阅读说明：本技术 一种培育花青素含量降低和开花时间推迟的转基因植物的方法 (Method for cultivating transgenic plant with reduced anthocyanin content and delayed flowering time ) 是由 林浩 季文楷 牛丽芳 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种培育花青素含量降低和开花时间推迟的转基因植物的方法。EAF2蛋白的氨基酸序列如SEQ ID NO：2所示。实验证明,向突变体eaf2中导入重组质粒35S::EAF2,得到互补株；与突变体eaf2相比,互补株的叶片中花青素含量显著降低,开花节数增加,开花时间推迟。由此可见,EAF2蛋白可以调控花青素合成、开花节数和开花时间。本发明具有重要的应用价值。(The invention discloses a method for cultivating a transgenic plant with reduced anthocyanin content and delayed flowering time. The amino acid sequence of the EAF2 protein is shown as SEQ ID NO: 2, respectively. Experiments prove that the mutant EAF2 is introduced with recombinant plasmid 35S, EAF2, to obtain a complementary strain; compared with the mutant eaf2, the anthocyanin content in the leaves of the complementary strain is obviously reduced, the number of flowering nodes is increased, and the flowering time is delayed. Therefore, the protein EAF2 can regulate and control anthocyanin synthesis, flowering node number and flowering time. The invention has important application value.)

The application of the EAF2 protein is at least one of A1) -A6):

A1) regulating and controlling anthocyanin synthesis;

A2) cultivating a transgenic plant with changed anthocyanin content;

A3) regulating and controlling the flowering time of the plants;

A4) cultivating transgenic plants with changed flowering time;

A5) regulating and controlling the number of flowering nodes of the plants;

A6) and (5) cultivating the transgenic plant with the changed flowering node number.

2. The use of claim 1, wherein: the EAF2 protein is a1) or a2) or a3) or a4) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2) and is related to plant anthocyanin synthesis, flowering node number and/or flowering time;

a4) protein which has 80% or more than 80% of identity with the protein shown in a1) or a2), is derived from medicago truncatula and is related to plant anthocyanin synthesis, flowering node number and/or flowering time.

3. Use of a nucleic acid molecule encoding the EAF2 protein of claim 1 or 2, at least one of a1) -a 6):

A1) regulating and controlling anthocyanin synthesis;

A2) cultivating a transgenic plant with changed anthocyanin content;

A3) regulating and controlling the flowering time of the plants;

A4) cultivating transgenic plants with changed flowering time;

A5) regulating and controlling the number of flowering nodes of the plants;

A6) and (5) cultivating the transgenic plant with the changed flowering node number.

4. Use according to claim 3, characterized in that: the nucleic acid molecule is a DNA molecule shown as b1) or b2) or b3) or b 4):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in b1) or b2), derived from medicago truncatula and encoding an EAF2 protein as defined in claim 1 or 2;

b4) a DNA molecule derived from medicago truncatula and encoding the EAF2 protein as claimed in claim 1 or 2, which hybridizes under stringent conditions to the nucleotide sequence defined in b1) or b 2).

5, H1) or H2):

H1) the plant breeding method A comprises the following steps: increasing the expression level and/or activity of the EAF2 protein of claim 1 or 2 in a plant, thereby reducing anthocyanin levels, increasing flowering node number and/or delaying flowering time;

H2) the plant breeding method B comprises the following steps: reducing the expression level and/or activity of the EAF2 protein of claim 1 or 2 in a plant, thereby increasing anthocyanin levels, reducing flowering node numbers and/or advancing flowering time.

6. The use according to any one of claims 1 to 4 or the method according to claim 5, wherein: the plant is any one of the following c1) to c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c5) medicago truncatula R108; c6) mutant eaf 2.

7. A method for breeding transgenic plant A comprises the following steps: increasing the expression level and/or activity of the EAF2 protein in claim 1 or 2 in a starting plant A to obtain a transgenic plant A; the transgenic plant A has reduced anthocyanin content, increased number of flowering nodes, and/or delayed flowering time compared to the starting plant A.

8. The method of claim 7, wherein: the expression level and/or activity of the EAF2 protein in the starting plant A are/is improved by introducing a nucleic acid molecule encoding the EAF2 protein into the starting plant A.

9. A method for breeding a transgenic plant B comprises the following steps: reducing the expression level and/or activity of the EAF2 protein in claim 1 or 2 in a starting plant B to obtain a transgenic plant B; the transgenic plant B has increased anthocyanin content, reduced flowering node number and/or earlier flowering time compared to the starting plant B.

