CN111733164B

CN111733164B - IbNAC56 gene for promoting anthocyanin synthesis and application thereof

Info

Publication number: CN111733164B
Application number: CN201910724870.6A
Authority: CN
Inventors: 张华�; 姚改芳; 胡康棣; 魏曾正; 唐君; 胡兰英; 李延红; 陈晓燕; 钟庭颖; 孙红叶
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2022-05-03
Anticipated expiration: 2039-08-07
Also published as: CN111733164A

Abstract

A method for promoting synthesis of anthocyaninIbNAC56Genes and the application thereof,IbNAC56the nucleotide sequence of the gene is shown in SEQ ID No. 1.IbNAC56The application of the gene in promoting the transfer of the purple sweet potato tuberous root anthocyanin provides a new gene resource for promoting the molecular breeding of the accumulation of the sweet potato tuberous root anthocyanin and a new genetic resource for implementing green agriculture, and the development and utilization of the genetic resource are beneficial to reducing the agricultural cost and realizing environmental friendliness.

Description

IbNAC56 gene for promoting anthocyanin synthesis and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to an NAC family member IbNAC56 gene which is separated and cloned from tuber roots of sweet potato Han Zi and is related to anthocyanin biosynthesis and application thereof.

Background

Anthocyanin is the most obvious of plant secondary metabolites, and has important biological effects on plant disease resistance, ultraviolet ray resistance, adaptation to severe environment and the like. Anthocyanin determines color of flower, fruit, and vegetable, and can protect plant from ultraviolet injury, low temperature, drought stress, and microbial pathogen. Has important function in preventing human nervous system, cardiovascular disease, cancer and diabetes, so the anthocyanin has great application value.

Most plant anthocyanidin synthesis is regulated by the MYB-bHLH complex or the MYB-bHLH-WD40 complex (MBW complex). In addition, transcription factors influence anthocyanin biosynthesis through interaction with the MBW complex. The NAC family is one of the largest plant-specific transcription factors in plant genomes, with NAC genes being expressed in different developmental stages and tissues. The NAC gene is involved in the growth and development, disease resistance and abiotic stress response of plants. At the same time, the NAC gene has also been shown to be involved in the regulation of anthocyanin biosynthesis. For example, in peach, a gene encoding a transcription factor for the NAC domain was found, which is expressed only in BLOOD fleshy peaches and is designated BLOOD (BL). The anthocyanin can be used as a heterodimer with PpNAC1 in a blood peach, so that the transcription of PpMYB10.1 is activated, and the accumulation of anthocyanin is promoted. Reports show that in Arabidopsis thaliana, the transcription levels and total anthocyanin content of several key genes related to flavonoid biosynthesis are remarkably increased in OX-ANAC078 plants under high illumination conditions, and are reduced in plants with the ANAC078 gene knocked out, which indicates that the ANAC078 protein plays an important role in regulating and controlling the expression of genes related to flavonoid biosynthesis, thereby causing anthocyanin pigmentation under high illumination. Furthermore, in arabidopsis thaliana, studies report a negative regulatory effect of ANAC032 on anthocyanin accumulation and anthocyanin biosynthesis (DFR, ANS/LDOX) gene expression against high sucrose, oxidative and abiotic stress.

Disclosure of Invention

The invention aims to provide an IbNAC56 gene for promoting anthocyanin synthesis and application thereof.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: an IbNAC56 gene for promoting anthocyanin synthesis, which is characterized in that: the nucleotide sequence is shown in SEQ ID No. 1.

The preferable technical scheme is as follows: the IbNAC56 gene promoting anthocyanin synthesis cooperates with at least one of the coaction factors IbMYB340 and IbbHLH3 to promote anthocyanin synthesis; the nucleotide sequence of the IbMYB340 gene is shown as SEQ ID No.5, the amino acid sequence of the encoded protein is shown as SEQ ID No.6, the nucleotide sequence of the IbbHLH3 gene is shown as SEQ ID No.7, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 8.

