences of the primers of the internal reference are as follows:

qActin-F:5'-TCAATGTGCCTGCCATGTATGT-3'；

qActin-R:5'-ACTCACACCGTCACCAGAATCC-3'。

III, RH1 and obtaining of encoding gene RH1 thereof

1. Total RNA of mature compound leaves of medicago truncatula R108 which are just unfolded is extracted and is reversely transcribed into cDNA.

2. And (3) taking the cDNA obtained in the step (1) as a template, and amplifying by using a primer pair consisting of a primer cacc40-CDS-F and a primer 40-CDS-R to obtain a PCR amplification product.

5'-caccATGGCGAATACAAGCGGCGT-3' parts of 40-CDS-F; (cacc is a linker sequence)

40-CDS-R:5’-TTAAAGATCTCGAAGAAATTCAA-3’。

3. Sequencing the PCR amplification product obtained in the step 2 to obtain a coding region sequence of the target gene, wherein the sequence is a sequence 2 in the sequence table, and the sequence is a protein shown in the sequence 1 in the coding sequence table. The protein shown in the sequence 2 of the sequence table is named as RH1, and RH1 consists of 242 amino acid residues. The gene encoding RH1 was designated as RH1 gene.

Example 2 functional verification of RH1 Gene

Construction of CRISPR/Cas9 genome editing vector

PCR amplification is carried out on MtU6-F1 and MtU6-R1 by taking pMtU6-Cas (Meng et al, 2017, Targeted mutagenesis by CRISPR/Cas9system in the model regulating medical stem and loop, Plant Cell Rep (2017)36: 371-374) as a template and utilizing primers to obtain a MtU6 promoter; PCR amplification is carried out by respectively using a primer pair 1 (consisting of a 40-sgRNA-F1 primer and an R2 primer) and a primer pair 2 (consisting of a 40-sgRNA-F2 primer and an R2 primer) to obtain two DNA fragments of sgRNA and scaffold which respectively code target fragments, namely a sgRNA1-scaffold fragment and a sgRNA2-scaffold fragment. Taking a mixture of MtU6 promoter and sgRNA1-scaffold fragment as a template, and carrying out PCR amplification by using a primer pair (consisting of MtU6-F1 and R2) to obtain pMtU6: sgRNA1-scaffold fragment; a mixture of MtU6 promoter and sgRNA2-scaffold fragment is used as a template, a primer pair (consisting of MtU6-F1 and R2) is utilized to carry out PCR amplification to obtain pMtU6, wherein sgRNA2-scaffold fragment is recombined to a linearized pFGC5941-cas9 vector skeleton obtained by XbaI digestion of pFGC5941-cas9 vector, and a recombinant vector is obtained.

The two recombinant vectors with correct sequences are named as CRISPR-1 and CRISPR-2 respectively through sequencing verification. The CRISPR-1 can express Cas9 and sgRNA1 targeting RH1 gene, the target sequence of sgRNA1 is GATGATAAAGTTATAGGCCG (400-419 bit of sequence 2 in the sequence table), the CRISPR-2 can express Cas9 and sgRNA2 targeting RH1 gene, and the target sequence of sgRNA2 is AATTCTCCCGGTTGATGTA (175-193 bit of sequence 2 in the sequence table). The primers used were as follows:

MtU6-F1:5'-gcttaggccttctagaATCCAACATTTCACTTGAGTTAACT-3'；

MtU6-R1:5'-AAACCCTGCTGTTCGTCTAG-3'；

40-sgRNA-F1：

5’-CTAGACGAACAGCAGGGTTTGATGATAAAGTTATAGGCCGgttttagagctagaaatag-3’；

40-sgRNA-F2：

5’-CTAGACGAACAGCAGGGTTTgAATTCTCCCGGTTGATGTAgttttagagctagaaatag-3’；

R2：5’-GGCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGTGC-3’。

II, obtaining CRISPR/Cas9 genome editing plant

1. And (3) respectively transforming the recombinant vectors CRISPR-1 and CRISPR-2 prepared in the step one into agrobacterium AGL1 to respectively obtain recombinant bacteria AGL1/CRISPR-1 and AGL 1/CRISPR-2.

