CN113621039B

CN113621039B - Anthocyanin synthesis related protein IbMYB113 and coding gene and application thereof

Info

Publication number: CN113621039B
Application number: CN202110955183.2A
Authority: CN
Inventors: 郭华春; 李茂兴; 李有涵; 王琼
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2023-03-17
Anticipated expiration: 2041-08-19
Also published as: CN113621039A

Abstract

The invention relates to an anthocyanin synthesis related protein IbMYB113, and a coding gene and application thereof. The amino acid sequence of the IbMYB113 protein is shown as SEQ ID NO. 2; the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The invention clones IbMYB113 protein and coding gene thereof from sweet potato red potato peelIbMYB113The gene is connected to a plant expression vector, and overexpression is obtained through agrobacterium-mediated genetic transformationIbMYB113The transgenic tobacco plant shows that the whole plant of the transgenic tobacco plant is accumulated, and the content of delphinidin and cyanidin is obviously increased by detecting through a high performance liquid chromatography. The IbMYB113 protein and the coding gene thereof provided by the invention have important theoretical significance and application value for promoting synthesis and accumulation of plant anthocyanin.

Description

Anthocyanin synthesis related protein IbMYB113 and coding gene and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to anthocyanin synthesis related protein IbMYB113 and coding gene cloning and application thereof.

Background

The sweet potato is one of important grain crops, economic crops and energy crops in the world, and the color of the potato peel mainly comprises white, light yellow, light red, purple, dark purple and the like; the color of the potato pulp mainly comprises white, yellow, orange, red, purple, dark purple, mixed colors and the like. The red and purple sweet potato varieties mainly contain anthocyanin, and are very popular with consumers due to beautiful appearance of the sweet potato pieces and excellent health care function.

Anthocyanin is a water-soluble natural pigment, widely present in higher plants, and the most important metabolite in flavonoid metabolic pathways. Over 635 anthocyanins have been found in nature, the main 6 common in plants, being pelargonidin, cyanidin, delphinidin, peonidin, petunianin and malvidin, respectively. For plants, anthocyanin participates in many important biological processes, and is accumulated in overground parts of plants such as floral organs, fruits, leaves and stems, so that the plant has bright color to attract entomophilous pollinators and fruit propagators, and the plant can be protected from ultraviolet injury, diseases and insect pests and the like; anthocyanin is accumulated in underground parts of plants such as tuberous root tuber crops, namely potato tubers and tuberous roots of sweet potatoes, is less influenced by environmental factors, has better light stability and heat stability, and can improve the resistance of the plants to biological stress and abiotic stress. For human, anthocyanins have a variety of health-care functions of resisting oxidation, resisting mutation, resisting inflammation, removing free radicals, reducing the risk of cancer, arthritis, cardiovascular diseases, diabetes and neurological diseases, protecting the functions of organisms and the like.

The biosynthesis of anthocyanin in plants mainly involves structural genes such as phenylalanine lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumaroyl CoA ligase (4 CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid-3 '-hydroxylase (F3' H), flavonoid-3 ',5' -hydroxylase (F3 '5'H), flavanonol reductase (DFR), anthocyanin synthase (ANS), glycosyltransferase (GTs), and the like, and three transcription factors such as MYB, bHLH, WD40, and the like. At present, the research on anthocyanin of the sweet potato mainly focuses on synthesis and accumulation of anthocyanin in the purple sweet potato, structural genes such as IbCHS, ibCHI, ibF3' H, ibDFR, ibANS, ibUFGT, ibGSTF4 and the like have been cloned, and an important transcription factor IbMYB1-2 is found. IbMYB1-2 can regulate and control the biosynthesis and accumulation of anthocyanin in purple sweet potato flesh, but is limited by polyploidy, genomic hybridization and complex genetic background of sweet potatoes, and a new anthocyanin synthesis regulation gene in the sweet potatoes is not found yet, for example, the regulation gene for the biosynthesis and accumulation of anthocyanin in sweet potato red potato peels is not clear yet and needs further research.

Disclosure of Invention

The invention aims to provide a anthocyanin synthesis related protein IbMYB113 and coding gene cloning and application thereof.

