CN111733163B

CN111733163B - IbMYB44 gene for regulating anthocyanin synthesis and recombinant expression vector and application thereof

Info

Publication number: CN111733163B
Application number: CN201910724867.4A
Authority: CN
Inventors: 姚改芳; 胡康棣; 张华�; 魏曾正; 唐君; 胡兰英; 钟庭颖; 孙红叶; 孙忱
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: CN111733163A

Abstract

Regulating anthocyanin synthesisIbMYB44A gene, a recombinant expression vector and application thereof,IbMYB44the nucleotide sequence of the gene is shown in SEQ ID No. 1.IbMYB44The application of the gene in regulation of the transport of the purple sweet potato tuberous root anthocyanin provides a new gene resource for molecular breeding for regulating 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

IbMYB44 gene for regulating anthocyanin synthesis and recombinant expression vector and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, relates to an IbMYB44 gene for regulating anthocyanin synthesis and construction and application of a recombinant expression vector thereof, and particularly relates to an IbMYB44 gene which is a MYB TF family member related to sweet potato tuber anthocyanin biosynthesis and is obtained by separating and cloning sweet potatoes and application of the gene.

Background

Sweet potato is the seventh most important crop in the world and the fourth most important crop in China, and provides a large amount of carbohydrates, proteins, vitamins and minerals for human beings. Purple sweet potatoes are popular due to attractive appearance and high anthocyanin content, and are health-care food, which have beneficial effects on preventing diseases such as cancer, diabetes and the like and improving liver functions. The purple sweet potato tuber contains a large amount of polyphenol, and anthocyanin is a common visible polyphenol-like substance in purple sweet potatoes. The purple sweet potato anthocyanin has a plurality of physiological functions, and has other physiological functions of resisting oxidation, regulating blood fat and blood sugar, eliminating inflammation, improving memory and the like.

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. Meanwhile, the color of flowers, fruits and vegetables can be determined, and the plants can be protected from ultraviolet ray damage and can resist low temperature, drought stress and invasion of microbial pathogens. Has important effects in preventing nervous system and cardiovascular diseases, cancer and diabetes of human, so the anthocyanin has great research value. Most plant anthocyanidin synthesis is regulated by the MYB-bHLH complex or the MYB-bHLH-WD40 complex (MBW complex).

In the MBW complex, the transcription factor MYBs plays an important role. For example, ATMYB75(PAP1), ATMYB90(PAP2), ATMYB113 and ATMYB114 in Arabidopsis, MDMYB10 and MDMYB110a in apple, PYMB10 and PYMYB114 in pear, and FAMYB10 in strawberry all appear to be involved in anthocyanin biosynthesis as activators. In addition to being activators, MYBs transcription factors that exert negative control on anthocyanin have also been widely reported, including Arabidopsis AtMYBL2 and grape VvMYBC2-L1(R3-MYBs) and strawberry FaMYB1, peach PpMYB18, and potato StMYB44(R2R 3-MYBs). Among these, the EAR motif (lxlxlxl or DLNXXP) is the most common transcription repression motif in plants.

Disclosure of Invention

The invention aims to provide an IbMYB44 gene for regulating anthocyanin synthesis, and a recombinant expression vector and application thereof.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: an IbMYB44 gene for regulating anthocyanin synthesis, the nucleotide sequence of which is shown in SEQ ID No. 1.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the protein encoded by the IbMYB44 gene synthesized by the anthocyanin is regulated, and 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 host bacterium of the IbMYB44 gene for regulating anthocyanin synthesis.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the primer pair of the IbMYB44 gene for regulating anthocyanin synthesis has a cloning right, wherein the sequence of an upstream primer IbMYB44-F1 is shown as SEQ ID No.3, and the sequence of a downstream primer IbMYB44-R1 is shown as SEQ ID No. 4.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the IbMYB44 gene for regulating anthocyanin synthesis is applied to regulating anthocyanin synthesis in purple sweet potato tubers.

The preferable technical scheme is as follows: the IbMYB44 gene for regulating anthocyanin synthesis is combined with the IbMYB340 gene to regulate anthocyanin synthesis in the purple sweet potato tuber; the nucleotide sequence of the IbMYB340 gene is shown in SEQ ID No.5, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 6.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a recombinant expression vector containing the IbMYB44 gene for regulating anthocyanin synthesis.

