CN112851779A - Method for cultivating transgenic plant with increased anthocyanin content - Google Patents

Abstract

The invention discloses a method for cultivating a transgenic plant with improved anthocyanin content. The method comprises the following steps: improving the expression quantity and/or activity of protein IbDDE in a receptor plant to obtain a transgenic plant; the transgenic plant has an increased anthocyanin content as compared to the recipient plant. The amino acid sequence of the protein IbDDE is shown as SEQ ID NO. 2. Experiments prove that the over-expression IbDDE gene can obviously promote the synthesis of anthocyanin in tobacco. Therefore, the protein IbDDE and the coding gene thereof can regulate and control the content of plant anthocyanin. The invention has important application value.

Description

Method for cultivating transgenic plant with increased anthocyanin content

Technical Field

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

Background

In recent years, with the continuous improvement of living standard of people, the nutritional value and the health care function of purple sweet potatoes (namely purple sweet potatoes) are more and more valued by people. The purple sweet potato not only has the nutrient components of common cultivated sweet potatoes, but also is rich in selenium, dietary fiber and anthocyanin. And the residue after the purple sweet potato is subjected to pigment extraction can be used for brewing alcohol, producing starch, making feed and the like, so that the purple sweet potato has higher comprehensive utilization value.

Anthocyanin is an important secondary metabolite in plants, is synthesized through enzyme reactions in cytoplasm and endoplasmic reticulum membrane, and is widely present in plant vacuoles in the form of glycoside, including blue, purple, carmine, red, orange and other colors. The accumulation of anthocyanin can prevent the photosynthetic system of the leaves from being damaged by ultraviolet rays, low temperature and transient strong light, improve the drought resistance and the invasion resistance of diseases and insects of plants, and attract the pollination of insects and the seed transmission. The anthocyanin contains a polyphenol structure, belongs to natural antioxidant active substances, can effectively remove free radicals, has strong inoxidizability, can prevent a human body biological enzyme system from being damaged, and has health-care functions of resisting mutation, resisting and preventing cancers, reducing blood pressure, softening blood vessels and the like. Anthocyanin is used as a natural pigment and is widely used as a food additive, so that anthocyanin has very important application value in various aspects of food, health care, crop improvement and the like. Therefore, obtaining anthocyanin synthesis genes to increase anthocyanin content in plants is the focus of research on genetic engineering for plant quality.

Disclosure of Invention

The purpose of the invention is to increase the anthocyanin content of plants.

The invention firstly protects the IbDDE protein derived from sweet potatoes, and the IbDDE protein can be 1) or 2) or 3) or 4) as follows:

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

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

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

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

Wherein, SEQ ID NO 2 consists of 393 amino acid residues.

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

TABLE 1 sequence of tags

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

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

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

The gene encoding the protein of 3) above can be obtained by deleting one or several amino acid residues of the codon in the DNA sequence shown in SEQ ID NO.1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

The invention also protects a nucleic acid molecule for coding the protein IbDDE.

The nucleic acid molecule for encoding the protein IbDDE can be a DNA molecule shown as (a1) or (a2) or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

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

(a3) a DNA molecule which has 75 percent or more homology with the nucleotide sequence limited by (a1) or (a2), is derived from the sweet potato and codes the protein IbDDE;

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

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

Wherein, SEQ ID NO.1 consists of 1182 nucleotides, and the nucleotides of SEQ ID NO.1 encode the amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence encoding the protein IbDDE of the 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 to have 75% or more identity to the nucleotide sequence of the protein IbDDE isolated in the present invention, as long as they encode the protein IbDDE, are derived from and identical to the nucleotide sequence of the present invention.

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

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

The recombinant vector containing any one of the nucleic acid molecules can be obtained by inserting the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing.

The recombinant vector can be specifically a recombinant plasmid pCB-IbDDE. The recombinant plasmid pCB-IbDDE can be obtained by replacing a small fragment between recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS with a DNA molecule shown in SEQ ID NO. 1.