The anthocyanin synthesis related protein cloned by the invention is named as IbMYB113, is derived from sweet potatoes (Ipomoea batatas), has an amino acid sequence shown as SEQ ID NO.2, and consists of 236 amino acid residues.

The coding gene of the IbMYB113 protein cloned by the invention is a nucleic acid molecule which codes an amino acid sequence shown in SEQ ID NO. 2.

Specifically, the coding gene of the IbMYB113 protein cloned by the invention can be a nucleotide sequence shown as SEQ ID NO. 1.

The invention provides an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the IbMYB113 protein coding gene.

The recombinant vector containing the IbMYB113 protein coding gene can be a recombinant plasmid obtained by inserting a nucleotide sequence shown in SEQ ID NO.1 into a multiple cloning site of an expression vector.

Specifically, the recombinant vector may be a recombinant plasmid pHELLSGATE12-IbMYB113 obtained by replacing a small fragment between restriction enzyme XhoI and XbaI sites of the plasmid pHELLSGATE12 with a nucleotide sequence shown in SEQ ID NO. 1.

The recombinant microorganism containing the IbMYB113 protein coding gene can be a recombinant engineering bacterium obtained by introducing the recombinant vector containing the IbMYB113 protein coding gene into agrobacterium or escherichia coli.

Specifically, the recombinant microorganism can be recombinant Agrobacterium GV3101-pHELLSGATE12-IbMYB113 obtained by introducing a recombinant plasmid pHELLSGATE12-IbMYB113 into Agrobacterium tumefaciens GV 3101.

The invention also provides the protein IbMYB113, or a coding gene of the IbMYB113 protein, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the coding gene of the IbMYB113 protein, and application of the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic plant cell line in regulation and control of plant anthocyanin synthesis, types and content. The plant is tobacco; the tobacco is Honghuadajinyuan.

The invention also provides the protein IbMYB113, or an encoding gene of the IbMYB113 protein, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the encoding gene of the IbMYB113 protein, and application of the protein IbMYB113, the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic plant cell line in culturing transgenic plants with increased anthocyanin synthesis and accumulation and promoting synthesis and accumulation of plant delphinidin and cyanidin. The plant is tobacco; the tobacco is Honghuadajinyuan.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: introducing coding genes or nucleic acid molecules of the IbMYB113 protein into 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 IbMYB113 protein is shown in SEQ ID NO. 2. The coding gene of the IbMYB113 protein can be a nucleotide sequence shown as SEQ ID NO. 1.

Specifically, the invention provides a method for cultivating transgenic plants, which comprises the following steps: replacing a small fragment between XhoI restriction enzyme cutting sites and XbaI restriction enzyme cutting sites in pHELLSGATE12 plasmid with an encoding gene or a nucleic acid molecule (can be a nucleotide sequence shown as SEQ ID NO: 1) of IbMYB113 protein to obtain a recombinant vector pHELLSGATE12-IbMYB113, and introducing the recombinant vector into a plant to obtain a transgenic plant over-expressed by IbMYB 113; compared with receptor plants, the transgenic plant has improved anthocyanin content, especially the synthesis and accumulation of delphinidin and cyanidin are obviously enhanced, and the content is obviously increased.

The IbMYB113 protein and the coding gene thereof provided by the invention are introduced into common cultivated tobacco 'Honghuadajinyuan' through agrobacterium-mediated genetic transformation to obtain a transgenic tobacco plant. Experiments prove that compared with a control, a transgenic plant over expressing the IbMYB113 protein coding gene shows full purple, and the content of delphinidin and cyanidin is obviously increased by High Performance Liquid Chromatography (HPLC) detection. The 'Honghuadajinyuan' is one of main cultivation varieties in the market, the tobacco leaves have better yield and quality, the transgenic tobacco with high anthocyanin content is generated through genetic improvement, and the transgenic tobacco can be directly used for producing delphinium pigment and cornflower pigment products and has wide application potential. Therefore, the IbMYB113 protein and the coding gene thereof provided by the invention have important theoretical significance and application value for promoting the synthesis and accumulation of plant anthocyanin.