The preferable technical scheme is as follows: the method is characterized in that the IbMYB44 gene for regulating anthocyanin synthesis in claim 1 is inserted between EcoR I and Xbal I sites by taking pSAK277 as a starting vector.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the recombinant expression vector of the IbMYB44 gene for regulating anthocyanin synthesis is applied to regulation of anthocyanin synthesis in purple sweet potato tubers.

The preferable technical scheme is as follows: the use of the IbMYB44 gene recombinant expression vector of claim 9 in combination with a IbMYB340 gene-containing recombinant vector for the synthesis of anthocyanin in sweet potato tubers.

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

the application of the IbMYB44 gene in regulation of sweet potato tuber anthocyanin transport provides a new gene resource for molecular breeding for regulating accumulation of sweet potato tuber anthocyanin, provides a new genetic resource for implementing green agriculture, and is beneficial to reducing agricultural cost and realizing environmental friendliness through development and utilization of the genetic resource.

2. According to the invention, the transcription factor IbMYB44 is used for inhibiting the synthesis of fruit anthocyanin when the tomato in the color breaking period passes through transformation, meanwhile, IbMYB44 and IbMYB340 are used for co-transforming tobacco leaves to regulate the accumulation of tobacco leaf anthocyanin, and biological function verification shows that the cloned IbMYB44 and IbMYB340 jointly regulate the function of sweet potato tuber anthocyanin transport.

Drawings

FIG. 1 shows that IbMYB44 inhibits pigment accumulation of tomato fruits in the color breaking period. Wherein a is empty and b-e are the infestations of different fruits. The photographs were taken 7 days after infection.

FIG. 2(a) shows that the IbMYB44 and IbMYB340 co-transformed tobacco leaves regulate the accumulation of anthocyanin. a-h are IbMYB 340: different ratios of the bacterial liquids of IbMYB 44. (b) And (4) measuring the total anthocyanin content of the tobacco leaves. (c) Measuring the L value of the anthocyanin accumulation area of the tobacco leaves by a colorimeter, wherein the L value represents the luminosity, and the higher the value is, the brighter the color is. (d) The accumulation of pigment near the accumulation of anthocyanin in the tobacco leaves is measured by a colorimeter, the ratio of a/b indicates the change of the pigment, and the change of the ratio of a/b from a negative value to a positive value indicates that the color of the tobacco leaves is changed from green to red. The error bars in (b-d) are the average of six measurements.

FIG. 3 shows that the IbMYB44 and IbMYB340 combined with a sweet potato promoter IbANS co-infect tobacco leaves to carry out a dual-luciferase experiment, and the activation effect of the IbANS promoter is verified.

FIG. 4 verifies the interaction between transcription factors by yeast two-hybrid (Y2H) and luciferase complementation experiments. (a) I-VI represent the different amino acid residues of IbMYB 340. (b) Y2H verifies the interaction site of IbMYB340 and IbMYB 44. (c) Luciferase complementation experiments demonstrated the interactive effect of IbMYB340 and IbMYB 44.

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. 1-4. 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: cloning of IbMYB44 gene in purple sweet potato and construction of recombinant vector

