CN114014918A

CN114014918A - Upstream regulatory factor IbEBF2 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato

Info

Publication number: CN114014918A
Application number: CN202111396451.8A
Authority: CN
Inventors: 张南南; 付丹文; 凌秋平; 高峰
Original assignee: Nanfan Seed Industry Research Institute Guangdong Academy Of Sciences
Current assignee: Nanfan Seed Industry Research Institute Guangdong Academy Of Sciences
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-02-08
Anticipated expiration: 2041-11-23
Also published as: ZA202208161B; CN114014918B

Abstract

The invention discloses an upstream regulatory factor IbEBF2 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potatoes. The invention takes purple sweet potato strain 'A5' as an experimental material, clones the promoter sequence of IbbHLH2, and successfully obtains the upstream regulatory factor IbEBF2 of the IbbHLH2 gene through a yeast single hybrid library screening experiment. The interaction between the IbbHLH2 promoter and the upstream regulatory factor IbEBF2 is proved by using a yeast single-hybridization rotation experiment and a dual-luciferase report system detection. Subcellular localization results showed that IbEBF2 localized to the nucleus. The self-activating activity experiment result shows that the IbEBF2 has the self-activating activity. The invention can enrich and deepen the basic theory of plant anthocyanin biosynthesis molecule regulation and control in theory, and can provide new ideas and clues for cultivation measures for improving the pigment content in the purple sweet potato tuberous root.

Description

Upstream regulatory factor IbEBF2 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato

Technical Field

The invention relates to the technical field of plant gene breeding, in particular to an upstream regulatory factor IbEBF2 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potatoes.

Background

Purple sweet potato is a new sweet potato variety with unique genetic character cultivated successfully in Japan, and the root tuber of the purple sweet potato is dark purple due to the abundant anthocyanin. The plant type of the purple sweet potato plant is creeping, the tendrils are long, the branch positions are upward, the leaves are dark green, the fibrous roots are more, the tubers are expanded and grow slowly, and the purple sweet potato plant is rich in protein and various amino acids. Compared with the common sweet potato, the content of trace elements such as selenium, iron, phosphorus and the like is about 25-30%. The yield per mu is 1000-1500 kg, and the disease resistance is strong and the adaptability is wide. In addition, the most important character of purple sweet potato is that its root tuber is rich in anthocyanin.

MYB proteins normally interact with bHLH and WD40 proteins in plant anthocyanin biosynthesis and are regulated by a synergistic effect of the formation of ternary complex MBW. Transcription factors involved in transcriptional expression regulation of plant anthocyanin synthase genes mainly include three types: MYB, bHLH (also known as MYC), and WD 40. The bHLH class of transcription factors have conserved basic-helix-loop-helix domains and are a large family of transcription factors that are widely found in eukaryotes. A typical bHLH domain contains about 18 hydrophilic basic amino acids at its N-terminus, followed by two hydrophilic and lipophilic alpha helices separated by a loop. The bHLH transcription factor forms homodimer or heterodimer through an HLH region and is combined with different parts of a target gene promoter, so that the bHLH transcription factor plays a role in regulating and controlling the transcription of a gene. The bHLH transcription factor is involved in a plurality of biological processes such as plant growth and development, signal transduction, secondary metabolism and the like. Among them, regulation of flavonoid and anthocyanin synthesis is one of the most important functions of plant bHLH transcription factors.

Previous studies found that bHLH class of transcription factors can regulate plant growth and development as well as anthocyanin anabolism. bHLH transcription factor genes related to anthocyanin biosynthesis regulation have been isolated from various plants such as dahlia, maize, Arabidopsis, petunia, and sweet orange. The bHLH transcription factor family members involved in anthocyanin synthesis regulation can be divided into 2 types according to the evolutionary characteristics: bHLH1 and bHLH 2. However, the bHLH family of transcription factors involved in anthocyanin regulation varies among plants of different species, and also differs in the regulatory pattern of anthocyanin synthesis.

The gene sequence of a transcription factor IbbHLH2 is obtained by cloning in the purple sweet potato, and the IbbHLH2 gene can regulate the expression of key enzyme genes in an anthocyanin synthesis pathway, so that the accumulation of anthocyanin in the purple sweet potato is influenced. Further research shows that the expression of IbbHLH2 gene and the synthesis and accumulation of anthocyanin are completely synchronous in different sweet potato varieties (lines) and root development periods thereof, which indicates that IbbHLH2 is a key transcription regulation factor in the anabolism of purple sweet potato anthocyanin. Because the molecular weight of plant anthocyanin is small, the analysis of the chemical structure is relatively easy, and different metabolites can present different colors, the regulation mechanism and the signal transmission process of plant anthocyanin synthesis become an ideal system for researching plant gene expression and regulation and interaction between genes and the environment.