2. The recombinant bacteria AGL1/CRISPR-1 and AGL1/CRISPR-2 obtained in the step 1 are respectively inoculated on a YEP solid culture medium containing 50mg/mL rifampicin antibiotic and 50mg/mL kanamycin antibiotic, the culture is carried out at 28 ℃ until single colonies grow out, the single colonies are picked up to a YEP liquid culture medium containing 50mg/L rifampicin antibiotic and 50mg/L kanamycin antibiotic, and the culture is carried out overnight at 28 ℃ and 200rpm in a shaking way.

3. After completing step 2, inoculating 500 μ L of the bacterial liquid into 5mL YEP liquid medium containing 25mg/L rifampicin antibiotic and 50mg/L kanamycin antibiotic, adding 5 μ L100 mg/mL acetosyringone, performing shaking culture at 28 deg.C and 200rpm until OD is reached_600nmThe bacterial solution was centrifuged at 3800rpm for 15min at 0.8 to collect the bacterial cells.

4. Suspending the thallus obtained in step 3 by using a callus induction liquid culture medium containing 100mg/L acetosyringone, and adjusting OD of the bacteria solution_600nmAnd (5) obtaining the staining solution when the concentration is 0.2.

5. And (3) taking the first compound leaf of the rh1 mutant growing for about 4 weeks, rinsing the first compound leaf with 75% ethanol aqueous solution for about 10s, then disinfecting the first compound leaf with 5% sodium hypochlorite for 5min, washing the first compound leaf with sterile water for at least 5 times on a super clean bench, cutting the leaf, then placing the cut leaf into the staining solution obtained in the step (4), and infecting the cut leaf for 15 min.

6. After the step 5 is completed, the infected leaves are transferred to a callus induction solid culture medium, 2mg of glufosinate-ammonium is added into every 1L of the culture medium for antibiotic screening, and the white embryogenic callus is cultured for four weeks until the white embryogenic callus appears (the culture medium is replaced once in two weeks).

7. After step 6, the white embryogenic callus was transferred to differentiation medium, 2mg of glufosinate was added to 1L of medium for antibiotic selection, and cultured for four weeks to differentiate green embryoid (medium was changed once for two weeks).

8. And (3) after the step (7) is completed, transferring the green embryoid to a rooting culture medium, replacing the culture medium once in two weeks, transferring the rooting and leaf growing medium to vermiculite until the rooting and leaf growing medium becomes seedling, and respectively obtaining CRISPR-1 and CRISPR-2 transgenic plants.

Third, phenotype and genotype determination of CRISPR/Cas9 genome editing plants

And (3) the plant to be detected: the medicago truncatula rh1 mutant, CRISPR-1 transgenic plant, CRISPR-2 transgenic plant and wild-type medicago truncatula R108.

1. Observing the developed compound leaf phenotype of the plant to be detected. The results are shown in FIG. 3. The results show that the leaves of the rh1 mutant, which have undergone genome editing, disappear from the scarlet heart of the rh1 mutant, and the base of the leaflet has no red circle, or the red heart becomes smaller.

2. And (3) detecting the relative content of anthocyanin: and (3) detecting the relative content of the anthocyanin of each plant to be detected according to the method for measuring the relative content of the anthocyanin in the step one in the embodiment 1. The results show that the relative content of anthocyanin in the medicago truncatula rh1 mutant is obviously higher than that in the CRISPR-1 transgenic plant, the CRISPR-2 transgenic plant and the wild-type medicago truncatula R108.

3. Extracting the genome DNA of the transgenic plant with the diminished or reduced scarlet heart, performing PCR amplification by adopting 40-cas-JD-F and 40-cas-JD-R primer pairs, and sequencing the obtained PCR product. 40-cas-JD-F and 40-cas-JD-R primer sequences:

40-cas-JD-F:5’-GCCTTTATGCGTCACCTATCT-3’；

40-cas-JD-R:5’-CTCCCCATGTCCCATAAACTG-3’。

the result of the alignment with the genome sequence of the RH1 gene is shown in FIG. 3, and different strains are found to have different genome sequence deletions, and the result shows that the CRISPR/Cas9 genome editing plant RH1 gene detection is positive.