1. Extracting RNA from sweet potato tubers: the extraction of the Total RNA of the sweet potato adopts a Plant Total RNA Isolation KitPlus (Foregene, RE-05022) kit, and the operation is carried out according to an operation instruction provided by the kit, and the method specifically comprises the following steps: placing 500mg of freeze-dried root tuber of sweet potato with chestnut flavor in mortar, and performing subsequent experimental proceduresRNA was obtained as described in the kit instructions. Then, 2. mu.L of RNA was run on agarose gel electrophoresis, and the concentration of RNA was estimated from the band brightness and stored in a refrigerator at-80 ℃ until use. The method specifically comprises the following steps: putting 500mg of freeze-dried root tuber of 'chestnut-flavor' 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. Synthesis of first Strand cDNA Using Prime Script^TMRT Master Mix (Takara) reverse transcription kit (according to the kit provided instructions for operation). 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 is carried out to obtain the first strand cDNA of chestnut flavor for amplifying the full length of IbMYB44 gene. The amplification gene primer pair is IbMYB 44-F1: 5'-ACTAGTGGATCCAAAGAATTCATGGCGAGTATTAGTCCTAATGGG-3' (SEQ ID No. 3); IbMYB 44-R1: 5'-TCATTAAAGCAGGACTCTAGACTACTCTAATCGGTTAACTCCGACG-3' (SEQ ID No. 4)). 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, the DNA fragment was recovered by using an agarose gel recovery kit (purchased from Tiangen, China), and the procedure was as described in the instruction manual. A part of the recovered and purified DNA solution is sent to Shanghai Yingjun biotechnology limited company for sequencing, and sequencing results show that the total length of the gene is 1182bp, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1. The amino acid sequence of the protein coded by the IbMYB44 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, 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. The transformation was carried out by thermal shock method (see molecular cloning, A laboratory Manual, third edition, science Press, 2002)Escherichia coli DH 5. alpha. was transformed, positive clones were selected on LB solid plates containing 50mg/L spectinomycin, and 5 positive clones were picked and sequenced. The correct plasmid containing the gene sequence of interest was designated ItfERF71a-pSAK 277. The recombinant vector was introduced into Agrobacterium GV3101 by freeze-thaw method. 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; the pellet was resuspended in ice-chilled 100. mu.l YEB broth and added to 5. mu.l each of 1.0. mu.g/. mu.l pSAK277-IbMYB 34010. mu.l ice-chilled 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 template cloned by the helper factor IbMYB340 is purple potato cDNA, and the forward primer sequence amplified by PCR is as follows: 5'-actagtggatccaaagaattcATGGTGGGAGCTGCTGAGAA-3', respectively; the reverse primer sequence was 5'-tcattaaagcaggactctagaTCAGAAAATAAACCCTCCATTCCA-3', and the amplification reaction system included 200ng cDNA, 2 × Phanta Max Buffer 25. mu.L, 10mM dNTP 1. mu.L, Phanta Max Super-Fidelity DNA Polymerase (1U/. mu.L), 10. mu.M 2. mu.L of the above primers, plus ddH in 50. mu.L₂O 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

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 a constructed recombinant vector is named as pSAK277-IbMYB 340; the recombinant vector was introduced into Agrobacterium GV3101 by freeze-thaw method. 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; the pellet was resuspended in ice-chilled 100. mu.l YEB broth and added to 5. mu.l each of 1.0. mu.g/. mu.l pSAK277-IbMYB 34010. mu.l ice-chilled 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.

Example 2: the IbMYB44 related by the invention inhibits the pigment accumulation of tomato fruits in the color breaking period

As shown in FIG. 1, where (a) is blank control, it was found from (b) - (e) that IbMYB44 can inhibit pigment accumulation in tomato fruits in the breaker stage.

Example 3: the IbMYB44 gene and the coaction factor IbMYB340 co-transform and regulate the accumulation of anthocyanin in tobacco leaves

The application of the IbMYB44 gene and the IbMYB340 gene related to anthocyanin synthesis in regulating anthocyanin synthesis in tobacco leaves; the nucleotide sequence of the IbMYB340 gene is shown in SEQ ID No.5, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 6.

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 IbMYB44-pSAK277 plasmid was picked and cultured by inversion at 28 ℃ for 2d, and Agrobacterium GV3101 was resuspended in 10ml of a medium containing 10mM MgCl ₂200 μm acetosyringone and 10mM MES (2- (N-morpholine) -ethanesulfonic acid, ph 5.6) were added to a buffer solution until the OD600 value became 0.8-1.0, and suspension culture was carried out at 25 ℃ for 4-5 hours for infection to obtain a bacterial solution of IbMYB 44. And then injecting a proper amount of bacterial liquid into the back of the 2-3 weeks old tobacco leaves along two ends of the axis by using an injector, then placing the tobacco at 16 ℃ for dark induction for 12h, taking out the tobacco leaves, placing the tobacco leaves in a long-day (16h) tissue culture room for culture for 4-7 days to observe phenotype, photographing and sampling for subsequent experiments.

The second embodiment: a single colony of Agrobacterium GV3101 carrying the plasmid of the plant expression vector pSAK277-IbMYB340 was picked and cultured by inversion at 28 ℃ for 2d, and Agrobacterium GV3101 was resuspended in 10ml of a medium containing 10mM MgCl ₂200 mu m acetosyringone and 10mM MES (2- (N-morpholine) -ethanesulfonic acid, ph 5.6) buffer solution until the OD600 value is 0.8-1.0, and suspension culture is carried out for 4-5 hours at 25 ℃ for infection to obtain the IbMYB340 bacterial solution. Injecting an IbMYB44 bacterial liquid and an IbMYB340 bacterial liquid into the back of the 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 16 ℃ and inducing for 12 hours in a dark placeAnd taking out and placing a tissue culture room with long sunlight (16h) for culturing for 4-7 days to observe the phenotype, photographing and sampling for subsequent experiments.