Drawings

FIG. 1 is a seedling phenotype diagram of WT and IbMYB113-OE transgenic tobacco plants, WT being a tobacco plant transformed with the empty vector pHELLSGATE12, and IbMYB113-OE being a tobacco plant transformed with the expression vector pHELLSGATE12-IbMYB113.

FIG. 2 is an HPLC assay of anthocyanin from WT and IbMYB113-OE transgenic tobacco plants.

FIG. 3 is a graph of anthocyanin levels in WT and IbMYB113-OE transgenic tobacco plants, with Nd indicating no detection.

Detailed Description

The invention is described in further detail below with reference to the attached drawings and specific embodiments, which are only for better illustrating the invention but not for limiting the invention.

The experimental procedures used in the following examples are conventional unless otherwise specified. The experimental materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1. Sweet potato anthocyanin synthesis related protein IbMYB113 and cloning of coding gene thereof

The cloning steps of the IbMYB113 gene are as follows:

1. the method adopts Trizol reagent produced by Beijing all-purpose gold biotechnology limited company to extract the total RNA of the sweet potato, and comprises the following specific operation steps:

(1) taking a proper amount of sweet potato red potato peel, quickly grinding into powder in a mortar by using liquid nitrogen;

(2) transferring about 0.1g of the ground sample into a 1.5mL centrifuge tube, adding 1.0mL Trizol reagent, carrying out vortex oscillation for 15sec, and standing at room temperature for 5min;

(3) adding 0.2mL of chloroform, carrying out vortex oscillation for 15sec, and standing at room temperature for 3min;

(4) centrifuging at 4 deg.C and 12000rpm for 15min, collecting supernatant about 550 μ L to another new 1.5mL centrifuge tube, adding 0.5mL isopropanol, mixing, and standing at room temperature for 10min;

(5) centrifuging at 4 deg.C and 12000rpm for 10min, removing supernatant, and collecting precipitate;

(6) adding 1.0mL of 75% alcohol, eluting and precipitating for 1 time, centrifuging at 4 deg.C and 7500rpm for 5min, removing supernatant, and leaving white precipitate;

(7) air-drying the clean bench for 5-10min to completely remove residual alcohol;

(8) adding 40 μ L DEPC water, and performing metal bath at 56 deg.C for 2min to dissolve precipitate;

(9) 4 μ L of the DNA was subjected to 1.5% agarose gel electrophoresis to detect the RNA quality, and the remainder was stored in a refrigerator at-80 ℃.

Synthesis of first Strand of cDNA

Produced by Nanjing NuoZan Biotech GmbH

III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) Kit (R312-01) for cDNA first Strand Synthesis, the specific operation steps are as follows:

(1) adding 3 μ L of total RNA of sweet potato extracted in the last step into RNase-free centrifuge tube, and adding RNase-free ddH ₂ Supplementing O to 8 μ L, metal bath at 65 deg.C for 5min, rapidly cooling on ice, and standing on ice for 2min;

(2) adding 2 μ L of 5 XgDNA wiper Mix, gently blowing and stirring with a pipette, and carrying out metal bath at 42 deg.C for 2min;

(3) the following reagents were added separately: mu.L of 10 XTT Mix, 2. Mu.L of HiScript III Enzyme Mix, 1. Mu.L of Oligo (dT) ₂₀ VN、5μL RNase-free ddH ₂ O, lightly blowing and uniformly mixing by using a pipette gun, and carrying out 45min at 50 ℃ and 5sec at 85 ℃ on a PCR instrument;

(4) mu.L of the suspension was diluted 10-fold in a 0.5mL centrifuge tube for subsequent experiments and stored in a-20 ℃ freezer.