10. The method of any of claims 7 to 9, wherein:

the starting plant A is any one of c1), c2), c3), c4) and c 6);

the starting plant B is any one of c1), c2), c3), c4) and c 5);

c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c5) medicago truncatula R108; c6) mutant eaf 2.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for cultivating a transgenic plant with reduced anthocyanin content and delayed flowering time.

Background

The plant flowering mark is switched from a nutrition stage to a reproduction stage, and plays an important role in the processes of plant progeny reproduction and ecological adaptation; wherein the early and late flowering of the crops have important influence on the high yield, stable yield, quality and regional distribution of the crops. Breeders have been working on obtaining excellent germplasm with ideal flowering time, using appropriate flowering time as one of the key agronomic traits. The analysis of the flowering time of plants has been a research hotspot of multiple disciplines such as plant molecular genetics, evolutionary biology, ecology, crop breeding and the like. At present, the molecular mechanism for regulating and controlling the flowering time of leguminous forage is not clear yet and needs to be further researched.

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.

Disclosure of Invention

The invention aims to promote the synthesis of plant anthocyanin.

The invention firstly protects the application of the EAF2 protein, which can be at least one of A1) -A6):

A1) regulating and controlling anthocyanin synthesis;

A2) cultivating a transgenic plant with changed anthocyanin content;

A3) regulating and controlling the flowering time of the plants;

A4) cultivating transgenic plants with changed flowering time;

A5) regulating and controlling the number of flowering nodes of the plants;

A6) and (5) cultivating the transgenic plant with the changed flowering node number.

In the above application, the protein of EAF2 may be a1) or a2) or a3) or a4) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2) and is related to plant anthocyanin synthesis, flowering node number and/or flowering time;

a4) protein which has 80% or more than 80% of identity with the protein shown in a1) or a2), is derived from medicago truncatula and is related to plant anthocyanin synthesis, flowering node number and/or flowering time.

Wherein, SEQ ID NO: 2 consists of 140 amino acid residues.

To facilitate purification and detection of the protein, the protein may be identified in the sequence set forth by SEQ ID NO: 2, and the amino terminal or the carboxyl terminal of the EAF2 protein consisting of the amino acid sequence shown in the table 1 is connected with the label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The EAF2 protein can be synthesized artificially, or synthesized by first synthesizing the coding gene and then performing biological expression.

The coding gene of the EAF2 protein can be obtained by converting SEQ ID NO: 1 to the 5 'end and/or the 3' end of the DNA sequence shown in Table 1.

The invention also protects the application of the nucleic acid molecule for coding any one of the EAF2 proteins, which can be at least one of A1) -A6):

A1) regulating and controlling anthocyanin synthesis;

A2) cultivating a transgenic plant with changed anthocyanin content;

A3) regulating and controlling the flowering time of the plants;

A4) cultivating transgenic plants with changed flowering time;

A5) regulating and controlling the number of flowering nodes of the plants;

A6) and (5) cultivating the transgenic plant with the changed flowering node number.

In the above application, the nucleic acid molecule may be a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from medicago truncatula and codes any one of the EAF2 proteins;

b4) a DNA molecule which is derived from medicago truncatula and encodes any of the EAF2 proteins and is hybridized with the nucleotide sequence defined by b1) or b2) 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 423 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

The nucleotide sequence encoding the EAF2 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 which are artificially modified and have 75% or more identity to the nucleotide sequence of the EAF2 protein isolated according to the present invention, as long as they encode the EAF2 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 the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 2, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the EAF2 protein. 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 nucleic acid molecule encoding the EAF2 protein can be specifically a gene encoding the EAF2 protein and is named as EAF2 gene.

In any of the above applications, the regulation of plant anthocyanin synthesis can be promotion of plant anthocyanin synthesis or inhibition of plant anthocyanin synthesis.

In any of the above applications, the cultivation of the transgenic plant with the modified anthocyanin content can be cultivation of a transgenic plant with an increased anthocyanin content or cultivation of a transgenic plant with a reduced anthocyanin content.

In any of the above applications, the "regulating the plant flowering time" may be advancing the plant flowering time or retarding the plant flowering time.

In any of the above applications, the "transgenic plant with altered flowering time" may be a transgenic plant with an earlier flowering time or a transgenic plant with a later flowering time.

In any of the above applications, the "controlling the number of flowering nodes of the plant" may be an increase in the number of flowering nodes of the plant or a decrease in the number of flowering nodes of the plant.