The result shows that the separately transformed IbMYB340 has more obvious anthocyanin content accumulation, and the anthocyanin content accumulation in the tobacco gradually becomes less with the increase of the proportion of the IbMYB44 in the staining solution (figure 3). The anthocyanin-accumulated tobacco leaves were tested for pigment content and color change by spectrophotometer and colorimeter (FIG. 3), and the results were consistent with the phenotypic chart. Therefore, the IbMYB44 gene and the IbMYB340 co-transformed tobacco leaves can regulate the synthesis of anthocyanin in the tobacco leaves.

Example 4: the IbMYB44 and the IbMYB340 are combined with a sweet potato promoter IbANS to co-infect tobacco leaves to carry out a dual-luciferase experiment, and the activation effect of the IbANS promoter is verified.

Mixing the IbANS promoter with transcription factors IbMYB44 and IbMYB340 according to the ratio of 1: 9, mixing and injecting tobacco leaves, wherein the IbMYB44 and the IbMYB340 are mixed with bacteria liquid according to different ratios of experimental design. Sampling and detecting LUC: REN value, and the activation effect of the co-transformation of the IbMYB44 and the IbMYB340 on an IbANS promoter is verified, as shown in figure 3, the fact that the activity of the IbANS promoter is gradually reduced along with the increase of the ratio of the IbMYB44 in the mixed bacterial liquid can be found, and therefore anthocyanin synthesis in tobacco leaves is regulated.

Example 5: yeast two-hybrid validation of the interaction between IbMYB44 and IbMYB340

To verify the presence of interaction between IbMYB44 and IbMYB340, the full length sequence of IbMYB340 CDS and the C-or N-terminal residue amino acid sequence were cloned separately and inserted into pGBKT7 vector to verify self-activating activity, respectively, by yeast two-hybrid assay. Meanwhile, the CDS full-length sequence of the IbMYB44 is cloned and inserted into a pGADT7 vector so as to detect whether protein interaction exists with the IbMYB 340. Firstly, the IbMYB44 and the IbMYB340 are co-transformed to grow on a culture medium with two deletions of SD-Trp-Leu, and then transformants are transferred to a culture medium with four deletions of SD-Trp-Leu-His-Ade for screening, and the result shows that the amino acid sequence (IV) of the CDS of the IbMYB340 shows strong transcriptional activation activity. When yeast was co-transformed, the amino acid sequence (IV) and IbMYB44 could grow not only in the medium lacking SD-Trp-Leu, but also in the medium lacking SD-Trp-Leu-His-Ade, indicating that there is an interaction site between IbMYB340 and IbMYB44 (b in FIG. 4).

Meanwhile, in order to further verify the interaction effect between the IbMYB44 and the IbMYB 340. We cloned and inserted the full-length sequence of IbMYB340 CDS into Nluc vector, and IbMYB44 cloned and inserted its full-length sequence into CLuc vector. And (3) carrying out a luciferase complementation experiment on the tobacco leaves co-transformed by 1: 1 after the successfully constructed vector is transformed into the agrobacterium GV3101, and sampling to detect the Luc fluorescence value. As a result, NLuc-IbMYB340+ CLuc-IbMYB44 showed a higher fluorescence value, while no more significant luciferase activity was detected in the control group, indicating that there was an interaction effect between IbMYB340 and IbMYB44 (FIG. 4).

Example 6: host bacterium of IbMYB44 gene for regulating anthocyanin synthesis

And taking the GV3101 agrobacterium stored at-80 ℃ to be infected at room temperature or palm for a moment until the part of the agrobacterium is melted. And is inserted into ice in a state of ice-water mixture. 0.1ug of pSAK277-IbMYB44 recombinant plasmid was added to 50ul of competence, and the mixture was stirred by hand into the bottom of the tube, and then kept on ice for 5 minutes, kept in liquid nitrogen for 5 minutes, and then kept in a 37 ℃ water bath for 5 minutes and ice-cooled for 5 minutes. Adding 700ul of LB without antibiotics, and culturing for 2-3 hours at 28 ℃ with shaking. And (3) centrifuging at 6000rpm for one minute to collect bacteria, reserving about 100ul of supernatant, slightly blowing and beating the resuspended bacteria liquid, coating the resuspended bacteria liquid on an LB plate containing spectinomycin and rifampicin (80 ul of 100mg/ml solid LB and 250ul of 10mg/ml rifampicin are added to each 100ml solid LB, and inversely placing the mixture in a 28-degree incubator for culturing for 2-3 days.