The preferable technical scheme is as follows: the IbNAC56 gene promoting anthocyanin synthesis and at least one of IbMYB340 and IbbHLH3 co-act to promote anthocyanin accumulation in tobacco leaves or strawberry fruits.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a protein coded by IbNAC56 gene promoting anthocyanin synthesis, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a primer pair for cloning IbNAC56 gene promoting anthocyanin synthesis, which is characterized in that: the sequence of the upstream primer IbNAC56-F is shown as SEQ ID No.3, and the sequence of the downstream primer IbNAC56-R is shown as SEQ ID No. 4.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

1. through the application of the IbNAC56 gene in promoting the transport and accumulation of anthocyanin, a new gene resource is provided for promoting molecular breeding of accumulation of tuberous root anthocyanin of sweet potato, a new genetic resource is provided for implementing green agriculture, and the development and utilization of the genetic resource are beneficial to reducing agricultural cost and realizing environmental friendliness.

2. The transcription factor IbNAC56 and at least one of auxiliary factors IbMYB340 and IbbHLH3 are co-transformed to promote the large-scale accumulation of anthocyanin in tobacco leaves and strawberry fruits, and experiments prove that the clone IbNAC56 and at least one of the auxiliary factors IbMYB340 and IbbHLH3 have the function of promoting the transport and accumulation of anthocyanin in combination.

Drawings

FIG. 1 is an appearance diagram of IbNAC56 gene on tobacco leaf anthocyanin accumulation.

Fig. 2 shows the effect of the ibac 56 gene on tobacco lamina a/b values.

FIG. 3 shows the effect of the IbNAC56 gene on anthocyanin accumulation in tobacco leaves.

FIG. 4 is an appearance diagram of IbNAC56 gene on the accumulation of anthocyanin in strawberry fruits.

Fig. 5 shows the effect of the ibac 56 gene on strawberry fruit a/b values.

FIG. 6 shows the effect of the IbNAC56 gene on anthocyanin accumulation in strawberry fruits.

FIG. 7 is a graph showing the effect of the IbNAC56 gene on the expression level of a anthocyanin metabolism pathway-associated gene.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-7. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.

Example 1: IbNAC56 gene for promoting anthocyanin synthesis and application thereof

Extracting RNA from tuberous root of sweet potato 'Hanzi' by using Plant TotalRNArelationship KitPlus (Foregene, RE-05022) kit and PrimeScript^TMThe first strand cDNA was synthesized by RTMasterMix (Takara) reverse transcription kit, and the IbNAC56 gene full length was amplified using purple potato cDNA as a template.

The operation method comprises the following steps: 1. extracting RNA from tuberous roots of sweet potato 'Hanzi'; the method adopts a Plant TotalRNAI relationship KitPlus (Foregene, RE-05022) kit, and the operation is carried out according to an operation instruction provided by the kit, and specifically comprises the following steps: putting 500mg of freeze-dried root tuber of purple sweet potato in a mortar, adding liquid nitrogen, and fully grinding to fine powder; sucking 500 mu of LBufferPSL1 into a 2mL centrifuge tube, adding 10 mu of beta-mercaptoethanol, and uniformly mixing; scraping a proper amount (about 50mg) of ground powder by using a blue gun head cooled by liquid nitrogen, transferring the powder into BufferPSL1, and uniformly mixing the powder by vortex oscillation; standing at room temperature for 5min, adding 100 μ LBuffERPS, and mixing gently; transferring all liquid into DNA-cleaning column, centrifuging at 12000rpm for 2min, removing filter column, and collecting supernatant in the tube; carefully transferring 300mL of the supernatant into a new 2mL centrifuge tube, adding 450mLBuffer PSL2, and gently mixing; transferring 500 μ L of the mixed solution into RNA-only column, centrifuging at 12000rpm for 1min, and removing the waste liquid; adding 500 mu LBuffERPRW1 into RNA-only column, centrifuging for 1min, and discarding waste liquid; adding 700 μ L of anhydrous ethanol, centrifuging for 1min, and removing waste liquid; adding 700 μ LBufferPRW2 into RNA-only column, centrifuging at 12000rpm for 1min, discarding waste liquid, and repeating the step once; centrifuging at 12000rpm for 2min, and discarding the collection tube; the RNA-only column was transferred to a new 2mL centrifuge tube, and 60. mu.L of RNase-freedH preheated at 65 ℃ was dropped into the center of the membrane₂O, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and collecting RNA. mu.L of RNA was run through agarose gel electrophoresis, RNA concentration was estimated from band intensity, and stored in a freezer at-80 ℃ until use.