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

The starting microorganism can be agrobacterium or escherichia coli. The agrobacterium may specifically be agrobacterium tumefaciens. The agrobacterium tumefaciens may specifically be agrobacterium tumefaciens EHA 105.

The recombinant microorganism comprising any of the nucleic acid molecules described above may specifically be EHA 105/pCB-IbDDE. The EHA105/pCB-IbDDE can be a recombinant agrobacterium obtained by introducing a recombinant plasmid pCB-IbDDE into the agrobacterium EHA 105.

The invention also provides application of any one of the protein IbDDE, any one of the nucleic acid molecules or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules in regulation and control of plant anthocyanin content.

The invention also provides application of any one of the protein IbDDE, any one of the nucleic acid molecules or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules in cultivation of transgenic plants with changed anthocyanin content.

In any of the above applications, the regulating plant anthocyanin content can be increasing plant anthocyanin content.

In any of the above applications, the cultivated transgenic plant with altered anthocyanin content may be a cultivated transgenic plant with increased anthocyanin content.

In the use of any of the above, the plant may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) wild type Arabidopsis thaliana Col-0.

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

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

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

In the above method, the nucleic acid molecule encoding the protein IbDDE may be a DNA molecule represented by (a1), or (a2), or (a3), or (a 4):

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

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

(a3) a DNA molecule which has 75 percent or more homology with the nucleotide sequence limited by (a1) or (a2), is derived from the sweet potato and codes the protein IbDDE;

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

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

Wherein, SEQ ID NO.1 consists of 1182 nucleotides, and the nucleotides of SEQ ID NO.1 encode the amino acid sequence shown in SEQ ID NO. 2.

The introduction of a nucleic acid molecule encoding the protein IbDDE into a recipient plant can be specifically effected by introducing a recombinant vector containing any of the above-mentioned nucleic acid molecules into a recipient plant.

The recombinant vector containing any one of the nucleic acid molecules can be specifically a recombinant plasmid pCB-IbDDE. The recombinant plasmid pCB-IbDDE can be obtained by replacing a small fragment between recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS with a DNA molecule shown in SEQ ID NO. 1.

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

In any of the methods described above, the plant may be any of c1), c2), c3), c4), c8), c9), and c10) as follows: c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c8) a plant of the Solanaceae family; c9) tobacco; c10) w38 tobacco.

IbDDE gene is introduced into W38 tobacco to obtain IbDDE gene-transferred tobacco. Experiments prove that compared with W38 tobacco, the anthocyanin content of the IbDDE gene-transferred tobacco is obviously increased; namely, the over-expression IbDDE gene can obviously promote the synthesis of anthocyanin in tobacco. Therefore, the protein IbDDE and the coding gene thereof can regulate and control the content of plant anthocyanin. The invention has important application value.

Drawings

FIG. 1 shows the molecular identification results of 10 IbDDE gene-mimicked tobaccos.

FIG. 2 shows real-time quantitative PCR detection of expression levels of IbDDE genes in 10 transgenic tobacco plants.

FIG. 3 shows the comparison of the color of 5 flowers of IbDDE transgenic tobacco.

FIG. 4 is the measurement of anthocyanin content in 5 IbDDE transgenic tobacco.

Detailed Description

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

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

The sweet potato line Jingshu 6-5 is described in the following documents: ZHao HY, ZHang SS, Wang FB, ZHao N, He SZ, Liu QC, ZHai H.comprehensive transfer analysis of pure-fly sweet potato products identifications into the molecular mechanism of anticancer in biosyntheses. frontiers of Agricultural Science and Engineering, 2018, doi.org/10.15302/J-FASE-2018219. The public can obtain from sweet potato genetic breeding research laboratory of Chinese agriculture university to repeat the experiment, and can not be used as other purposes.