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.

Sequence listing

<110> university of fertilizer industry

<120> IbMYB44 gene for regulating anthocyanin synthesis, recombinant expression vector and application thereof

<140> 2019107248674

<141> 2019-08-07

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<170> PatentIn version 3.5

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tcgctgatca gtaaatcgat tccggggaga tcggggaagt cgtgccggtt gcggtggtgt 180

aatcagctgt cgccgcaggt ggagcaccgg gcgtttacgg cggaggagga tgatactata 240

atccgggcgc atgctcggtt tgggaacaag tgggcgacca tagctaggtt gttggctggg 300

cggaccgaca acgcgattaa gaaccactgg aactctacct tgaagaggaa gtgctcgtcc 360

atgagcgccg atgaagggaa tgatctggcg gataggcttc aacagcagcc gttgaagaga 420

tcggtgagtg ccggggccgc ggttacgctc tcgggtctgc acttcaatcc ggggagtcct 480

tccgggtcgg atgttagcga atcgagtttg ccggttatgt ccccgtctca cgtgtttaaa 540

ccgatagcta gaacaggtgg agttctacca ccaccggtag agacgcctcc gcctccgccg 600

cctccaccgc cagcgaacga ccctcctact tcgctcagcc tctctctccc aggagttgac 660

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ctattttctt ccgctattca tacacctccc cctccaccgc ccctgccggt gccgtttcaa 780

cagccactag aaaaattcga tcttggagga ggcgcgccac cgccgatggc ttgtccaata 840

ccgccaaagg aggcggtgcc ggctccagct cagcaagacc gagtttttct cccgttcagc 900

caggagctac tggcggtgat gcaggacatg ataaagacgg aggtgcggaa ctacatgatg 960

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Met Ala Ser Ile Ser Pro Asn Gly Arg Arg Lys Asp Met Asp Arg Val