2. First Strand cDNA Synthesis Using PrimeScript RTMasterMix (Takara) reverse transcription kit (according to the instructions provided in the kit)Do so). The reverse transcription system is 10 mu L: 5 XPrimeScriptR^TMasterMix2μL，totalRNA2μL，RNase-FreeddH₂O6 μ L. The reaction conditions are 37 ℃ and 15 min; 5s at 85 ℃; 4 ℃ and infinity. The obtained cDNA can be stored in a refrigerator at-20 ℃. The reverse transcription of the first strand cDNA was used to amplify the full length of the IbNAC56 gene. The amplification gene primer pair is IbNAC 56-F: 5'-ACTAGTGGATCCAAAGAATTCATGGAGAGCACCGATTCATCG-3', respectively; IbNAC56-R: 5'-TCATTAAAGCAGGACTCTAGATTAAGAGTACCAATTGATGCCGG-3'. Ultra-fidelity DNA polymerase

Super-Fidelity DNA Polymerase (P505-d1) was purchased from Novowed Biotech. The amplified reaction system contained 200ng of cDNA in 50. mu.L, 2 XPphanta Max Buffer 25. mu.L, 10mM dNTP 1. mu.L, Phanta Max Super-Fidelity DNA Polymerase (1U/. mu.L), 1. mu.L, 10. mu.M 2. mu.L of the above primers, plus ddH₂O to 50. mu.L. The PCR reaction was performed on an eppendorf amplification machine according to the following procedure: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 90 sec, 35 thermal cycles, extension at 72 ℃ for 5min, and storage at 4 ℃. One single PCR band product was generated.

After the PCR product was detected by 1% agarose gel electrophoresis, DNA fragments were recovered using AxyGEN miniprep kit (purchased from Hangzhou, Inc., Cin, technologies, Aisijin) according to the instructions of AxyGEN miniprep kit. A part of the recovered and purified DNA solution is sent to Shanghai Yingjun biotechnology limited for sequencing, and the sequencing result shows that the total length of the IbNAC56 gene is 1062bp, and the nucleotide sequence is shown as SEQ ID NO. 1. The amino acid sequence of the protein coded by the IbNAC56 gene is shown in SEQ ID No. 2.

A portion of the purified DNA solution was recovered and ligated with a double-digested (EcoRI/XbaI) linear pSAK277 vector to prepare a recombinant enzyme

II One Step Cloning Kit (cat # C112-01) was purchased from Novowed Biotech and was conducted according to the procedures described. The total volume of the ligation reaction system was 10. mu.L, includingIncluding 2. mu.L of 5 × CE II Buffer, 50-200 ng of linearized cloning vector (100 ng in this example), 50-200 ng of insert amplification product (100 ng in this example), and 1. mu.L of