In any of the above applications, the "breeding of transgenic plants with altered flowering nodes" may be breeding of transgenic plants with increased flowering nodes or breeding of transgenic plants with decreased flowering nodes.

In the use of any of the above, the plant may be any of the following c1) to c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c5) medicago truncatula R108; c6) mutant eaf 2.

The invention also protects H1) or H2).

H1) The plant breeding method A comprises the following steps: increasing the expression level and/or activity of any of the above-described EAF2 proteins in a plant, thereby reducing anthocyanin levels, increasing flowering node numbers, and/or delaying flowering time.

H2) The plant breeding method B comprises the following steps: reducing the expression level and/or activity of any of the EAF2 proteins in a plant, thereby increasing anthocyanin content, reducing flowering node number and/or advancing flowering time.

In the above method, the plant may be any one of the following c1) to c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c5) medicago truncatula R108; c6) mutant eaf 2.

The invention also provides a method for cultivating the transgenic plant A, which comprises the following steps: increasing the expression quantity and/or activity of any one of the EAF2 proteins in the starting plant A to obtain a transgenic plant A; the transgenic plant A has reduced anthocyanin content, increased number of flowering nodes, and/or delayed flowering time compared to the starting plant A.

In the method, the effect of improving the expression level and/or activity of the EAF2 protein can be achieved by using methods known in the art, such as multiple copies, promoter change, regulatory factor change, transgene change and the like, of the 'improvement of the expression level and/or activity of any one of the EAF2 proteins in the starting plant A'.

In the above method, the "increasing the expression level and/or activity of any of the above-mentioned EAF2 proteins in the starting plant a" may be performed by introducing a nucleic acid molecule encoding an EAF2 protein into the starting plant a.

In the above method, the nucleic acid molecule encoding the EAF2 protein may be a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from medicago truncatula and codes any one of the EAF2 proteins;

b4) a DNA molecule which is derived from medicago truncatula and encodes any of the EAF2 proteins and is hybridized with the nucleotide sequence defined by b1) or b2) 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 423 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In the above method, the "introduction of a nucleic acid molecule encoding an EAF2 protein into a starting plant a" may be specifically carried out by introducing a recombinant vector containing any of the nucleic acid molecules described above into the starting plant a. The "recombinant vector containing any one of the nucleic acid molecules" can be specifically a recombinant plasmid 35S:: EAF 2.

The construction process of the recombinant plasmid 35S is as follows:

(1) taking a pMDC83 vector as a template, and adopting a primer cacGFP-F: 5'-caccATGGGTAAAGGAGAACTTTTCA-3' and primers HA-R +6 Gly: 5'-GCCACCCCCTCCGCCACCGGCATAATCAGGCACATCG-3', and PCR amplification was performed to obtain a GFP fragment of about 800 bp.

(2) As set forth in SEQ ID NO: 1 as a template, and adopting a primer 6X Gly-NFY-B4-F: 5'-GGTGGCGGAGGGGGTGGCATGGCTGAAACGGAGGAG-3' and primer MtNF-YB 4-R: 5'-TCATTGGTCATCATCAACACCATC-3', and PCR amplifying to obtain NF-YB4 fragment of about 420 bp.

(3) The GFP fragment and the NF-YB4 fragment are mixed to be used as a template, and a primer pair consisting of a primer cacGFP-F and a primer MtNF-YB4-R is adopted for PCR amplification to obtain a GFP-NF-YB4 fragment of about 1200 bp.

(4) And carrying out BP reaction on the GFP-NF-YB4 fragment and a vector pENTR/D-TOP0(Invitrogen) to obtain an intermediate vector pENTR/D-GFP-NF-YB 4.

(5) And (3) carrying out LR reaction on the intermediate vector pENTR/D-GFP-NF-YB4 and the pMDC83 vector to obtain a recombinant plasmid 35S, namely EAF 2.

Any one of the starting plant formazan may be any one of c1), c2), c3), c4) and c 6); c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c6) mutant eaf 2.

When the starting plant A is the mutant EAF2, the transgenic plant A can be specifically the complementary strain 35S in the embodiment, EAF2/EAF2-2, 35S, EAF2/EAF2-6 or 35S, EAF2/EAF 2-8.

The invention also provides a method for cultivating the transgenic plant B, which comprises the following steps: reducing the expression level and/or activity of any one of the EAF2 proteins in the starting plant B to obtain a transgenic plant B; the transgenic plant B has increased anthocyanin content, reduced flowering node number and/or earlier flowering time compared to the starting plant B.