1 5 10 15

Lys Gly Pro Trp Ser Pro Glu Glu Asp Glu Leu Leu Gln Gln Leu Val

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

35 40 45

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

50 55 60

Pro Gln Val Glu His Arg Ala Phe Thr Ala Glu Glu Asp Asp Thr Ile

65 70 75 80

Ile Arg Ala His Ala Arg Phe Gly Asn Lys Trp Ala Thr Ile Ala Arg

85 90 95

Leu Leu Ala Gly Arg Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser

100 105 110

Thr Leu Lys Arg Lys Cys Ser Ser Met Ser Ala Asp Glu Gly Asn Asp

115 120 125

Leu Ala Asp Arg Leu Gln Gln Gln Pro Leu Lys Arg Ser Val Ser Ala

130 135 140

Gly Ala Ala Val Thr Leu Ser Gly Leu His Phe Asn Pro Gly Ser Pro

145 150 155 160

Ser Gly Ser Asp Val Ser Glu Ser Ser Leu Pro Val Met Ser Pro Ser

165 170 175

His Val Phe Lys Pro Ile Ala Arg Thr Gly Gly Val Leu Pro Pro Pro

180 185 190

Val Glu Thr Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Asn Asp Pro

195 200 205

Pro Thr Ser Leu Ser Leu Ser Leu Pro Gly Val Asp Ser Ser Ser Asp

210 215 220

Val Ser Pro Arg Leu Thr Glu Ser Thr Gln Pro Ile Ser Pro Ile Gln

225 230 235 240

Leu Phe Ser Ser Ala Ile His Thr Pro Pro Pro Pro Pro Pro Leu Pro

245 250 255

Val Pro Phe Gln Gln Pro Leu Glu Lys Phe Asp Leu Gly Gly Gly Ala

260 265 270

Pro Pro Pro Met Ala Cys Pro Ile Pro Pro Lys Glu Ala Val Pro Ala

275 280 285

Pro Ala Gln Gln Asp Arg Val Phe Leu Pro Phe Ser Gln Glu Leu Leu

290 295 300

Ala Val Met Gln Asp Met Ile Lys Thr Glu Val Arg Asn Tyr Met Met

305 310 315 320

Gly Val Glu Pro Gln Gln Pro Gln Pro Ser Gln Gln Gln Arg Tyr His

325 330 335

Gln His His His Gln Gln His Gln Gln Gln Gln Gln Phe Gln Phe Gln

340 345 350

Gln Gln Gln Gln Leu Gln Asn Gly Ile Gly Arg Gly Met Cys Leu Gln

355 360 365

Arg Ala Thr Ser Asn Asp Gly Leu Arg Tyr Ala Ala Ala Ala Thr Val

370 375 380

Asn Arg Val Gly Val Asn Arg Leu Glu

385 390

<210> 3

<211> 45

<212> DNA

<213> Artificial sequence

<400> 3

actagtggat ccaaagaatt catggcgagt attagtccta atggg 45

<210> 4

<211> 46

<212> DNA

<213> Artificial sequence

<400> 4

tcattaaagc aggactctag actactctaa tcggttaact ccgacg 46

<210> 5

<211> 635

<212> PRT

<213> Ipomoea batatas

<400> 5

Ala Thr Gly Gly Thr Gly Gly Gly Ala Gly Cys Thr Gly Cys Thr Gly

1 5 10 15

Ala Gly Ala Ala Ala Gly Thr Ala Gly Gly Gly Thr Gly Gly Ala Gly

20 25 30

Gly Ala Ala Ala Gly Gly Gly Cys Cys Ala Thr Gly Gly Ala Cys Gly

35 40 45

Cys Cys Cys Ala Ala Gly Gly Ala Ala Gly Ala Cys Ala Ala Gly Cys

50 55 60

Thr Thr Cys Thr Thr Gly Gly Cys Gly Ala Thr Thr Ala Thr Gly Thr

65 70 75 80

Thr Ala Gly Cys Thr Thr Gly Cys Ala Thr Gly Gly Thr Gly Ala Ala

85 90 95

Gly Gly Ala Ala Gly Ala Thr Gly Gly Ala Gly Cys Thr Cys Thr Gly

100 105 110

Thr Gly Gly Cys Thr Ala Gly Ala Thr Gly Thr Gly Cys Ala Gly Gly

115 120 125

Thr Thr Thr Gly Ala Ala Thr Thr Gly Cys Ala Thr Gly Cys Thr Thr

130 135 140

Thr Ala Thr Gly Gly Gly Gly Cys Ala Ala Cys Ala Ala Ala Thr Gly

145 150 155 160

Gly Thr Cys Ala Ala Cys Thr Ala Thr Ala Gly Cys Ala Ala Gly Ala

165 170 175

Thr Ala Cys Thr Thr Gly Cys Cys Ala Gly Gly Ala Ala Gly Ala Ala

180 185 190

Cys Ala Gly Ala Cys Ala Ala Thr Gly Ala Gly Ala Thr Ala Ala Ala

195 200 205

Gly Ala Ala Cys Thr Ala Cys Thr Gly Gly Ala Gly Ala Ala Cys Thr

210 215 220

Cys Ala Thr Thr Thr Cys Ala Ala Gly Ala Ala Gly Ala Ala Ala Cys

225 230 235 240

Cys Thr Gly Cys Ala Ala Cys Thr Gly Gly Gly Ala Ala Gly Ala Cys

245 250 255

Thr Ala Gly Cys Gly Ala Ala Ala Ala Gly Cys Ala Ala Gly Ala Thr

260 265 270

Ala Gly Gly Ala Gly Ala Ala Ala Gly Ala Ala Cys Cys Gly Cys Ala

275 280 285

Gly Ala Ala Ala Gly Ala Gly Ala Ala Ala Thr Gly Ala Ala Gly Ala

290 295 300

Gly Ala Ala Ala Gly Thr Ala Ala Thr Ala Ala Ala Cys Gly Ala Cys