3. According to the transcriptome sequencing result of the sweet potato red potato skin, a g17108 transcript is obtained through screening and is used as a candidate gene related to anthocyanin synthesis, a KEGG functional annotation is a MYB transcription factor, the full-length cDNA sequence of the g17108 transcript is found through homologous sequence comparison, and a forward primer and a reverse primer are designed for PCR amplification. The primer is synthesized by Shanghai biological engineering Co., ltd, and the sequence of the primer is as follows:

forward Primer (Forward Primer): 5'-GTATAGCATACCCTGTCATGG-3'

Reverse Primer (Reverse Primer): 5'-ATAAAGATTTCGTACACGACTT-3'

4. And 3, carrying out PCR amplification reaction by using the cDNA obtained in the step 2 as a template and the primer pair designed in the step 3. The DNA polymerase is produced by Beijing Quanji Biotech limited

DNA Polymerase。

The PCR reaction system is as follows:

the PCR reaction procedure was as follows:

the PCR amplification product was detected by 1.2% agarose gel electrophoresis to obtain a fragment of about 720bp in length.

5. Purifying the PCR product using a PCR product purification kit (centrifugal column type) produced by Shanghai Czeri bioengineering, inc.; the purified PCR product uses a T vector PCR product quick connection kit produced by Shanghai biological engineering Limited company to clone the T vector, and the lengths of the inserted fragments in the T vector are confirmed to be consistent by a colony PCR method; the positive colonies were picked up in LB liquid culture containing ampicillin, cultured overnight at 37 ℃ and 200rpm, and used as those produced by Beijing Quanjin Biotechnology Ltd

Plasmid MiniPrep Kit (EM 101) is used for extracting plasmids and sending the plasmids to Shanghai Biotechnology engineering Co., ltd for sequencing. The above experimental procedures were performed according to the kit instructions.

The sequencing result shows that the PCR amplification product has the nucleotide sequence shown in SEQ ID NO.1, the nucleotide sequence shown in SEQ ID NO.1 is named as IbMYB113 gene, the length is 711bp, the coded protein is named as IbMYB113 protein or protein IbMYB113, and the amino acid sequence is shown in SEQ ID NO.2 and consists of 236 amino acid residues.

Example 2 application of IbMYB113 protein of sweet potato in regulation and control of synthesis and accumulation of plant anthocyanin

1. Obtaining of IbMYB113 transgenic whole purple tobacco

1. Plant expression vector construction

Produced by Nanjing NuoZan Biotech GmbH

II One Step Cloning Kit (C112) for constructing a plant expression vector, the specific steps are as follows:

(1) preparation of a linearized vector: the vector pHELLSGATE12 was double digested with restriction enzymes XhoI and XbaI, and the linearized vector fragment of about 13798bp was recovered.

(2) Obtaining an insert: taking the plasmid DNA of the IbMYB113 obtained in the embodiment 1 as a template, introducing homologous sequences at two ends of a linearized vector, namely a forward primer OE-F:5'-TTGGAGAGGACACGCTCGAGATGGCTAATTCATCATGTGC-3' (the upstream vector end homology sequence is underlined), reverse primer OE-R:5'-TCATTAAAGCAGGACTCTAGTTAGCTTAAAAGTTGGGAAA-3' (the homologous sequence at the tail end of the downstream vector is underlined), and PCR amplification is carried out to obtain an IbMYB113 gene segment containing the homologous sequence at the tail end of the linearized vector.

(3) And (3) recombinant transformation: carrying out recombination reaction on the linearized vector obtained in the step (1) and the IbMYB113 gene fragment containing the terminal homologous sequence of the linearized vector obtained in the step (2) to obtain a recombinant product; the recombinant product was transformed into DH 5. Alpha. E.coli competent cells and cultured overnight to form hundreds of white single colonies.

(4) Obtaining a plant expression vector: selecting several white colonies generated in step (3) for colony PCR identification, inoculating positive colonies into LB liquid culture medium containing spectinomycin, culturing at 37 deg.C and 200rpm overnight, and culturing with Beijing Quanjin Biotechnology Ltd

Plasmid is extracted by a Plasmid MiniPrep Kit (EM 101) to obtain a recombinant Plasmid pHELLSGATE12-IbMYB113 which is a plant expression vector, the plant expression vector is stored in a refrigerator at the temperature of-20 ℃, and the recombinant Plasmid is sent to Shanghai biological engineering GmbH for sequencing. The sequencing result shows that the recombinant plasmid pHELLSGATE12-IbMYB113 is a recombinant plasmid obtained by replacing a small fragment between restriction enzyme XhoI and XbaI sites of pHELLSGATE12 plasmid by the nucleotide shown by SEQ ID NO.1 in a sequence table. The plant expression vector pHELLSGATE12-IbMYB113 expresses IbMYB113 protein shown by SEQ ID NO.2 in the sequence table.