In the above method, the "reducing the expression level and/or activity of any of the above EAF2 proteins in the starting plant b" can be achieved by methods known in the art, such as DNA insertion, RNA interference, homologous recombination, gene site-directed editing, and the like, to inhibit the expression level or activity of the EAF2 protein.

In the above method, the "reducing the expression level and/or activity of any of the above EAF2 proteins in the starting plant b" may be obtained by inserting DNA into an exon of the EAF2 gene.

Any one of the starting plants B can be any one of c1), c2), c3), c4) and c 5); c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) alfalfa tribulus; c5) medicago truncatula R108.

The transgenic plant B can be specifically a mutant eaf 2.

Experiments prove that the mutant EAF2 is introduced with recombinant plasmid 35S, EAF2, to obtain a complementary strain; compared with the mutant eaf2, the anthocyanin content in the leaves of the complementary strain is obviously reduced, the number of flowering nodes is increased, and the flowering time is delayed. Therefore, the protein EAF2 and the coding gene thereof can regulate and control anthocyanin synthesis, flowering node number and flowering time. The invention has important application value.

Drawings

FIG. 1 shows the flowering phenotype, statistical results of flowering time and number of flowering segments, and leaf phenotype of Medicago truncatula R108 and mutant eaf 2.

FIG. 2 shows the result of the anthocyanin content detection of Medicago truncatula R108 and mutant eaf 2.

FIG. 3 shows the results of measuring the expression level of EAF2 gene in the complementary strain.

FIG. 4 shows the flowering phenotype identification, the flowering node number statistics and the leaf phenotype identification of the complementation strains.

FIG. 5 shows the result of detecting the anthocyanin content of the complementary strain.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

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

The Medicago truncatula R108 is supplied by The Nobel Foundation (website: https:// www.nobelprize.org/The-Nobel-prize-organization/The-Nobel-Foundation /).

Both the pMDC83 vector and Agrobacterium tumefaciens AGL1 are provided by the Biotechnology institute of Chinese academy of agricultural sciences (i.e., the Applicant), and are available to the public from the Applicant.

YEP liquid medium: dissolving peptone 10g, yeast extract 10g and sodium chloride 5g with appropriate amount of distilled water, adding distilled water to constant volume of 1L, and autoclaving at 121 deg.C for 15 min.

Callus induction liquid medium: dissolving macroelement mother liquor 100mL, microelement mother liquor 1mL, organic element mother liquor 1mL, ferric salt mother liquor 20mL, inositol 100mg, sucrose 30g, auxin 4mg and cytokinin 0.5mg with appropriate amount of distilled water, then diluting to 1L with distilled water, adjusting pH to 5.8, and autoclaving at 121 ℃ for 15 min.

Callus induction solid medium: dissolving macroelement mother liquor 100mL, microelement mother liquor 1mL, organic element mother liquor 1mL, ferric salt mother liquor 20mL, inositol 100mg, sucrose 30g, auxin 4mg, cytokinin 0.5mg, cephalosporin 200mg, timentin 250mg, hygromycin B5 mg and Phytagel 3.2g with appropriate amount of distilled water, then fixing the volume to 1L with distilled water, adjusting the pH value to 5.8, and autoclaving at 121 ℃ for 15 min.

Differentiation medium: dissolving macroelement mother liquor 100mL, microelement mother liquor 1mL, organic element mother liquor 1mL, ferric salt mother liquor 20mL, inositol 100mg, sucrose 20g, cephalosporium 200mg, timentin 250mg, hygromycin B5 mg and Phytagel 3.2g with a proper amount of distilled water, then fixing the volume to 1L with distilled water, adjusting the pH value to 5.8, and sterilizing at 121 ℃ for 15min under high pressure.

Rooting culture medium: 2.215g of Murashige & Skoog basic Medium with Vitamins (product of Phytotechnology Laboratories, product number 16B0519138A), 15g of sucrose and 7g of agar powder were dissolved in an appropriate amount of distilled water, then the volume was adjusted to 1L with distilled water, the pH was adjusted to 5.8, and autoclaving was carried out at 121 ℃ for 15 min.

Mother liquor of iron salt: 37.3mg of disodium ethylene diamine tetraacetate and 27.8mg of ferrous sulfate heptahydrate are dissolved by using a proper amount of distilled water, and then the volume is fixed to 1L by using the distilled water.

Macroelement mother liquor: magnesium sulfate heptahydrate 1.85g, potassium nitrate 28.3g, ammonium sulfate 4.63g, calcium chloride dihydrate 1.66g and potassium dihydrogen phosphate 4g were dissolved in an appropriate amount of distilled water, and then a constant volume of 1L was obtained with distilled water.