The W38 tobacco is described in the following documents: jiang T, ZHai H, Wang FB, Zhou HN, Si ZZ, He SZ, Liu QC cloning and engineering of a Salt Tolerance-Associated Gene Encoding Trehase-6-Phosphate Synthase in Sweetpototo, Journal of Integrated age 2014, 13 (8): 1651-1661. The public can obtain from sweet potato genetic breeding research laboratory of Chinese agriculture university to repeat the experiment, and can not be used as other purposes.

The vector pCAMBIA3301 is a product of Youbao biology, and the product number is VT 1386. The vector pBI121 is a product of Youbao biology, and the product number is VT 1388. The plant total RNA extraction kit is a Transzol Up plant total RNA extraction kit (full-scale gold, catalog number ET 111). The pEASY-Blunt simple vector is a product of Beijing all-purpose gold biotechnology, Inc., and the product catalog number is CB 111-01. QuantScript RT Kit Quant cDNA Kit is a product of Tiangen Biochemical technology (Beijing) Co., Ltd., and has a product catalog number KR 103.

Example 1 obtaining of IbDDE Gene

The IbDDE gene is obtained by the following steps:

1. extracting the total RNA of the young leaf of the sweet potato strain Jingshu 6-5 by using a plant total RNA extraction Kit, and carrying out reverse transcription on the total RNA by using a QuantScript RT Kit Quant cDNA Kit to obtain first-strand cDNA.

2. Taking the cDNA obtained in the step 1 as a template, and adopting a primer O-F: 5'-ATGGCTCGTTTGGGTATAAGTAG-3' and primer O-R: 5'-CTAACTATTCCATTGGTTGAACATT-3', PCR amplification is carried out to obtain 1182bp PCR amplification product and sequencing is carried out.

The result shows that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 1. The gene shown in SEQ ID NO.1 is named as IbDDE gene, the coded protein is named as IbDDE protein or protein IbDDE, and the amino acid sequence is shown in SEQ ID NO. 2.

Example 2 application of IbDDE protein in increasing plant anthocyanin content

Construction of recombinant plasmid

1. The vector pCAMBIA3301 was double-digested with restriction enzymes HindIII and EcoRI, and the vector backbone 1 of about 11256bp was recovered.

2. The vector pBI121 was double-digested with the restriction enzymes HindIII and EcoRI, and the fragment 1 comprising about 3032bp was recovered.

3. And connecting the fragment 1 with a vector framework 1 to obtain the recombinant plasmid pCBGUS.

4. The recombinant plasmid pCBGUS was double digested with restriction enzymes BglII and PmlI, recovering vector backbone 2 of about 12388 bp.

5. Artificially synthesizing a double-stranded DNA molecule shown in SEQ ID NO. 1. Taking the double-stranded DNA molecule as a template, and taking a primer IbDDE-F1-BglII: 5' -GAAGATCTATGGCTCGTTTGGGTATAAGTAG-3' (recognition sequence for restriction enzyme BglII is underlined) and primer IbDDE-R1-PmlI: 5' -GCCACGTGCTACTATTCATTGGTTGACATTTGG-3' (the recognition sequence of the restriction enzyme PmlI is underlined) and PCR amplification is carried out to obtain the double-stranded DNA molecule containing the recognition sequence of the restriction enzyme.

6. And (3) connecting the double-stranded DNA molecule containing the recognition sequence of the restriction enzyme obtained in the step (5) to a pEASY-Blunt simple vector to obtain an intermediate vector.

7. The intermediate vector was double digested with restriction enzymes BglII and PmlI, recovering fragment 2 of about 1200 bp.

8. And connecting the fragment 2 with a vector framework 2 to obtain a recombinant plasmid pCB-IbDDE.

The recombinant plasmid pCB-IbDDE was sequenced. According to the sequencing result, the structure of the recombinant plasmid pCB-IbDDE is described as follows: the small fragment between the recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS is replaced by a DNA molecule shown in SEQ ID NO.1 to obtain the recombinant plasmid. The recombinant plasmid pCB-IbDDE expresses IbDDE protein shown in SEQ ID NO. 2.