Disclosure of Invention

The invention aims to solve the technical problem of screening an upstream regulatory factor for promoting the expression of IbbHLH2 transcription factor of purple sweet potato.

In order to solve the technical problems, the invention firstly extracts RNA from sweet potato tuberous roots by a Trizol method, and synthesizes double-stranded cDNA by reverse transcription by a SMART technology to construct a purple sweet potato yeast single-hybrid cDNA library.

Further, DNA of purple sweet potato root tuber is used as a template, and TaKaRa high fidelity enzyme is used

Max DNA Polymerase amplifies promoter DNA fragments of IbbHLH2 with ends with different enzyme cutting sites at two ends, constructs a promoter IbbHLH2 into a pAbAi vector, and the specific sequence of a PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 is as follows:

PIbbHLH 2-F: 5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and

PIbbHLH2-R：5'-TCTCATACCACCACACCCTAGTGG-3'。

after the constructed pAbAi-PIbbHLH2 bait vector is subjected to self-activation detection, the minimum inhibitory concentration of the self-activation AbA is determined to be 300 ng/mL.

According to the invention, the bait strain is prepared into Y1HGold competent cells, library plasmids are transferred into pAbAi-PIbbHLH2 induced competent cells, binding proteins are screened through a yeast single hybrid screening library, and an upstream regulatory factor IbEBF2 expressed by IbbHLH2 gene of purple sweet potato is obtained through screening.

Therefore, the first purpose of the invention is to provide an upstream regulatory factor IbEBF2 of the IbbHLH2 transcription factor of purple sweet potato, the amino acid sequence of which is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide a coding gene of the upstream regulatory factor IbEBF2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.

The third purpose of the invention is to provide a recombinant vector or a recombinant bacterium containing the coding gene.

It is a fourth object of the present invention to provide a transgenic cell line or expression cassette comprising the above-described encoding gene.

The fifth purpose of the invention is to provide an amplification primer of the upstream regulatory factor IbEBF2, wherein the specific sequence of the amplification primer is as follows:

IbEBF 2-F: 5'-ATGGCGTCCAGTGATCAAACG-3' and

IbEBF2-R：5'-TTACAGGATCTGAGCAGCAGTGTT-3'。

the sixth purpose of the invention is to provide the application of the upstream regulatory factor IbEBF2 in promoting the expression of IbbHLH2 transcription factor of purple sweet potato.

The seventh purpose of the invention is to provide the application of the upstream regulatory factor IbEBF2 in promoting the biosynthesis of plant anthocyanin.

The eighth purpose of the invention is to provide the application of the upstream regulatory factor IbEBF2 in the breeding of plant high anthocyanin varieties.

Further, the plant is purple sweet potato.

The upstream regulatory factors which promote the expression of IbbHLH2 are screened from a cDNA library by a yeast single-hybridization method, including IbERF10 and IbEBF2, but the action sites of different upstream regulatory factors are different. The IbbHLH2 promoter is divided into four segments, with the IbERF10 site in the fourth segment (located behind the promoter) and the IbEBF2 site in the first segment (located in front of the promoter).

Further, in order to further verify that the selected upstream regulatory factor IbEBF2 is combined with a promoter IbbHLH2, IbEBF2 is constructed into a pGADT7 yeast recombinant expression vector, ecori and BamH i in the vector are selected as enzyme cutting sites into which target fragments are inserted, primer sequences are synthesized and shown in table 2, and a yeast single hybridization experiment is performed in a way that a pGADT7-IbEBF2 yeast recombinant expression vector plasmid and a pabali-PIbbHLH 2 bait vector are jointly transformed into Y1 hgod yeast, and the results show that: the positive control p53AbAi + AD53 transformed strain was able to grow on SD/-Leu/AbA medium, while the negative control pAbAi-PIbbHLH2+ pGADT7 no-load transformed strain was not able to grow on SD/-Leu/AbA medium. Thus, the yeast single-hybridization experiment can effectively detect whether the protein is combined on the promoter. While pAbAi-PIbbHLH2+ pGADT7-IbEBF2 was able to grow on SD/-Leu/AbA medium (FIG. 1), indicating that the IbEBF2 protein binds to the promoter IbbHLH 2.

The invention detects whether the upstream regulatory factor IbEBF2 has self-activation activity again, constructs a pGBKT7-IbEBF2 fusion expression vector, transfers the fusion expression vector into yeast Y2HGold after successful construction, uniformly coats the transformed bacterial liquid on a tryptophan defect culture medium for growth, picks up a positive single clone point to culture on a histidine defect culture medium, and shows that the yeast strain transformed by pGBKT7-IbEBF2 can grow on the histidine defect culture medium and can enable X-alpha-Gal to show blue (figure 2). Indicating that the IbEBF2 protein has self-activating activity.