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 are dissolved by using a proper amount of distilled water, and then the volume is fixed to 1L by using the distilled water.

Organic element mother liquor: 500mg of nicotinic acid, 500mg of thiamine hydrochloride and 500mg of pyridoxine hydrochloride were dissolved in an appropriate amount of distilled water, and then the volume was made 1L with distilled water.

Mutant eaf2 was provided by a library of Medicago truncatula Tnt1 insertion mutants (website: https:// media-mutant. noble. org/mutant/index. php) and mutant eaf2 was numbered NF16632 in the library of Medicago truncatula Tnt1 insertion mutants. Mutant EAF2 is an alfalfa mutant of tribulus terrestris obtained by inserting Tnt1 into the first exon of the EAF2 gene (the EAF2 gene comprises 3 exons and two introns) according to the information recorded in the alfalfa Tnt1 insertion mutant library.

EXAMPLE 1 cloning of EAF2 Gene

Phenotypic analysis of mutant eaf2

And (3) vernalizing 30 mutant eaf2 seeds or medicago truncatula R108 seeds at 4 ℃ for 7d, planting the seeds into nutrient soil, alternately culturing in light and dark (16h of light/8 h of dark), observing the phenotype and counting.

The growth status of partial mutant eaf2 and Medicago truncatula R108 in light-dark alternate culture for 40 days is shown in A in FIG. 1 (WT is Medicago truncatula R108, eaf2 is mutant eaf 2). The results show that mutant eaf2 all flowered and alfalfa R108 from Zygophyllum did not flower when cultured alternately in dark and light for 40 days.

Counting the flowering time and the number of nodes of each medicago truncatula (namely the growth days and the number of nodes of the medicago truncatula when the first flower completely opens), and then taking an average value. The results are shown in B of figure 1 (WT is medicago truncatula R108, eaf2 is mutant eaf2, which indicates a very significant difference, P < 0.01). The results show that mutant eaf2 has significantly earlier flowering times and significantly reduced numbers of flowering nodes compared to alfalfa R108.

The positive and negative phenotypes of some alfalfa leaves are shown in FIG. 1C (WT is alfalfa R108, eaf2 mutant eaf2, Ad positive, Ab negative). The results showed that mutant eaf2 had more red spots on both the front and back of the compound leaves compared to alfalfa R108.

Secondly, detecting the anthocyanin content of leaves

1. Preparation of the Standard Curve

(1) Cyanidin 3-O-glucoside chloride standard (product of Sigma-Aldrich company, product number 52976) is taken and diluted with water to obtain standard solutions with different concentrations.

(2) And (3) respectively detecting the absorbance values (namely A530 values) of the standard solutions obtained in the step (1) at 530nm by using an ultraviolet-visible spectrophotometer.

(3) And (3) after the step (2) is finished, respectively taking the concentration of the standard solution as an abscissa and the corresponding A530 value as an ordinate, and drawing a standard curve.

The standard curve is: y is 0.024 x-0.0009; r²0.9988. Where x is the value of A530.

2. Detection of anthocyanin content

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

(1) taking just developed mature compound leaves (about 50mg) from a medicago truncatula plant (medicago truncatula R108 or mutant eaf2) to be detected with basically consistent growth state, and weighing (recording as m); freezing the leaves with liquid nitrogen for 20min, and grinding into powder; then respectively adding 500 μ L hydrochloric acid/methanol solution (prepared by mixing 999 μ L methanol and 1 μ L hydrochloric acid), shaking, mixing, and rotating overnight at 4 deg.C in the dark to obtain the extract of herba Medicaginis to be detected.

(2) Taking the extracting solution of the medicago truncatula to be detected, centrifuging for 15min at 2500g, and obtaining the supernatant of the medicago truncatula to be detected.

(3) And (3) detecting the absorbance value (namely A530 value) of the supernatant of the medicago truncatula to be detected at 530nm by using an ultraviolet-visible spectrophotometer.

(4) Substituting the absorbance value obtained in the step (3) into a standard curve to obtain the anthocyanin content in the supernate of the medicago truncatula; and further obtaining the anthocyanin content in the alfalfa leaves of the caltrops to be detected.

The results are shown in FIG. 2(WT is Medicago truncatula R108, eaf2 is mutant eaf 2; data are mean. + -. standard deviation of data, indicating significant differences, P < 0.05). The results show that compared with alfalfa R108, the anthocyanin content in the leaves of the mutant eaf2 is remarkably increased.