And II, performing treatment. Ligation was performed at 37 ℃ for 30 min. After the reaction is finished, the reaction is immediately placed in an ice water bath for cooling for 5min, and the reaction product can be directly converted. Transformation Escherichia coli DH 5. alpha. was transformed by heat shock method (see molecular cloning laboratory Manual, third edition, science publishers, 2002), positive clones were selected on LB solid plates containing 50mg/L spectinomycin, and 5 positive clones were selected and sequenced. The correct plasmid containing the gene sequence of interest was designated IbNAC56-pSAK 277. The recombinant vector IbNAC56-pSAK277 was introduced into Agrobacterium GV3101 by freeze-thawing. Selecting single colony of Agrobacterium GV3101, inoculating to 20ml YEB liquid culture medium with 30 μ g/ml Gentamicin, culturing at 28 deg.C and 200rpm for 1 day; sucking 200 μ l of the bacterial liquid, adding 20ml YEB liquid culture medium with 30 μ g/ml Gentamycin, and culturing at 28 deg.C to OD₆₀₀0.3-0.4; centrifuging 2ml of bacterial solution at 4 ℃ and 10000rpm for 10min, discarding the supernatant, resuspending the precipitate with 800 μ L of ice-precooled 0.01mol/L Tris-Cl (pH7.0), centrifuging 10min, and discarding the supernatant; resuspend pellet with ice-precooled 100. mu.l YEB broth and add to 5. mu.l each of 1.0. mu.g/. mu.l pSAK277-itfMYBb 10. mu.l ice-precooled LTE (10mmol/L Tris-Cl, 1mmol/L Na₂EDTA, pH8.0), mixing gently, immediately placing the mixture in liquid nitrogen for 5min, and quickly transferring to a 37 deg.C water bath for 25 min; 100. mu.L of LB liquid medium left at room temperature was added, pre-expressed at 28 ℃ for 1.5 hours, and plated on YEB solid medium supplemented with 60. mu.g/ml Gentamycin and 60. mu.g/ml Kanamycin, and cultured at 28 ℃ for 1-2 days until single colonies appeared.

The cloning template of the auxiliary factor IbMYB340 is also cDNA of sweet potato 'Han purple' tuberous root, and the forward primer sequence of PCR amplification is as follows: 5'-ACTAGTGGATCCAAAGAATTCATGGTGGGAGCTGCTGAGAA-3', respectively; the reaction system in which the reverse primer sequence was 5'-TCATTAAAGCAGGACTCTAGATCAGAAAATAAACCCTCCATTCCA-3' amplified was 50. mu.L including 200ng of cDNA, 2X Phanta Max Buffer 25. mu.L, 10mM dNTP 1. mu.L, Phanta Max Super-FidelityDNA Polymerase (1U/. mu.l) 1. mu.L, 10. mu.M 2. mu.L of the above primer, plus ddH₂O to 50. mu.L. The PCR reaction was performed on an eppendorf amplification machine according to the following procedure: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 90 sec, 35 thermal cycles, extension at 72 ℃ for 5min, and storage at 4 ℃. Detecting the PCR amplified product by 1% agarose gel electrophoresis, recovering DNA fragment with AxyGEN small gel recovery kit (purchased from Hangzhou, Inc. of Aisijin biotechnology), recovering purified DNA solution, performing ligation reaction with linear pSAK277 vector of double enzyme digestion (EcoRI/XhoI), and recombining enzyme

II One Step Cloning Kit (cat # C112-01) was purchased from Novowed Biotech and was conducted according to the procedures described. The total volume of the ligation reaction system was 10. mu.L, including 2. mu.L of 5 × CE II Buffer, 50-200 ng of linearized cloning vector (100 ng in this example), 50-200 ng of insert amplification product (100 ng in this example), and 1. mu.L