The expression level of the RH1 gene in the RH1 mutant is obviously higher than that of the wild-type medicago truncatula R108 by detecting the expression level of the RH1 gene in the RH1 mutant, the wild-type medicago truncatula R108, the transgenic plant of CRISPR-1 and the transgenic plant of CRISPR-2. The primers and the reference gene used for detecting the expression level of RH1 gene were the same as those used in example 1.

By observing the phenotype, the leaves of the homozygote plant obtained by the rh1 mutant through genome editing disappear from the scarlet heart of the rh1 mutant, the base of the leaflet has no red circle, and the red heart of the heterozygote plant obtained by the rh1 mutant through genome editing becomes small; the relative content of anthocyanin in homozygote plants is obviously lower than that of hybrid plants and the alfalfa rh1 mutant of the tribulus, and the relative content of anthocyanin in the heterozygote plants is obviously lower than that of the alfalfa rh1 mutant of the tribulus. The experiments show that the RH1 gene can improve the anthocyanin content of medicago truncatula.

Example 3 elevated levels of Medicago truncatula anthocyanin transgenic for RH1 Gene

In this example, RH1 gene was introduced into wild medicago truncatula R108 to further illustrate that RH1 gene can increase the anthocyanin content of medicago truncatula, and the specific method is as follows:

first, construction of recombinant vector

Taking the genome DNA of the mutant rh1 as a template, and carrying out PCR amplification on 40-PRO-EcoRI-F and 40-3' UTR-PstI-R by using a primer pair to obtain a DNA fragment shown in a sequence 3 in a sequence table; the DNA fragment between EcoRI and PstI recognition sequences of pCAMBIA2300 was replaced with the DNA fragment shown in sequence 3, and the resulting recombinant vector was named pCAMBIA2300-RH1pro:: RH 1. pCAMBIA2300-RH1pro RH1 can express protein shown in sequence 1 in a sequence table. The primer sequences used were:

40-PRO-EcoRI-F:5'-ccatgattacgaattcACTCGTATGTGGGGATTTTGA-3'；

40-3'UTR-PstI-R:5'-ttgtatatcactgcagTCTTTCTTTTCCACTGCATTGC-3'。

wherein, the 1 st-3178 th site of the sequence 3 is a sequence which starts RH1 gene expression at the upstream of RH1 gene, the 3179 st-6011 st site is a genome sequence of RH1 gene, and the 6012 st-7654 th site is a sequence which stops RH1 gene expression at the downstream of RH1 gene. The exon sequences in the genome sequence of the RH1 gene are 3179-3296 th site, 5254-5383 th site and 5534-6011 th site of the sequence 3 respectively.

Second, construction of transgenic plants

According to the method of the second step in the example 2, the CRISPR-1 and CRISPR-2 are replaced by pCAMBIA2300-RH1pro, RH1, the RH1 mutant is replaced by wild-type medicago truncatula R108, and other steps are not changed to prepare RH1 gene-transferred plants. And (3) adding 50mg of kanamycin into each 1L of culture medium by utilizing a callus induction solid culture medium and a differentiation culture medium of the RH-transferred 1 gene plant for screening to obtain a positive RH-transferred 1 gene plant.

Identification of transgenic plants

And (3) identifying the positive RH1 transgenic plant obtained in the second step on the RNA level, and using wild medicago truncatula R108 as a control by the following method:

the method for detecting the RH1 gene expression level in the example 2 is used for detecting the RH1 gene expression level in the positive RH1 transgenic plant, and the result shows that the RH1 gene expression level in the positive RH1 transgenic plant is obviously increased relative to the wild-type medicago truncatula R108, wherein the RH1 gene expression levels in the two positive RH1 transgenic plants are 835 and 1172 times of the wild-type medicago truncatula R108.

Fourth, identification of phenotype of transgenic plant

1. The whole plant of the positive RH1 gene is changed from green to purple, and the base of the leaf is darker than the edge.

2. And (3) detecting the relative content of anthocyanin: the anthocyanin relative content and the RH1 gene expression level of the positive transgenic RH1 gene plant in the third step are respectively detected according to the anthocyanin relative content measuring method and the RH1 gene expression level detecting method in the first step of the example 1, and wild-type medicago truncatula R108 and RH1 mutant are used as a control.