Cloning of EAF2 Gene

1. Extracting total RNA of mature compound leaves just developed from medicago truncatula R108 plants, and then carrying out reverse transcription to obtain cDNA of the medicago truncatula R108.

2. After the step 1 is finished, taking cDNA of the medicago truncatula R108 as a template, and adopting a primer MtNF-YB 4-F: 5'-ATGGCTGAAACGGAGGAG-3' and primer MtNF-YB 4-R: 5'-TCATTGGTCATCATCAACACCATC-3' to obtain 423bp PCR product.

3. Sequencing the PCR amplification product obtained in the step 2.

Sequencing results show that the PCR amplification product contains SEQ ID NO: 1. SEQ ID NO: 1, namely EAF2 gene, encodes the DNA molecule shown in SEQ ID NO: 2, EAF2 protein.

Example 2 acquisition of complementary strains, phenotypic identification and anthocyanin content detection

Firstly, construction of recombinant plasmid 35S, EAF2

1. Taking a pMDC83 vector as a template, and adopting a primer cacGFP-F: 5'-caccATGGGTAAAGGAGAACTTTTCA-3' and primers HA-R +6 Gly: 5'-GCCACCCCCTCCGCCACCGGCATAATCAGGCACATCG-3', and PCR amplification was performed to obtain a GFP fragment of about 800 bp.

2. Using the PCR amplification product obtained in step three 2 of example 1 as a template, primer 6 × Gly-NFY-B4-F: 5'-GGTGGCGGAGGGGGTGGCATGGCTGAAACGGAGGAG-3' and a primer MtNF-YB4-R to obtain NF-YB4 fragments of about 420 bp.

3. The GFP fragment and the NF-YB4 fragment are mixed to be used as a template, and a primer pair consisting of a primer cacGFP-F and a primer MtNF-YB4-R is adopted for PCR amplification to obtain a GFP-NF-YB4 fragment of about 1200 bp.

4. And carrying out BP reaction on the GFP-NF-YB4 fragment and a vector pENTR/D-TOP0(Invitrogen) to obtain an intermediate vector pENTR/D-GFP-NF-YB 4.

5. And (3) carrying out LR reaction on the intermediate vector pENTR/D-GFP-NF-YB4 and the pMDC83 vector to obtain a recombinant plasmid pMDC83-GFP-NF-YB4, which is marked as recombinant plasmid 35S, and EAF 2.

II, obtaining recombinant agrobacterium

The recombinant plasmid 35S, EAF2, is introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, which is named AGL1/35S, EAF 2.

Third, obtaining of complementary Strain

1. Preparation of the invaded dye liquor

(1) AGL 1/35S-EAF 2 single colony was inoculated in YEP liquid medium containing 50mg/mL rifampicin and 50mg/mL kanamycin, and shake-cultured overnight at 28 ℃ and 200rpm to obtain culture broth 1.

(2) After the completion of the step (1), 500. mu.L of the culture broth 1 was inoculated into 5mL of YEP liquid medium, 5. mu.L of an aqueous solution of acetosyringone having a concentration of 100mg/mL was added thereto, and shaking culture was carried out at 28 ℃ and 200rpm to obtain OD_600nm0.8 for the culture broth 2.

(3) And (3) after the step (2) is finished, taking the culture bacterial liquid 2, centrifuging at 3800rpm for 15min, and collecting thalli.

(4) After the step (3) is completed, the thalli are taken and resuspended by a callus induction liquid culture medium containing 100mg/L acetosyringone to obtain OD_600nmValue 0.2 of the aggressive dye liquor.

2. Acquisition of complementary Strain

(1) The first compound leaf of mutant eaf2, which grew for 4 weeks, was rinsed with 75% (v/v) ethanol water for 10s, then soaked with 5% (m/v) sodium hypochlorite solution for 5min, and then washed with sterile water at least 5 times in a super clean bench, and finally the leaf was cut into 4-5 incisions.

(2) And (3) after the step (1) is finished, placing the small leaf blocks in a staining solution, and standing for 15 min.

(3) After the step (2) is completed, the small leaf blocks are transferred to a callus induction solid culture medium and cultured in the dark at 24 ℃ for 4 weeks (the culture medium is replaced once every 2 weeks) to obtain white embryogenic callus.

(4) After the step (3) is completed, the white embryogenic callus is transferred to a differentiation medium, and is alternately cultured in light and dark at 24 ℃ for 4 weeks (the medium is replaced every 2 weeks), so that green embryoid bodies are differentiated.