And II, performing treatment. Ligation was performed at 37 ℃ for 30 min. After the reaction is finished, the reaction is immediately placed in an ice water bath for cooling for 5min, and the reaction product can be directly converted. Transformation Escherichia coli DH 5. alpha. was transformed by heat shock (see molecular cloning laboratory Manual, third edition, science publishers, 2002), positive clones were selected on LB solid plates containing 50mg/L spectinomycin, and 5 positive clones were selected for sequencing (accomplished by Shanghai Yingjun Biotechnology Co., Ltd.). Sequencing results show that the full length of the IbMYB340 gene is 720bp, the nucleotide sequence of the IbMYB340 gene is shown in SEQ ID NO.5, and the constructed recombinant vector is named as pSAK277-IbMYB 340. The constructed recombinant vector pSAK277-IbMYB340 is transformed into an agrobacterium strain GV3101 by a chemical transformation method, 0.01-1 mu g of plasmid DNA is added into each 100 mu L of competence, the mixture is uniformly mixed by dialing the bottom of a tube with a hand, and is kept stand on ice for 5min, liquid nitrogen for 5min, a water bath at 37 ℃ for 5min and an ice bath for 5min in sequence; adding 700 μ L LB liquid culture medium without antibiotic, and shake culturing at 28 deg.C for 2-3 hr; finally, the mixture is centrifuged at 6000rpm for one minute to collect the bacteria, and an appropriate amount of supernatant is left to be coated on the suspensionOn LB plates with the corresponding antibiotic, followed by cultivation in an inverted position at 28 ℃ for 2d, and the Agrobacterium is resuspended in 10ml of 10mM MgCl₂200 mu m acetosyringone and 10mM MES (2- (N-morpholine) -ethanesulfonic acid, pH 5.6) buffer solution until OD600 value is 0.8-1.0, suspension culture is carried out for 4-5 hours at 25 ℃ to obtain IbMYB340 bacterial solution for infection.

The IbNAC56 gene is applied to promoting anthocyanin to accumulate in tobacco leaves:

the application of the IbNAC56 gene combined with the IbMYB340 gene or the IbbHLH3 gene for promoting anthocyanin synthesis in tobacco leaves; the nucleotide sequence of the IbMYB340 gene is shown as SEQ ID No.5, the amino acid sequence of the encoded protein is shown as SEQ ID No.6, the nucleotide sequence of the IbbHLH3 gene is shown as SEQ ID No.7, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 8.

The constructed recombinant vector is used for instantly transforming tobacco leaves through agrobacterium-mediated transformation.

The first implementation mode comprises the following steps: a single colony of Agrobacterium GV3101 carrying the plant expression vector IbNAC56-pSAK277 plasmid was picked and cultured in an inverted culture at 28 ℃ for 2d, and Agrobacterium GV3101 was resuspended in 10ml of 10ml containing 10mM MgCl₂200 μm acetosyringone and 10mM MES (2- (N-morpholine) -ethanesulfonic acid, ph 5.6) to OD₆₀₀The value is 0.8-1.0, and the bacterial liquid IbNAC56 is obtained after the bacterial liquid is used for infection after suspension culture for 4-5 hours at 25 ℃. Injecting bacterial liquid at the back of 2-3 weeks old tobacco leaves along two ends of an axis by using an injector, then placing the tobacco at 16 ℃ in a dark place for inducing for 12h, taking out the tobacco, placing the tobacco in a long-day (16h) tissue culture room for culturing for 4-7 days under the illumination, observing the phenotype and taking a picture.

The second embodiment: injecting liquid IbNAC56 bacteria and IbMYB340 bacteria into the back of 2-3 weeks old tobacco leaves along two ends of an axis by using an injector according to the ratio of 1: 1, then placing the tobacco in a 16 ℃ dark induction room for 12h, taking out, placing in a long-day (16h) tissue culture room for culturing for 4-7 days, observing the phenotype, photographing and sampling for subsequent experiments.

The third embodiment is as follows: the preparation method of the IbbHLH3 bacterial liquid is the same as that of the IbNAC56 bacterial liquid. Injecting liquid IbbHLH3 bacteria, IbMYB340 bacteria and IbNAC56 bacteria into the back of 2-3 weeks old tobacco leaves along two ends of an axis by using an injector according to the ratio of 1: 1: 1, then placing the tobacco in a 16 ℃ dark induction room for 12h, taking out, placing in a long-day (16h) tissue culture room for culturing for 4-7 days, observing the phenotype, photographing and sampling for subsequent experiments.