2. Plant expression vector transformation agrobacterium tumefaciens

(1) Removing 100 μ L of GV3101 competent cells prepared in advance from-80 deg.C, removing the plant expression vector pHELLSGATE12-IbMYB113 obtained in step 1 above at-20 deg.C, and thawing on ice;

(2) adding 5 μ L plant expression vector pHELLSGATE12-IbMYB113 into 100 μ L GV3101 competent cell, and gently blowing and stirring;

(3) sequentially carrying out ice bath for 20min, liquid nitrogen quick freezing for 1min, water bath at 37 ℃ for 5min, and standing on ice for 3min;

(4) adding 1mL of LB liquid culture medium without antibiotics, and culturing at 28 ℃ and 200rpm for 4h;

(5) centrifuging at 5000rpm for 1min, collecting thallus, discarding supernatant, and keeping about 100 μ L of supernatant to resuspend thallus;

(6) uniformly coating the bacterial liquid on an LB solid culture medium plate containing 50 mu g/mL rifampicin (Rif) and 50 mu g/mL spectinomycin (Spe), and performing inverted culture at 28 ℃ for 2-3 days;

(7) selecting a white single colony for colony PCR identification, inoculating a positive colony into an LB liquid culture medium containing 50 mu g/mL rifampicin (Rif) and 50 mu g/mL spectinomycin (Spe), culturing at 28 ℃ and 200rpm until the OD600 value of a bacterial liquid reaches 0.8-1.0 to obtain the recombinant agrobacterium GV3101-pHELLSGATE12-IbMYB113, and storing in a refrigerator at 4 ℃ for later use.

3. Agrobacterium-mediated genetic transformation of tobacco

(1) Preparing seeds for cultivating tobacco 'Honghuadajinyuan', disinfecting the surfaces of 75% alcohol and 2% sodium hypochlorite, sowing the seeds in an MS solid culture medium, and culturing for 4-5 weeks at 25 ℃ in 16 h/8 h in darkness;

(2) selecting strong and tender green tobacco leaves, placing in a culture dish sterilized in advance, cutting off leaf veins and leaf edges by using a scalpel, uniformly marking wounds, and cutting enough tobacco leaf discs of 2cm multiplied by 2 cm;

(3) preparing an MS liquid culture medium containing 100 mu M acetosyringone (As), properly diluting the recombinant agrobacterium GV3101-pHELLSGATE12-IbMYB113 obtained in the step 2 to an OD600 value of 0.5-0.6, and transferring the tobacco leaf disc into the diluted recombinant agrobacterium for infection and soaking for 8min;

(4) sucking the redundant bacterial liquid on the tobacco leaf disc by using sterile filter paper, neatly placing the front side of the tobacco leaf disc on a co-culture medium (MS +1.0 mu g/mL 6-BA +0.1 mu g/mL NAA +100 mu MAs), and co-culturing for 48h under a dark condition;

(5) transferring the co-cultured tobacco leaf discs to a screening regeneration medium (MS +1.0 mu g/mL of 6-BA +0.1 mu g/mL of NAA +400mg/L of cefotaxime (Cef) +100mg/L of kanamycin (Kan)), culturing at 25 ℃, irradiating for 16 h/dark for 8h, inducing to generate callus, and subculturing for 1 time every 30d until the callus is differentiated into buds;

(6) and cutting the regeneration bud, subculturing on a rooting culture medium (MS +200mg/L Cef +50mg/L Kan), and culturing until a complete plant is grown to obtain a transgenic tobacco plant.