The result shows that the relative content of the anthocyanin of the RH1 mutant is obviously higher than that of the wild-type medicago truncatula R108, and the relative content of the anthocyanin of the plant with the positive RH1 transgenic gene is respectively obviously higher than that of the wild-type medicago truncatula R108 and the RH1 mutant. The RH1 gene expression level has the same trend in these plants. The RH1 gene is shown to improve the anthocyanin content of medicago truncatula.

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> anthocyanin synthesis related protein and application thereof in regulation and control of plant anthocyanin content

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 241

<212> PRT

<213> Medicago truncatula

<400> 1

Met Ala Asn Thr Ser Gly Val Arg Lys Gly Ala Trp Thr Tyr Glu Glu

1 5 10 15

Asp Asn Cys Leu Lys Ala Tyr Ile Leu Lys His Gly Val Gly Lys Trp

20 25 30

His Leu Ile Pro Glu Arg Thr Gly Leu Asn Arg Cys Arg Lys Ser Cys

35 40 45

Arg Leu Arg Trp Val Asn Tyr Leu Asn Pro Tyr Ile Asn Arg Glu Asn

50 55 60

Phe Ser Lys Asp Glu Val Asp Leu Ile Leu Arg Leu His Asn Leu Leu

65 70 75 80

Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro Gly Arg Thr Ala

85 90 95

Asn Asp Val Lys Asn Tyr Trp Asn Thr Tyr Leu Arg Lys Lys Val Glu

100 105 110

Ser Glu Ala Lys Glu Lys Glu Lys Ser Asn Glu Thr Met Lys Ala His

115 120 125

Glu Val Ile Lys Pro Arg Pro Ile Thr Leu Ser Ser Arg Ser His Phe

130 135 140

Val His Asp Ser Asn Lys Asp Arg Tyr Val Pro Ile Tyr Gln Asp Asp

145 150 155 160

Ser Ser Glu Thr Met Val Pro Ser Gln Ile Gly Gly Asp Tyr Ala Ser

165 170 175

Ala Val Gln Pro Ser Leu Gly Asn Asn Val Gln Thr Pro Cys Ala Met

180 185 190

Trp Ser Asp Ser Leu Trp Asp Met Gly Ser Ser Glu Lys Ile Gly Ser

195 200 205

Cys Ser Ser Leu Gln Glu Val Asn Asn Phe Asn Met Asp Phe Pro Asp

210 215 220

Asp Ser Phe Trp Asp Phe Asn Phe Ser Asp Phe Glu Phe Leu Arg Asp

225 230 235 240

Leu

<210> 2

<211> 726

<212> DNA

<213> Medicago truncatula

<400> 2

atggcgaata caagcggcgt tagaaaaggt gcatggacat atgaagaaga caactgtctc 60

aaggcttaca ttctcaagca tggtgtagga aaatggcatt taattcctga aagaacagga 120

ttgaatagat gtcgtaaaag ttgtagattg aggtgggtaa attatttaaa tccctacatc 180

aaccgggaga atttttctaa ggatgaagtt gatttgattc taaggttaca caacctccta 240

ggcaatagat ggtcattgat tgctggaagg cttccaggta gaacagctaa tgatgtgaaa 300

aactattgga atacatattt acgcaagaag gttgaatcag aagcaaaaga aaaagagaaa 360

tctaatgaaa caatgaaagc tcatgaggtt attaaacctc ggcctataac tttatcatct 420

cgttcacatt ttgttcatga ttcaaataaa gatcgttatg ttcccatata tcaagatgat 480

tcatcagaga ctatggttcc aagtcaaatt ggtggagatt atgcctctgc tgtacaacca 540

agtcttggta ataatgtcca aacaccgtgt gcaatgtggt cagacagttt atgggacatg 600

gggagcagtg agaaaattgg ctcatgctct tcattacaag aggtgaacaa cttcaacatg 660

gattttcctg atgactcctt ttgggatttc aacttttctg attttgaatt tcttcgagat 720

ctttaa 726

<210> 3

<211> 7654

<212> DNA

<213> Medicago truncatula

<400> 3

actcgtatgt ggggattttg agattcatct atattgatgt aataatagat tcatgacatg 60

tatatttgga gtactctggt ttatatccga tgcgactatt gagatatata tgcatgcatg 120

ttgagttttg aattttgaat aatgacgtag catctatttc ttgaataaac atattatttg 180