305 310 315 320

Ala Cys Cys Ala Ala Ala Cys Cys Ala Cys Ala Ala Gly Ala Gly Ala

325 330 335

Cys Gly Ala Thr Gly Ala Gly Thr Ala Ala Thr Ala Ala Thr Gly Ala

340 345 350

Thr Thr Cys Thr Thr Cys Gly Thr Gly Cys Ala Thr Cys Ala Cys Cys

355 360 365

Gly Cys Cys Gly Cys Cys Gly Cys Cys Gly Cys Thr Cys Cys Gly Ala

370 375 380

Thr Gly Gly Gly Ala Gly Ala Thr Ala Ala Thr Ala Ala Cys Cys Ala

385 390 395 400

Gly Cys Thr Gly Gly Gly Cys Gly Gly Thr Ala Cys Gly Thr Cys Ala

405 410 415

Gly Cys Cys Ala Ala Cys Ala Cys Thr Ala Cys Gly Ala Cys Gly Thr

420 425 430

Cys Gly Thr Thr Gly Thr Ala Thr Cys Ala Thr Gly Ala Ala Gly Ala

435 440 445

Cys Ala Thr Thr Gly Ala Ala Thr Cys Cys Thr Gly Gly Gly Thr Ala

450 455 460

Gly Ala Cys Ala Gly Cys Thr Thr Thr Gly Cys Cys Ala Thr Gly Gly

465 470 475 480

Ala Thr Ala Thr Gly Gly Ala Thr Gly Gly Thr Thr Thr Gly Thr Gly

485 490 495

Gly Gly Gly Ala Gly Gly Gly Gly Ala Ala Thr Thr Ala Thr Gly Gly

500 505 510

Ala Ala Cys Cys Thr Ala Gly Ala Cys Cys Ala Thr Gly Ala Thr Gly

515 520 525

Ala Cys Ala Gly Thr Thr Ala Thr Cys Cys Thr Gly Ala Ala Gly Cys

530 535 540

Gly Gly Cys Ala Thr Thr Ala Thr Thr Ala Gly Ala Gly Cys Ala Gly

545 550 555 560

Gly Gly Cys Cys Ala Cys Gly Thr Gly Ala Thr Thr Cys Ala Ala Ala

565 570 575

Ala Thr Cys Cys Thr Thr Gly Thr Gly Gly Thr Thr Thr Thr Gly Gly

580 585 590

Ala Gly Cys Thr Gly Ala Thr Cys Ala Thr Gly Cys Cys Gly Thr Thr

595 600 605

Ala Ala Cys Cys Thr Cys Thr Gly Gly Ala Ala Thr Gly Gly Ala Gly

610 615 620

Gly Gly Thr Thr Thr Ala Thr Thr Thr Thr Cys

625 630 635

<210> 6

<211> 202

<212> PRT

<213> Ipomoea batatas

<400> 6

Met Val Gly Ala Ala Glu Lys Val Gly Trp Arg Lys Gly Pro Trp Thr

1 5 10 15

Pro Lys Glu Asp Lys Leu Leu Gly Asp Tyr Val Ser Leu His Gly Glu

20 25 30

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

35 40 45

Tyr Gly Ala Thr Asn Gly Gln Leu Gln Asp Thr Cys Gln Glu Glu Gln

50 55 60

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

65 70 75 80

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

85 90 95

Glu Met Lys Arg Lys Thr Thr Pro Asn His Lys Arg Arg Val Ile Met

100 105 110

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> 41

<212> DNA

<213> Artificial sequence

<400> 7

actagtggat ccaaagaatt catggtggga gctgctgaga a 41

<210> 8

<211> 45

<212> DNA

<213> Artificial sequence

<400> 8

tcattaaagc aggactctag atcagaaaat aaaccctcca ttcca 45

Claims

1.IbMYB44Application of gene in promoting synthesis of anthocyanin in tomato fruits, and application of gene in promoting synthesis of anthocyanin in tomato fruitsIbMYB44The nucleotide sequence of the gene is shown in SEQ ID No. 1.

2.IbMYB44The application of the gene in promoting anthocyanin synthesis in tobacco leaves is characterized in that:IbMYB44gene associationIbMYB340The application of the gene in promoting anthocyanin synthesis in tobacco leaves; the above-mentionedIbMYB44The nucleotide sequence of the gene is shown as SEQ ID No.1IbMYB340The gene nucleotide sequence is shown as SEQ ID No. 5.

3. ComprisesIbMYB44Application of recombinant expression vector of gene in promoting synthesis of anthocyanin in tomato fruits, and application of recombinant expression vector of gene in promoting synthesis of anthocyanin in tomato fruitsIbMYB44The nucleotide sequence of the gene is shown in SEQ ID No. 1.

4. ComprisesIbMYB44Recombinant expression vector combination of genesIbMYB340Application of recombinant expression vector of gene in promoting synthesis of anthocyanin in tobacco leaves, and preparation method of recombinant expression vectorIbMYB44The nucleotide sequence of the gene is shown as SEQ ID No.1IbMYB340The gene nucleotide sequence is shown as SEQ ID No. 5.