Furthermore, in order to verify the interaction between the promoter IbbHLH2 and the upstream transcription factor IbEBF2, the invention constructs IbEBF2 on an overexpression vector pGreenII 002962-SK, extracts a recombinant bacterial liquid with successful sequencing, then transfers the recombinant bacterial liquid and a PIbbHLH2+ pGreenII 0800 LUC recombinant plasmid into an Arabidopsis thaliana protoplast, selects Sac I and Xho I in a pGreenII 002962-SK vector and Kpn I and Nco I in a pGreenII 0800-LUC vector as enzyme cutting sites of an inserted target fragment, and constructs vector primer sequences shown in Table 2. The results showed that IbEBF2 was able to increase the activity of IbbHLH2 promoter (fig. 3), suggesting that IbEBF2 was able to promote expression of IbbHLH 2.

Furthermore, in order to clarify the function of the upstream regulatory factor IbEBF2, the invention constructs a fusion protein of IbEBF2 and Green Fluorescent Protein (GFP) and locates the action site of IbEBF 2. By constructing a subcellular localization expression vector, selecting BamHI and HindII in the vector as enzyme cutting sites for inserting a target fragment, designing a specific primer containing an initiation codon and not containing a termination codon, and synthesizing a primer sequence shown in Table 2. After transient transformation of Arabidopsis protoplasts, subcellular localization was observed using confocal laser microscopy, pCambia1300-GFP was used as a positive control. The GFP protein in the empty vector was expressed in the respective structures of Arabidopsis protoplasts, and the IbEBF2 protein was expressed in the nucleus (FIG. 4), indicating that IbEBF2 is a typical transcription factor.

The invention has the beneficial effects that: through a yeast single hybrid library screening experiment, the upstream regulatory factor IbEBF2 is successfully obtained in the screening of the upstream regulatory factor of the promoter IbbHLH 2. The invention results can enrich and deepen the basic theory of plant anthocyanin biosynthesis molecular regulation theoretically; in application, the method can provide a new genetic marker for breeding purple sweet potato high anthocyanin varieties, screen out appropriate operating elements or modification targets for molecular breeding of the purple sweet potato high anthocyanin varieties, and meanwhile, can provide a new thought and clue for cultivation measures for improving the pigment content in purple sweet potato tuberous roots.

Drawings

FIG. 1 shows the results of gyration validation of the IbbHLH2 promoter and its upstream regulatory factor IbEBF 2. The positive control is p53AbAi + AD-53, the negative control is PIbbHLH2-1-pAbAi + AD, the positive colony (PIbbHLH2-pAbAi + IbEBF2-AD) indicates that the corresponding upstream regulatory factor protein (IbEBF2) can be combined on the IbbHLH2 promoter, and the ratio of AD: pGADT 7.

FIG. 2 shows the detection of the self-activating activity of the upstream regulatory factor IbHLH 2 promoter IbEBF 2.

FIG. 3 shows the interaction of the IbbHLH2 promoter with its upstream regulatory factor IbEBF 2.

FIG. 4 shows the subcellular localization of the IbbHLH2 promoter upstream regulatory factor IbEBF2 in Arabidopsis protoplasts (20 μm ruler). A: green fluorescence plot; b: chloroplast autofluorescence; c: a bright field map; d: and (4) overlaying the graph.

Detailed Description

The present invention will be further described with reference to the following examples, wherein the test methods in the following examples are all conventional test methods unless otherwise specified, and the test reagents and consumables described in the following examples are all available from conventional biochemical reagents company, unless otherwise specified.

Example 1: construction of purple sweet potato Yeast Single hybrid cDNA library

(1) RNA was extracted from the tuberous root of purple sweet potato (line A5) by Trizol method, and double-stranded cDNA was synthesized by reverse transcription using SMART technique.

(2) The amplified cDNA is purified with TaKaRa MiniBEST DNA Fragment Purification Kit to obtain dH₂And (4) dissolving out the O.

(3) Performing column treatment on the cDNA after enzyme digestion by the restriction enzyme SfiI, performing PCI/CI purification treatment, and finally obtaining ddH₂And (4) dissolving out the O.

(4) pGADT7-SfiI vector (clontech, cat # 630490) was ligated with the appropriate post-column cDNA using the DNA ligation Kit. Purifying and refining the connecting liquid to obtain a primary cDNA library.

(5) Transferring a small amount of primary library ligation solution into competent cells E.coli HST08 by an electrotransformation method; after the identification is correct, coating a proper amount of bacterial liquid on an LB plate containing Amp resistance, and culturing for 12h at 37 ℃; the primary library capacity was calculated by the number of colonies growing on the plate.