(5) And (4) after the step (4) is finished, transferring the green embryoid to a rooting culture medium, carrying out light-dark alternate culture at 24 ℃ (the culture medium is replaced every 2 weeks), transferring the rooting and leaf growing into vermiculite, and transferring into nutrient soil until the seedling is formed, namely the complementary plant.

10 complementation strains were obtained in total.

3. Real-time fluorescent quantitative detection of relative expression quantity of EAF2 gene in complementary strain

The medicago truncatula to be tested is 10 complementary strains, medicago truncatula R108 and a mutant eaf2 respectively.

(1) And respectively extracting total RNA of the leaves of the medicago truncatula to be detected, and then carrying out reverse transcription to obtain cDNA of the medicago truncatula to be detected.

(2) And respectively taking cDNA of medicago truncatula to be detected as a template, and detecting the relative expression quantity of the EAF2 gene (the Actin gene is an internal reference gene) by real-time quantitative PCR.

The reaction system is 20 mu L, and comprises 10 mu L TransStart Tip Green qPCR SuperMix, 0.5 mu L upstream primer aqueous solution (with the concentration of 10 mu M), 0.5 mu L downstream primer aqueous solution (with the concentration of 10 mu M), 0.5 mu L cDNA of the medicago truncatula to be detected and 8.5 mu L ddH₂And (C) O.

TransStart Tip Green qPCR Supermix (2X) is a product of all-purpose gold.

The reaction procedure is as follows: 5min at 94 ℃; 94 ℃ for 10s, 60 ℃ for 20s, 72 ℃ for 20s, 40 cycles.

The upstream primer for detecting the EAF2 gene is 5'-AGGAGCTTCCCAAAACTATCG-3', and the downstream primer is 5'-AGATTCTGGCACTTTCGGAG-3'.

The upstream primer for detecting the Actin gene is 5'-TCAATGTGCCTGCCATGTATGT-3', and the downstream primer is 5'-ACTCACACCGTCACCAGAATCC-3'.

The results of partial tests are shown in FIG. 3(WT is Medicago truncatula R108, EAF2 is mutant EAF2, 35S:: EAF2/EAF2-2, 35S:: EAF2/EAF2-6 and 35S:: EAF2/EAF2-8 is a complementary strain; indicating that there is a very significant difference, P < 0.01). The results show that compared with alfalfa R108, the expression level of EAF2 gene in the mutant EAF2 is obviously reduced, the expression level of EAF2 gene in each complementary strain is increased to different degrees, wherein the expression level of EAF2 gene in 3 complementary strains is obviously increased, and the three complementary strains are sequentially named as 35S: : EAF2/EAF2-2, 35S: : EAF2/EAF2-6 and 35S: : EAF2/EAF 2-8.

Fourth, phenotypic identification of the complementation plants

30 seeds to be tested (mutant EAF2 seeds, Medicago truncatula R108 seeds, 35S:: EAF2/EAF2-2 seeds and 35S:: EAF2/EAF2-6 seeds or 35S:: EAF2/EAF2-8 seeds) are taken, vernalized at 4 ℃ for 7 days, planted in nutrient soil, alternately cultured in light and dark (16h of light/8 h of dark), observed for phenotype and counted.

The growth states of 35S:: EAF2/EAF2-2, mutant EAF2 and Medicago truncatula R108 in light and dark alternate culture for 40 days are shown in A in FIG. 4 (WT is Medicago truncatula R108, EAF2 is mutant EAF2, 35S:: EAF2/EAF2 is 35S:: EAF2/EAF 2-2). The results show that when the mutant EAF2 is cultured alternately in dark and light for 40 days, the mutants have bloomed, and the medicago truncatula R108 and 35S, EAF2/EAF2-2, 35S, EAF2/EAF2-6 and 35S, EAF2/EAF2-8 have not bloomed.

Counting the flowering time and the number of flowering nodes of each medicago truncatula (namely the number of growing days and the number of nodes of the medicago truncatula when the first flower completely opens), and then taking an average value. Partial statistics are shown in B of fig. 4 (WT is medicago truncatula R108, eaf2 is mutant eaf2, which indicates a very significant difference, P < 0.01). The results show that compared with alfalfa R108, mutant eaf2 has significantly reduced number of flowering nodes and significantly advanced flowering time; 35S, EAF2/EAF2-2, 35S, EAF2/EAF2-6 and 35S, EAF2/EAF2-8 and medicago truncatula R108 have no obvious difference in the number of flowering nodes and the flowering time.