catgttttat tgctttaata gaaatagggt gttacaattt tcatccaatt aagctaaaaa 240

ctcatggtta ttgtaacacc ctctttgaat tatttgtttt atttaattat ttaattgagt 300

ctagaattta ttaagaagag tttagaatat atttatatga ttatgtaatt tattaagtgg 360

cttattatat tatttaatag aataagatta gaaaataaaa taatattaag tttaggggtt 420

gttgtgagat tttagagagt tttggggaag aaagagaaat aagataaaat agaaataaag 480

attataaata ggagagacct agtttagaaa aatagaatgt acgaccaatt ttggagaaaa 540

gggagaaaaa gccaagagaa gggagaagca catagaggct gcgattttat tattctaagg 600

taagggtgag actagcattc aattctatta atctatataa ttctgatgat tttatttagc 660

aaatgtagga ttgaattaga gaagttggga attagggttg gaatgtagaa ttgaattgtg 720

gaaagaagta gataatatga tggattaggg tgataaagga tgtttagatt gtagattaat 780

cacagactaa gtttccaatt agtattgggg tttgagtcga tggaaatagg gattttcgtt 840

tgaatagttg gtgtctaaat gttttgcaaa taagagatta tgtgaggttt ggaaaatcga 900

tttttggtgg ttgaaacttg aatctttttg tagattatga tagggctaag tgttaggagc 960

gtgtaggcaa aaatggcatc gaaaacgggt taacggtttg atttttatgt tcgaaaacgt 1020

gaaggttgaa aatctgaagt cgtctgtcaa atctggaaac atcgtgccac gatgggagac 1080

caacgtgcca cgatgatgtc ataccagaaa ccttaaaaat gaaattttct tcactccaaa 1140

tgcttccaaa cttcttcata agtgtaggga ctttatccta accatctaga gatgttttta 1200

ggaaataaat aacgttgtat aaagggttta atgagttgtt gaaagttgaa ccttggggta 1260

gttatgaata ttatttacaa tattaatgat gatctgtgaa gctgatttat gatgatttga 1320

tgttgtttat aatgtgatat aattgtatat gcattacttg aattatatgt gaatatttga 1380

gatgttgttt acttgcgttc gatattatta tatcatgatg ttgtgcatac tgcctatgtt 1440

gctgattgtt aattaactaa gctgcatgag tcagtctaag tcgttgaaga tgttgatgtt 1500

ccaaattatt ggactattat aagaggttgt tgaattaaga tgtttaagaa gtcgagtcca 1560

tgcattatct tttcagtgtt gggggtttga tgccttggag ctttgtactc accacgttgg 1620

aggtttagct caattagcga taggggcttg attccctaga gtataataat actcagatag 1680

cggtaggggc ttgatgctcc gtattggtac cacatgcata taagaggtct aagttgcata 1740

atcgagttgg agtcgcattt gtcgagtcaa ggatgtttaa gttgataagt tgtgaagttg 1800

taattgytta tacaaactat gattgaagtg ataaatgata tattactatc tataatgaat 1860

attaagatga tttgatgttg attaaatata attgttgaat tctatgctta atattattgt 1920

tttgtgaaat ctcacccctt ctgcttggaa atgttgctct tcgtatgagt aacttgcagg 1980

tattaaagat tagtaggagt ggtagctcaa ttgtcttagg gctctgatat gtaacgggat 2040

ggaaaattgt tgctatgttt atcattcgtt tatgtatcaa ttttggaaac atgttgaagc 2100

ttttattgtc attgaattca atctgctgca aagtttaatt attacattgt tggaatttga 2160

aggataatta tttaattatt ccatttataa tttttaagaa aagaagtgta acatttcgtt 2220

taaggtgttt tactctgatt aatttatgaa atttttatgt tgggaaaaca tggtgttata 2280

gctatgaact taatataatc aaacattgaa attgatgtaa caacaacaag attctaccac 2340

ttttcacata gtcaaactta tcacctcatt tagagcatga aaatcaactt aaataatcat 2400

caattttaca aattaacata aacaaacatg acccaaacac tagaaaagtg attatttatt 2460

atgagaatca aacctatccc cttacctcaa tctcctaaaa gcttaagtta gagtttcact 2520

ctcctcttct ttctcttcta agatattctt atttcttccc cttctcttta aatttgttat 2580

ttgacggaaa tcaaaggagg taatggtgtg tcgttaagaa aaggtattaa caaatacaca 2640

gacttaaaat tttcttaata aatttgatta caatatctat catttccatt tggaaaaact 2700

tatgacatga atgcacaacc ctaatttctt agtagagtta tccttatata atctcactaa 2760

atagcttagg gatgtattat taattacttg aaccttctct agtattatta ctaagtcagt 2820