Second, obtaining of IbDDE transgenic tobacco

1. The recombinant plasmid pCB-IbDDE is transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which is named as EHA 105/pCB-IbDDE.

2. The IbDDE gene-simulated tobacco was obtained by transferring EHA105/pCB-IbDDE to W38 tobacco by Agrobacterium-mediated method (see: Wang LJ, He SZ, ZHai H, Liu DG, Wang YN, Liu QC: Molecular cloning and functional characterization of a salt-associated gene IbNFU1 from sweet potato. journal of integrated Agriculture, 2013, 12 (1): 27-35). The method comprises the following specific steps:

(1) w38 tobacco leaves growing in sterile medium for about 4 weeks were placed in a sterile petri dish, the main veins and the leaf margins were removed, and then cut into tobacco leaf disks of 1cm by 1 cm.

(2) Placing the tobacco leaf disc with the front side facing upwards in a pre-culture medium (MS solid medium containing 1.0mg/L6-BA and 0.1mg/L NAA), and performing dark culture at 28 deg.C for 2-3d to obtain pre-cultured tobacco leaf disc.

(3) Soaking the pre-cultured tobacco leaf discs in OD_600nm0.4-0.6 of agrobacterium infection solution for 5-10min, then sucking off the redundant bacterial liquid on the tobacco leaf disc, placing the tobacco leaf disc on a co-culture medium (MS solid medium containing 1.0mg/L6-BA and 0.1mg/L NAA) on the front side, and culturing for 2-3d at 28 ℃ in the dark.

(4) After the step (3) is completed, the tobacco leaf disc is sequentially cleaned by an MS liquid culture medium containing 600mg/L CS, an MS liquid culture medium containing 300mg/L CS and an MS liquid culture medium containing 150mg/L CS, then, redundant liquid on the tobacco leaf disc is sucked by clean filter paper sterilized at high temperature and high pressure, and the tobacco leaf disc is placed on a regeneration culture medium (an MS solid culture medium containing 15mg/L hygromycin, 400mg/L CS, 1.0mg/L6-BA and 0.1mg/L NAA) and is subjected to illumination culture (the illumination intensity is 3000lux) for 30d at the temperature of 28 ℃ to obtain a regeneration bud.

(5) After the step (4) is completed, cutting 1cm of regeneration bud, then placing the regeneration bud on a rooting culture medium (1/2 MS solid culture medium containing 1.0mg/L6-BA, 0.1mg/L NAA, 400mg/L cephalexin and 15mg/L hygromycin) until a complete plant is grown, and obtaining the IbDDE gene transfer-simulated tobacco.

The obtained 10 IbDDE gene-like tobacco were sequentially named L1-L10.

3. Molecular identification

The tobacco to be detected is W38 tobacco, L1, L2, L3, L4, L5, L6, L7, L8, L9 or L10.

(1) Extracting genome DNA of tobacco leaves to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of a primer O-F and a primer O-R to obtain a PCR amplification product; taking water as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer O-F and a primer O-R to obtain a PCR amplification product as a negative control; and (3) carrying out PCR amplification by using the recombinant plasmid pCB-IbDDE as a template and a primer pair consisting of a primer O-F and a primer O-R to obtain a PCR amplification product as a positive control.

(2) After completion of step (1), each PCR amplification product was subjected to 1% (w/v) agarose gel electrophoresis, followed by judgment as follows: if the PCR amplification product obtained by taking the genome DNA of a certain IbDDE gene-simulated tobacco as a template contains 1182bp DNA fragments (the same as the fragments of a positive control), the IbDDE gene-simulated tobacco is a positive plant; otherwise, the plant is not a positive plant.