4. Phenotype identification and anthocyanin content determination of over-expression IbMYB113 transgenic tobacco

According to step 3, tobacco transformed with pHELLSGATE12 empty vector is used as control, i.e. wild type tobacco WT, and tobacco transformed with pHELLSGATE12-IbMYB113 expression vector is used for comparative analysis, i.e. IbMYB113-OE; observing the phenotype, the tobacco leaf discs begin to induce callus at about 30d on the screening regeneration medium, and after 3 subcultures, the induced callus is differentiated into shoots, and the shoots are transferred to the rooting medium. As shown in figure 1, the whole WT tobacco is green, the whole IbMYB113-OE tobacco is purple, and compared with WT tobacco, the IbMYB113-OE tobacco shows obvious anthocyanin synthesis and accumulation.

Culturing on rooting culture medium for 30-60 days to obtain transgenic tobacco seedling. The types and the contents of anthocyanin of WT and IbMYB113-OE transgenic tobacco plants are detected by referring to the high performance liquid chromatography which is a standard of agricultural industry of the people's republic of China and is used for determining anthocyanin in NY/T2640-2014 plant-derived food, and the average value is obtained by repeating the experiment for three times. As shown in FIG. 2, no peak pattern of anthocyanins was detected in WT tobacco seedlings, two kinds of anthocyanins, delphinidin and cyanidin, were detected in IbMYB113-OE tobacco seedlings, and the peak area of cyanidin was larger than that of delphinidin. The content is calculated according to the peak area, and the result is shown in figure 3, no anthocyanin (No detect, nd) is detected in the WT tobacco seedlings, the average content of delphinidin and cyanidin in the IbMYB113-OE tobacco seedlings is higher than 25mg/100g, the total anthocyanin content reaches 59mg/100g, and the anthocyanin content of the IbMYB113-OE tobacco seedlings is remarkably increased compared with that of the WT tobacco seedlings.

The experimental result shows that the over-expression IbMYB113 gene can obviously promote the synthesis and accumulation of tobacco anthocyanin, and more particularly the synthesis and accumulation of delphinidin and cyanidin in tobacco.

The invention clones and identifies new anthocyanin biosynthesis and regulation genes in the sweet potato red potato peel through molecular biology technologies such as transcriptome sequencing, molecular cloning, genetic transformation and the like, and performs gene function verification in the cultivated tobacco variety 'Honghuadajinyuan', thereby providing theoretical basis for plant anthocyanin biosynthesis regulation network and providing new gene resources for crop quality improvement and new variety cultivation of high anthocyanin crops.

The invention has been described in detail, it should be understood that the above-described embodiments are only for the purpose of better understanding, and further modifications may be made, and are not intended to limit the invention in any way. In general, any modification, variation or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Yunnan university of agriculture

<120> anthocyanin synthesis related protein IbMYB113, and coding gene and application thereof