aggtctcaat atatgcgggt cgtagtactt aaacttttac cagtattttt attctatata 2880

attttttgct aataaaaata aattaacttc tgtggctgag cgcgttcgac actttccgca 2940

aatcatatct attaaataca tacatacagt acctttatgg tggcaaagaa tgaaaaggac 3000

gtatcacaaa aaacaatcaa agaaaagtat tgtaatactc tcgtctctat gttctatatg 3060

cgaaataagt tttttttgtc ttttacccaa ccttactatt aaaggtcgta tatatttgta 3120

gcaaaagatc atgcattcat gcctatattt gtgtatgaac agttaaggaa aggtttgtat 3180

ggcgaataca agcggcgtta gaaaaggtgc atggacatat gaagaagaca actgtctcaa 3240

ggcttacatt ctcaagcatg gtgtaggaaa atggcattta attcctgaaa gaacaggtat 3300

aaagaatgtt ccttattcta ctacgtattt ggcttataat tagttaaaat aattaaacaa 3360

caatagagac aacaaatgta aaataacgat gcataattgt ttttcctaaa tacaataaca 3420

atttttattt ttttatacaa atcatttgct agaggtatgc ttatatcaca aacaatcgaa 3480

ataaaaattg aaaactaata ggagtattca taacccaaat ggatattttt caaaaaaaaa 3540

aaaaaaaaaa accaaatgga tcaaattaga tcaaatttaa aaaagaaata aatcaatcag 3600

tatgatgttt taatgaataa atatataggt tataatgtat attaattttt taaaaaatag 3660

tatgaagttt tattaatcca ttatcttttg tttttaattt tttcttacta tttttatgaa 3720

tgaaattaat agtagaaaat atcatattat attatatttt ttggactagc aaacgagaag 3780

atatgaatat aataaaaaat agggatcata gtttaaagaa tactaaagaa tacccatttt 3840

tttttaggga tattatattt ttaaataatt gtaacatata acctaatttt ttcatcccta 3900

aaaaaaaacc tacttttttc agaaaactcc atgttaatga tgtttgcaac gactacagtt 3960

caatagcttt agaaaaaaag gaacttgtta gtgtcaatat aaatcaaaac gaataatgga 4020

tgagtgccac gatttatccc aaatcagtta attaaataag aagtgagttg tctctataag 4080

catagttcag ttggcaggga catacattgt tatatgcatg gactggggtt tgaaccctga 4140

acacctttac ttattcatct tgaaaaaagt gaattctagt cactaaacca cttgttcatc 4200

ttgaaaaagt gaattcaaaa gtgcagcaat tcggttaaaa gcttaattca aagtcactaa 4260

actcttcggt taattcttat gattttagat ttatggaaac gagtaatttc ccatgtaaac 4320

tctaagtttt tttttttttt tttttttttt tttttttttt ttttttttta agtttgaact 4380

ctggaccttg catatattat ccattgtccc taccaactga gctaaactca caagaaccgt 4440

aaactctatg tttcttaaac tagtacgagt tgaagaaaaa aaaaacactg ataacatttg 4500

gtaaattctc gattctaata atgatttact cacaaggtta cgattataca ttaatttagt 4560

ttttagagta atgctaatag tatctcagga cattagttac gaaaataaaa atagaatttt 4620

ttcattaaaa acaccttttt tttttgtcaa aaaaaaaaaa agagttgaac atctcacttt 4680

ttacggcttt ttaatccaag atttctatat taattttact gaataatgct ctagggacac 4740

tagttagaat ttttctagtt tttatacata gatttttgga aaatattcgc cagtatttga 4800

tataattatt tatgtgttgc aatatatacc ttagttttaa aaagtaaatt tatggtttta 4860

gtgtagtagt gagggacaac tatatactcg cctttatgcg tcacctatct atcgaattat 4920

cgaaaaaata ggcaattatc atatagttat tatttatcta tgttttaatt ttttatttcg 4980

ctaatttgtt ttagttattc ttaaatattt atcagtttga aaataattat ggcttttgat 5040

agagatgaaa tgtttaatta ttaattatta ttattattaa cttaatgatt ttattttatc 5100

tatatgtgtg ttgttttgat attatgtaaa ctctaacgag tttataagtt gagtctacat 5160

gttgaattta tgtagactta actctaacaa ctttggtcta atgacataaa ctgaacaaga 5220

attttgaaaa tatttgttgc atgtacttgt caggattgaa tagatgtcgt aaaagttgta 5280

gattgaggtg ggtaaattat ttaaatccct acatcaaccg ggagaatttt tctaaggatg 5340

aagttgattt gattctaagg ttacacaacc tcctaggcaa taggtaagct tcaaaggtca 5400

gatcaaatat tgtttgtgta tgctagcgct gctgaggata tttatgcttg tgtgtatcta 5460