The detection results are shown in FIG. 1(M is DNA Marker, W is negative control, P is positive control, WT is wild type Arabidopsis thaliana). The results show that the PCR amplification products of L1-L10 and the positive control both contain 1182bp DNA fragments, and the PCR amplification products of the negative control and W38 tobacco do not contain about 1182bp DNA fragments. Therefore, L1-L10 are all positive plants, namely IbDDE transgenic tobacco.

4. Real-time quantitative PCR detection of IbDDE gene expression level in L1-L10

The tobacco to be detected is W38 tobacco, L1, L2, L3, L4, L5, L6, L7, L8, L9 or L10.

(1) Extracting total RNA of tobacco seedlings to be detected, and reversing the total RNA by using reverse transcriptase to obtain cDNA; and detecting the relative expression quantity of the IbDDE gene in the cDNA (taking the tobacco Actin gene as an internal reference gene) by real-time quantitative PCR (polymerase chain reaction).

The primer for detecting the IbDDE gene is IbDDE-F: 5'-CTACTATTCATTGGTTGACATTTGGTT-3' and IbDDE-R: 5'-GCTCCACAATTTTATAAGACAAGTGAT-3' are provided.

The primer for detecting the Actin gene is NtActin-F: 5'-GAGGAATGCAGATCTTCGTG-3' and NtActin-R: 5'-TCCTTGTCCTGGATCTTAGC-3' are provided.

The results of the partial detection are shown in FIG. 2. The result shows that the IbDDE gene is a tobacco foreign gene and is hardly expressed in W38 tobacco, but the IbDDE gene is expressed in different degrees in L1-L10.

5 strains (namely L1, L2, L3, L4 and L5) with the expression level of the IbDDE gene which is obviously higher than that of W38 tobacco are randomly selected for subsequent experiments.

Determination of anthocyanin content in IbDDE transgenic tobacco

The tobacco to be detected is W38 tobacco, L1, L2, L3, L4 or L5.

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

1. transplanting the tobacco with good growth condition and consistent size to a greenhouse, and observing the color of the flowers after blooming.

The results are shown in FIG. 3. The results showed that the IbDDE transgenic tobacco (e.g., L1, L2, L3, L4 and L5) was significantly redder in color than W38 tobacco.

2. After step 1, the flower of tobacco to be tested is taken and the Anthocyanin content is detected by referring to the method of Li and the like (Li S, Wang W, Gao J, et al. MYB75 phosphorylation by MPK4 is required for light-induced Anthocynin amplification. plant Cell,2016,28(11): 2866-2883).

The results are shown in FIG. 4. The result shows that compared with W38 tobacco, the content of anthocyanin in IbDDE transgenic tobacco (such as L1, L2, L3, L4 and L5) is obviously increased.

The result shows that the over-expression IbDDE gene can obviously promote the synthesis of anthocyanin in tobacco.

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

<110> university of agriculture in China

<120> a method for breeding transgenic plants with increased anthocyanin content

<160> 2

<170> PatentIn version 3.5

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<213> Ipomoea batatas

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atggctcgtt tgggtataag tagaactgct gaaagaagaa aaaggattgt ggctgctttg 60

actgtttttt tagagatgta tactgttgtt agtaccttag tacttttaat ggcatcagca 120

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gttaaaaatc gagtcattca atacaatttt agaaggtcag gtgagagtat cagtcggacg 420

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Met Ala Arg Leu Gly Ile Ser Arg Thr Ala Glu Arg Arg Lys Arg Ile