<130> 2021.06.15

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 711

<212> DNA

<213> Ipomoea batatas

<400> 1

atggctaatt catcatgtgc atggtcggga gtgagaaaag gtgcatggtc cgaagaagaa 60

gataatcttt tgaggaagtg cattcagaaa tatggtgaag gaaaatggca tcttattccc 120

tttagagctg ggttgaatag gtgcagaaaa agttgcagat taagatggct gaactatctt 180

cgtccagata taaagagagg cgacttcaaa ttggatgaag ttgatctaat tctgcgcctc 240

cataagctct taggcaacag gtggtcgctt attgccggca gaattccggg aagaacagca 300

aacgatgtga agaatttctg gaacacccat attcagaaaa aggtatttgc catggcagcc 360

gcttcatccg ataattggaa gggcaaagcc ccagaaatga gggaaaacac cgttgttagg 420

cctcgacctc ggagattatc ctatcggacg ccattgaccg gaaaagctac cgctgttatc 480

tgtgatgctc aaatccaagg acataagata ccaacatcgg agttggtgat ggaaaatttg 540

caagaaaaca acacaatcac gtcagaatta gaaacaacaa cgtcaaacga caaagtgcag 600

tggtgggagg attttctgtt cgacaatgaa ggaagcactt gcgtgaacca aggacaagtc 660

ggttgggcta actttacaat tgatatggac ctttcccaac ttttaagcta a 711

<210> 2

<211> 236

<212> PRT

<213> Ipomoea batatas

<400> 2

Met Ala Asn Ser Ser Cys Ala Trp Ser Gly Val Arg Lys Gly Ala Trp

1 5 10 15

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

20 25 30

Glu Gly Lys Trp His Leu Ile Pro Phe Arg Ala Gly Leu Asn Arg Cys

35 40 45

Arg Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp Ile

50 55 60

Lys Arg Gly Asp Phe Lys Leu Asp Glu Val Asp Leu Ile Leu Arg Leu

65 70 75 80

His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Ile Pro

85 90 95

Gly Arg Thr Ala Asn Asp Val Lys Asn Phe Trp Asn Thr His Ile Gln

100 105 110

Lys Lys Val Phe Ala Met Ala Ala Ala Ser Ser Asp Asn Trp Lys Gly

115 120 125

Lys Ala Pro Glu Met Arg Glu Asn Thr Val Val Arg Pro Arg Pro Arg

130 135 140

Arg Leu Ser Tyr Arg Thr Pro Leu Thr Gly Lys Ala Thr Ala Val Ile

145 150 155 160

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

165 170 175

Met Glu Asn Leu Gln Glu Asn Asn Thr Ile Thr Ser Glu Leu Glu Thr

180 185 190

Thr Thr Ser Asn Asp Lys Val Gln Trp Trp Glu Asp Phe Leu Phe Asp

195 200 205

Asn Glu Gly Ser Thr Cys Val Asn Gln Gly Gln Val Gly Trp Ala Asn

210 215 220

Phe Thr Ile Asp Met Asp Leu Ser Gln Leu Leu Ser

225 230 235

Claims

1. The anthocyanin synthesis related protein IbMYB113 is characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

2. The gene encoding an anthocyanin synthesis-associated protein IbMYB113 as recited in claim 1, wherein the gene comprises: is a nucleic acid molecule which codes for the amino acid sequence shown as SEQ ID NO. 2.

3. The coding gene of claim 2, wherein: the nucleotide sequence is shown in SEQ ID NO. 1.

4. An expression cassette, a recombinant vector or a recombinant microorganism comprising a gene encoding the IbMYB113 protein of claim 2 or 3.

5. The recombinant vector of claim 4, wherein: the recombinant vector is pHELLSGATE12-IbMYB113 obtained by replacing a small fragment between XhoI and XbaI restriction sites of a plasmid pHELLSGATE12 with an encoding gene of IbMYB113 protein.

6. Use of the protein IbMYB113 of claim 1, or the gene encoding the IbMYB113 protein of claim 2 or 3, or the expression cassette of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, or the recombinant vector of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, or the recombinant microorganism of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, for regulating the synthesis, type and content of plant anthocyanins.

7. Use of the protein IbMYB113 of claim 1, or the gene encoding the IbMYB113 protein of claim 2 or 3, or the expression cassette of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, or the recombinant vector of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, or the recombinant microorganism of claim 4 containing the gene encoding the IbMYB113 protein of claim 2 or 3, for promoting the synthesis and accumulation of delphinidin and cyanidin in plants.

8. A method of breeding a transgenic plant, comprising: the method comprises the following steps:

introducing coding genes or nucleic acid molecules of the IbMYB113 protein into a receptor plant to obtain a transgenic plant; the amino acid sequence of the IbMYB113 protein is shown as SEQ ID NO. 2; the transgenic plant has an increased anthocyanin content as compared to the recipient plant.

9. The use of claim 6 or 7, or the method of claim 8, wherein: the plant is tobacco.

10. A method of breeding transgenic tobacco, comprising: replacing a small fragment between XhoI restriction enzyme cutting sites and XbaI restriction enzyme cutting sites in pHELLSGATE12 plasmid with a coding gene or a nucleic acid molecule of IbMYB113 protein as defined in claim 2 or 3 to obtain a recombinant vector pHELLSGATE12-IbMYB113, and introducing the recombinant vector into tobacco to obtain transgenic tobacco with over-expression of IbMYB113.