tctgtctata tatatatata tattagtcaa cattgaagtt aatttactta tacatacaat 5520

acatatcgta cagatggtca ttgattgctg gaaggcttcc aggtagaaca gctaatgatg 5580

tgaaaaacta ttggaataca tatttacgca agaaggttga atcagaagca aaagaaaaag 5640

agaaatctaa tgaaacaatg aaagctcatg aggttattaa acctcggcct ataactttat 5700

catctcgttc acattttgtt catgattcaa ataaagatcg ttatgttccc atatatcaag 5760

atgattcatc agagactatg gttccaagtc aaattggtgg agattatgcc tctgctgtac 5820

aaccaagtct tggtaataat gtccaaacac cgtgtgcaat gtggtcagac agtttatggg 5880

acatggggag cagtgagaaa attggctcat gctcttcatt acaagaggtg aacaacttca 5940

acatggattt tcctgatgac tccttttggg atttcaactt ttctgatttt gaatttcttc 6000

gagatcttta atttttgtga aatattatta gttctacgtt gaagattaac aaagtcgctt 6060

actgtttgtc aaaacaaaat ggcacgaaat aaaatgaatc aaaatgtaac atatgttcta 6120

tgttatattg catggcttga gtattttgat tttggaataa aagaatcttt gaataagaaa 6180

gatagtgatg ttgttgaaag ttggatggtt tttttggagg ggttatggtg aacagcaatc 6240

gaggggtgat tgccttttgt tttggtggtc actcacaagg aattgtgaag gtttgacttt 6300

gatttaggtg ttgaaactgg tttgtcctca gcttagatga tgtgtattgg tgtaattatg 6360

atttttcgtt tgggtgttca gtctatatat acagcgtctt cgttttgtag tttttgtcca 6420

tatatatatc tttgttcgtc ttgtataggg atttagttat taataaattt tgctgttaaa 6480

aaaaaaatag atctgatgat gatgatgaag ttgatgttac tagaaaccta aggtattcac 6540

ttcattcatc taatgtttct ttaaatttta ttgaacaaaa atgaactata ttggcgtgtt 6600

ggtactatgt gattatgtga tggtgatatt ttttatagca atcctatatc tataaaaaaa 6660

ataacatcaa caattcttca agaataaaga gcagccaatc acaatgcact atttacacaa 6720

tttaaggagg tgtggctgaa aaaaatggat attttgttgg gcggcagaat gaacaaaata 6780

gtttgttaag tgttgttttg attaagttta cttcatgaga tttattctct tgatctttaa 6840

ttataaatac tcattgagca acaattttgt ccctaatata gcctcttata gaatttagaa 6900

gatgtatttt tttttaatac aactaacttg atttgaaatt tgaaatcaaa tttaatacac 6960

atacacaaat gacaatttaa gcaaaaaaaa ttcattttct ttgtcccaaa atataagcaa 7020

aaaagacaaa cttttattct atttaatctt gtttttttta aaaatctctt tccaaagaaa 7080

aacttttgtt tattctcata aaattaaacg caaattacat tgaattttct ccctctttca 7140

ttttttccat aaccaatact caatgaaaac tgtttttaca tctttctata aaactttttt 7200

taaaaaaaac acaaaaaact atactacaaa ttcttatgtt ttagtttttt tagcaagtgt 7260

gatttttatt tttattttta taatctggga catagggagt atcaatattt tttttggtac 7320

aatcaactat atcaaattta tgtattaaaa acttgaacaa ggattaatat gttgatgaag 7380

ggtacatatt taaaagatga gaaaacatgg gagaacaaac cgaagagagg aaccttttcc 7440

gtacacaccc acccacatgg attgtgcaga agaatttcca gaaaagttat acataacaaa 7500

gcacacgaga atagataaat agttctgata tgttttatat gtaagtaatg tgtcaagcag 7560

agagaggagg tgtaaatggt taaatagttc tgaaatccta attatatgca ttagataaac 7620

ttgtgtatgt atgcaatgca gtggaaaaga aaga 7654

Claims

1. Any one of the following applications of the anthocyanin synthesis related protein:

D1) regulating and controlling the anthocyanin content of the medicago truncatula;

D2) preparing a product for regulating and controlling the anthocyanin content of the medicago truncatula;

D3) cultivating medicago truncatula with increased anthocyanin content;

D4) cultivating alfalfa with reduced anthocyanin content;

D5) regulating and controlling the leaf color of medicago truncatula;

D6) preparing a product for regulating and controlling the color of leaves of medicago truncatula;