No pigmentation was observed in the case of the individual transformants with empty pSAK277, IbNAC56 and IbbHLH3, and in the case of the individual transformant with little pigmentation in IbMYB340, in the case of the cotransformation of IbMYB340+ IbbHLH3, in the case of the IbMYB340+ IbbNAC 56, in the case of the additional transformant with increased pigmentation in the case of the IbMYB340+ IbbHLH3+ IbNAC56 (FIG. 1). The result of detecting the deposition of the pigment on the tobacco leaves by using a colorimeter shows that the ratio of a to b is the highest and is positive, namely IbMYB340+ IbbHLH3+ IbNAC56 co-transformation, the ratio of the singly transformed unloaded pSAK277 to the IbNAC56 to the IbbHLH3 is negative (figure 2), the total anthocyanin content is further determined by using a spectrophotometer, the contents of the co-transformed IbMYB340+ IbbHLH3+ IbNAC56 anthocyanin are the highest, the contents of the unloaded and singly transformed pSAK277 and IbNAC56 and IbbHLH3 are almost not detected, the singly transformed IbMYB340 has little pigment deposition, the singly transformed IbMYB340 has increased anthocyanin content, the co-transformed IbMYB340+ IbbHLH3 and the IbMYB340+ IbMYB 56 have increased anthocyanin content (figure 3).

Example 4: IbNAC56 gene applied to promotion of anthocyanin accumulation in strawberry fruits

Preparing a staining solution and performing subsequent operations, injecting the bacterial solution into strawberry fruits 2 weeks after flowering by using an injector, then placing the strawberries into a 16 ℃ dark place for induction for 12 hours, taking out the strawberries, placing the strawberries in a long sunshine (16h) tissue culture room for culture for 4-7 days, observing phenotypes and taking pictures (figure 4), wherein the deposition of pigments in strawberry receptacle is consistent with the result in tobacco (figure 4), a chromatometer detects the pigment deposition of a coloring part, the results show that the ratio of IbbHLH3 a/b of independent transformation unloaded pSAK277, IbbNAC 56 and IbbHLH 3/b is almost 0, the co-transformation ratios of IbMYB340 and IbbMYB 340+ IbbHLH3 and IbbMYB 340+ IbbHLH3+ IbNAC56 are gradually increased in turn (figure 5), and the result of the total content of the co-transformation IbMYB340+ IbbHLH3+ IbbNAC 56 is obviously higher than other combinations (figure 6); RT-qPCR analysis indicated that co-transformation of IbMYB340+ IbbHLH3+ IbNAC56 significantly up-regulated the expression levels of anthocyanin metabolism-associated genes, particularly ANS and RAP (anthocyanin-encoding transformation-associated gene GST) (FIG. 7). The other methods of this example were the same as example 3.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

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Ile Leu Arg Ala Ser Pro Pro Pro Pro Leu Arg Trp Glu Ile Ile Thr