1 5 10 15

Val Ala Ala Leu Thr Val Phe Leu Glu Met Tyr Thr Val Val Ser Thr

20 25 30

Leu Val Leu Leu Met Ala Ser Ala Ala Ser Leu Asn Ser Tyr Arg Pro

35 40 45

Ser Ile Arg Asn Tyr Cys Leu Asp Ser Tyr Tyr Lys Arg Glu Tyr Ile

50 55 60

Gln Arg Ile Leu Asn Asn Asn Glu Ser Cys Ile Ser Leu Leu Arg Met

65 70 75 80

Asn Leu Gln Ala Phe Ser Lys Leu Cys Glu Met Leu Glu Ser Leu Gly

85 90 95

Gly Leu Lys Pro Thr Arg Asn Met Ala Ile Glu Glu Gln Val Ala Ile

100 105 110

Phe Leu His Ile Leu Ala His His Val Lys Asn Arg Val Ile Gln Tyr

115 120 125

Asn Phe Arg Arg Ser Gly Glu Ser Ile Ser Arg Thr Phe His Lys Val

130 135 140

Leu Asn Ala Ile Met His Leu Gln Gly His Leu Phe Lys Thr Pro Glu

145 150 155 160

Pro Val Pro Ala Asn Cys Thr Asp Ser Arg Trp Lys Trp Phe Lys Asn

165 170 175

Cys Leu Gly Ala Leu Asp Gly Thr Phe Ile Lys Val Asn Val Pro Ser

180 185 190

Ser Asp Lys Pro Arg Tyr Arg Thr Arg Lys Gly Asp Ile Ala Thr Asn

195 200 205

Val Leu Gly Val Cys Thr Pro Asp Met Gln Phe Val Tyr Val Leu Ser

210 215 220

Gly Trp Glu Gly Ser Val Ala Asp Ser Arg Val Leu Arg Asp Ala Ile

225 230 235 240

Ser Arg Thr His Ala Leu Glu Val Pro His Gly Cys Tyr Tyr Leu Val

245 250 255

Asp Ala Gly Tyr Thr Asn Cys Glu Gly Phe Leu Ala Pro Phe Arg Gly

260 265 270

Gln Arg Tyr His Leu Asn Glu Trp Arg Gln Gly Tyr Gln Pro Thr Ser

275 280 285

Pro Gln Glu Phe Phe Asn Met Lys His Ala Ala Ala Arg Asn Val Ile

290 295 300

Glu Arg Cys Phe Gly Leu Leu Lys Ile Arg Trp Gly Ile Leu Arg Ser

305 310 315 320

Pro Ser Tyr Tyr Pro Ile Lys Thr His Asn Arg Ile Ile Ile Ala Cys

325 330 335

Cys Leu Leu His Asn Phe Ile Arg Gln Val Met Gln Val Asp Pro Met

340 345 350

Glu Ser Glu Leu Asp Ala Phe Asp His Val Glu Gly Glu Thr Asn Ser

355 360 365

Ile Asn Thr Val Asp Pro Ser Asp Ala Trp Thr Asn Trp Arg Met Glu

370 375 380

Leu Ala Asn Gln Met Ser Thr Asn Glu

385 390

Claims (10)

1. Protein IbDDE, 1) or 2) or 3) or 4) as follows:

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

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

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

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

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

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

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

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

(a3) a DNA molecule having 75% or more homology with the nucleotide sequence defined in (a1) or (a2), derived from sweetpotato, and encoding the protein IbDDE of claim 1;

(a4) a DNA molecule which is derived from sweetpotato and encodes the protein IbDDE of claim 1, and which hybridizes with the nucleotide sequence defined in (a1) or (a2) under stringent conditions.

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

5, b1) or b 2):

b1) use of the protein IbDDE of claim 1, or the nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3, for modulating anthocyanin content in plants;

b2) use of the protein IbDDE according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the nucleic acid molecule according to claim 2 or 3, for the production of transgenic plants with an altered anthocyanin content.

6. The use of claim 5, wherein: the plant is any one of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) wild type Arabidopsis thaliana Col-0.

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

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

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

10. The method of any of claims 6-8, wherein: the plant is any one of the following c1), c2), c3), c4), c8), c9) and c 10): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c8) a plant of the Solanaceae family; c9) tobacco; c10) w38 tobacco.

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