D7) culturing medicago truncatula with changed leaf color;

the anthocyanin synthesis related protein is A1) or A2) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) a1) at the N-terminus or/and the C-terminus.

2. Use of a biological material related to the protein involved in anthocyanin synthesis as set forth in claim 1, wherein the biological material comprises:

D3) cultivating medicago truncatula with increased anthocyanin content;

D4) cultivating alfalfa with reduced anthocyanin content;

D5) regulating and controlling the leaf color of medicago truncatula;

D7) culturing medicago truncatula with changed leaf color;

the biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the protein involved in anthocyanin synthesis of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) as follows:

b11) the coding sequence is a DNA molecule of a sequence 2 in a sequence table;

b13) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding the protein related to anthocyanin synthesis as claimed in claim 1;

B2) the expression cassette is a DNA molecule shown in a sequence 3 in a sequence table.

4. Any one of the following methods:

x1) cultivating the medicago truncatula with increased anthocyanin content, which comprises the steps of increasing the content of the protein related to anthocyanin synthesis in claim 1 in the receptor medicago truncatula, or increasing the activity of the protein related to anthocyanin synthesis in claim 1 in the receptor medicago truncatula, so as to obtain the medicago truncatula with increased anthocyanin content;

x2) cultivating the medicago truncatula with reduced anthocyanin content, which comprises reducing the content of the protein related to anthocyanin synthesis in claim 1 in the receptor medicago truncatula, or reducing the activity of the protein related to anthocyanin synthesis in claim 1 in the receptor medicago truncatula, so as to obtain the medicago truncatula with reduced anthocyanin content;

x3) cultivating the leaf color-changed medicago truncatula, which comprises changing the content of the protein related to anthocyanin synthesis in the claim 1 in the receptor medicago truncatula or changing the activity of the protein related to anthocyanin synthesis in the claim 1 in the receptor medicago truncatula, so as to obtain the destination medicago truncatula with the changed leaf color.

5. The method of claim 4, wherein: x1) by introducing a coding gene of the protein related to anthocyanin synthesis in claim 1 into the receptor medicago truncatula and expressing the coding gene;

x2) by inhibiting the expression of a gene encoding said protein involved in the synthesis of anthocyanidin according to claim 1 in said lucerne tribulus;

x3) by introducing a coding gene of the anthocyanin synthesis-related protein in the claim 1 into the receptor medicago truncatula and expressing the coding gene, or inhibiting the expression of the coding gene of the anthocyanin synthesis-related protein in the receptor medicago truncatula in the claim 1.

6. The method of claim 5, wherein: the coding gene is the nucleic acid molecule of B1) in claim 2 or 3.