115 120 125

Ser Trp Ala Val Arg Gln Pro Thr Leu Arg Arg Arg Cys Ile Met Lys

130 135 140

Thr Leu Asn Pro Gly Thr Ala Leu Pro Trp Ile Trp Met Val Cys Gly

145 150 155 160

Glu Gly Asn Tyr Gly Thr Thr Met Met Thr Val Ile Leu Lys Arg His

165 170 175

Tyr Ser Arg Ala Thr Phe Lys Ile Leu Val Val Leu Glu Leu Ile Met

180 185 190

Pro Leu Thr Ser Gly Met Glu Gly Leu Phe

195 200

<210> 7

<211> 555

<212> DNA

<213> purple sweet potato

<400> 7

ccaaatatca agagaggaga ctttaaagag gatgaagtag atcttatact tagacttcac 60

aggcttttgg gaaacaggtg gtcattgatt gctagaagac ttccaggaag aacagcgaat 120

gatgtgaaaa attattggaa cactcgattg cggatcgatt ctcgcatgaa aacgttgaaa 180

aataaatctc aagaaacgag aaagaccaat gtgataagac ctcagcccca aaaattcatc 240

aaaagttcat attacttaag cagtaaagaa ccaattctag aacatattca atcagcagaa 300

gatttaagta cgccatcaca aacgtcgtcg tcaacaaaga acggaaatga ttggtgggag 360

accttgttcg aaggcgagga tacttttgaa agggctgcat gtcccagcat tgagttagag 420

tcaaaagaag aatagtttcc tgaagaagga caaagtagaa gtgaattctc ctttagcatg 480

gacctttgga atcatgaaga actcttcaca actttttggt ttgatgatcg actgtcggca 540

agatcatgtg ccaat 555

<210> 8

<211> 244

<212> PRT

<213> purple sweet potato

<400> 8

Thr Leu Phe Glu Gly Glu Asp Thr Phe Glu Arg Ala Ala Cys Pro Ser

1 5 10 15

Ile Glu Leu Glu Glu Glu Leu Phe Thr Thr Phe Trp Phe Asp Asp Arg

20 25 30

Leu Ser Ala Arg Ser Cys Ala Asn Phe Pro Glu Glu Gly Gln Ser Arg

35 40 45

Ser Glu Phe Ser Phe Ser Met Asp Leu Trp Asn His Ser Lys Glu Glu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg Leu Arg Ile Asp Ser Arg Met Lys Thr Leu Lys Asn Lys Ser Gln

115 120 125

Glu Thr Arg Lys Thr Asn Val Ile Arg Pro Gln Pro Gln Lys Phe Ile

130 135 140

Lys Ser Ser Tyr Tyr Leu Ser Ser Lys Glu Pro Ile Leu Glu His Ile

145 150 155 160

Gln Ser Ala Glu Asp Leu Ser Thr Pro Ser Gln Thr Ser Ser Ser Thr

165 170 175

Lys Asn Gly Asn Asp Trp Trp Glu Met Glu Gly Tyr Asn Val Asn Leu

180 185 190

Ser Val Arg Lys Gly Ala Trp Thr Arg Glu Glu Asp Asn Leu Leu Arg

195 200 205

Gln Cys Ile Glu Ile His Gly Glu Gly Lys Trp Asn Gln Val Ser Tyr

210 215 220

Lys Ala Gly Leu Asn Arg Cys Arg Lys Ser Cys Arg Gln Arg Trp Leu

225 230 235 240

Asn Tyr Leu Lys

<210> 9

<211> 41

<212> DNA

<213> Artificial sequence

<400> 9

actagtggat ccaaagaatt catggtggga gctgctgaga a 41

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence

<400> 10

tcattaaagc aggactctag atcagaaaat aaaccctcca ttcca 45

Claims

1.IbNAC56The application of the gene in promoting the synthesis of anthocyanin in tobacco leaves is characterized in that: the above-mentionedIbNAC56Genes and coactivatorsIbMYB340AndIbbHLH3the combined action promotes the synthesis of anthocyanin in the tobacco leaves; saidIbNAC56The nucleotide sequence of the gene is shown as SEQ ID No.1IbMYB340The nucleotide sequence of the gene is shown as SEQ ID No.5IbbHLH3The gene nucleotide sequence is shown as SEQ ID No. 7.

2. IbNAC56The application of the gene in promoting the synthesis of anthocyanin in strawberry fruits is characterized in that: the above-mentionedIbNAC56Genes and coactivatorsIbMYB340AndIbbHLH3the synthesis of anthocyanin in the strawberry fruits is promoted under the combined action; saidIbNAC56The nucleotide sequence of the gene is shown as SEQ ID No.1IbMYB340The nucleotide sequence of the gene is shown as SEQ ID No.5IbbHLH3The gene nucleotide sequence is shown as SEQ ID No. 7.