The positive and negative phenotypes of some Medicago truncatula leaves are shown in FIG. 4C (WT is Medicago truncatula R108, EAF2 is mutant EAF2, 35S:: EAF2/EAF2 is 35S:: EAF2/EAF2-2, Ad is positive, Ab is negative). The results showed that mutant eaf2 had more red spots on both the front and back of the compound leaves compared to alfalfa R108; 35S, EAF2/EAF2-2, 35S, EAF2/EAF2-6 and 35S, EAF2/EAF2-8 and medicago truncatula R108 have no significant difference in the number of red spots on the front and back of the compound leaves.

It can be seen that the number of flowering nodes and the number of red spots on the compound leaves of the complementary strains (35S:: EAF2/EAF2-2, 35S:: EAF2/EAF2-6, 35S:: EAF2/EAF2-8) were restored to be consistent with those of Medicago truncatula R108.

Fifthly, detecting the anthocyanin content in the leaf of the complementary plant

The anthocyanin content in leaves of the medicago truncatula R108, the mutants EAF2 and 35S, EAF2/EAF2-2 and 35S, EAF2/EAF2-6 and 35S, EAF2/EAF2-8 is detected according to the method of the second step in the example 1.

The results of partial assays are shown in FIG. 5(WT is Medicago truncatula R108, EAF2 is mutant EAF2, 35S:: EAF2/EAF2 is 35S:: EAF2/EAF2-2, which indicates a very significant difference, P < 0.01). The result shows that compared with the alfalfa R108 from the tribulus, the anthocyanin content in the leaves of the mutant eaf2 is obviously improved; 35S, EAF2/EAF2-2, 35S, EAF2/EAF2-6 and 35S, EAF2/EAF2-8 and medicago truncatula R108 leaves have no significant difference in anthocyanin content.

It can be seen that the anthocyanin content of the complementary strains (35S:: EAF2/EAF2-2, 35S:: EAF2/EAF2-6 and 35S:: EAF2/EAF2-8) was restored to be consistent with Medicago truncatula R108.

The results show that the EAF2 protein can regulate and control the synthesis of plant anthocyanin, the number of flowering nodes and the flowering time.

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

<120> a method for breeding a transgenic plant having reduced anthocyanin content and delayed flowering time

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 423

<212> DNA

<213> Medicago truncatula

<400> 1

atggctgaaa cggaggagct tcccaaaact atcgtacgtc gtgtggtgaa ggagaagctt 60

tccacttgct ccgaagacgg cgacattgcc gttcacaaag acgctcttct tgcattctcc 120

gaaagtgcca gaatcttcat ccactacgtt tctgctacgg ctaatgatat atgtagggag 180

tcaaagaggc agattatcaa tgctgaagat gtgttcaaag ctcttgaaga aaccgagttt 240

gctgagtttg ttggccctct aaaagattct cttgaagagt ttaggaagaa gaatgctggg 300

aagaaagcgg cggtgccaaa aggaaaggga gatgagaagg gaaagggaga tgagaagaaa 360

aggaaaagaa aggcagaagg tgagccatct gataaaggcg atggtgttga tgatgaccaa 420

tga 423

<210> 2

<211> 140

<212> PRT

<213> Medicago truncatula

<400> 2

Met Ala Glu Thr Glu Glu Leu Pro Lys Thr Ile Val Arg Arg Val Val

1 5 10 15

Lys Glu Lys Leu Ser Thr Cys Ser Glu Asp Gly Asp Ile Ala Val His

20 25 30

Lys Asp Ala Leu Leu Ala Phe Ser Glu Ser Ala Arg Ile Phe Ile His

35 40 45

Tyr Val Ser Ala Thr Ala Asn Asp Ile Cys Arg Glu Ser Lys Arg Gln

50 55 60

Ile Ile Asn Ala Glu Asp Val Phe Lys Ala Leu Glu Glu Thr Glu Phe

65 70 75 80

Ala Glu Phe Val Gly Pro Leu Lys Asp Ser Leu Glu Glu Phe Arg Lys

85 90 95

Lys Asn Ala Gly Lys Lys Ala Ala Val Pro Lys Gly Lys Gly Asp Glu

100 105 110

Lys Gly Lys Gly Asp Glu Lys Lys Arg Lys Arg Lys Ala Glu Gly Glu

115 120 125

Pro Ser Asp Lys Gly Asp Gly Val Asp Asp Asp Gln

130 135 140