(6) And (4) carrying out overnight culture on the amplified colonies, and then carrying out plasmid extraction to obtain library plasmids.

Example 2: construction of pAbAi-PIbbHLH2 bait vector

(1) Taking purple sweet potato root tuber DNA as a template and TaKaRa high-protectionTrue enzyme

Max DNA Polymerase amplifies promoter DNA fragments of IbbHLH2 with different enzyme cutting site ends at two ends, and the sequence of a PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 is PIbbHLH 2-F: 5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and PIbbHLH 2-R: 5'-TCTCATACCACCACACCCTAGTGG-3' are provided. The reaction system (20. mu.L) was as follows:

the PCR reaction conditions are as follows:

(2) the promoter IbbHLH2 (the sequence is shown as SEQ ID NO. 3) is constructed into a pAbAi vector (Koehi Biotech limited, the product number is kl-zl-0879), and the steps are as follows: quickcut Using TaKaRa restriction enzyme^TMHind III and Quickcut^TMSma I carries out double digestion on the pAbAi plasmid, the sequence of a synthetic primer is shown as Pb2F/Pb2R in a table 2, the reaction condition is 37 ℃, the digestion is carried out for more than 3h, and the reaction system is as follows:

detecting the correct enzyme digestion product by electrophoresis, and cutting and recovering the gel.

The target fragment and the expression vector were ligated using Clon Express II One Step Cloning Kit (Vazyme) under 37 ℃ for 30 min. The reaction system is as follows:

(3) the ligation product was used for subsequent transformation of E.coli DH 5. alpha. competent cells.

Preparation of E.coli DH5 alpha competent cells (CaCl)₂Method):

(1) escherichia coli DH 5. alpha. was inoculated into 5mL of LB liquid medium and cultured overnight at 37 ℃ with shaking at 220 rpm.

(2) Transferring overnight cultured 2mL of the bacterial liquid to 100mL of LB liquid medium, and continuing the shaking culture to OD₆₀₀To about 0.5, and standing on ice for 30 min.

(3) 1mL of the bacterial solution was put into a new 1.5mL centrifuge tube, centrifuged at 4000rpm for 10min at 4 ℃ and the supernatant was aspirated off with a pipette.

(4) Pipette 1mL of precooled 0.1M CaCl with pipette₂Suspending, precipitating, slightly blowing, mixing, and standing on ice for 30 min.

(5) Centrifuging at 4 deg.C and 4000rpm for 10min, sucking the supernatant with a pipette, and sucking 0.2mL of precooled 0.1M CaCl with a pipette₂Suspending and precipitating, and standing on ice for 5h for transformation.

The ligation product was transformed into E.coli DH 5. alpha. competent cells:

(1)100 mu L of the prepared escherichia coli DH5 alpha competent cells are put into a new 1.5mL centrifuge tube, 10 mu L of DNA ligation product is added into an ultra-clean workbench, and the mixture is flicked, mixed evenly and placed on ice for 30 min.

(2) The conversion product was heat-shocked in a 42 ℃ water bath for 90s and removed immediately on ice for 5 min.

(3) Adding 1mL of LB liquid culture medium without resistance, and carrying out shaking culture at 37 ℃ and 180rpm for 60-90 min.

(4) Centrifuge at 5000rpm for 4min at room temperature, aspirate 900. mu.L of supernatant with pipette under sterile conditions, and resuspend the remaining 200. mu.L of liquid with gentle blowing.

(5) And uniformly coating the bacterial liquid in an LB solid culture medium containing Amp, standing for 30min and airing.

(6) And (4) carrying out inverted culture in an incubator at 37 ℃ for 12-16 h.

Screening and sequencing identification of positive clones:

resistant single colonies were picked from the culture dish with a sterile small gun head and cultured in LB liquid medium containing resistance at 37 ℃ for 4h with shaking at 220rpm, and 2. mu.L of the above-mentioned bacterial solution was taken as a template for colony PCR detection. 200 mu L of bacterial liquid of the positive strain which is amplified in the PCR reaction and has the same size with the target fragment is taken and sent to Shanghai Biotechnology Limited company for sequencing. And adding 20% of sterilized glycerol into the bacterial liquid with the correct sequencing into a 1.5mL centrifuge tube, storing the bacterial liquid in a refrigerator at the temperature of minus 80 ℃, and extracting the pAbAi-PIbbHLH2 bait plasmid.

Example 3: pAbAi-PIbbHLH2 bait strain self-activation detection

The pAbAi-PIbbHLH2 bait plasmid is transformed into Y1H yeast to obtain a bait strain. The lowest AbA concentration of the bait strain is tested by an auto-activation test to observe the growth of the bait strain on SD/-Ura solid culture medium, and the auto-activation test of the bait strain and the determination method of the lowest AbA concentration are as follows:

(1) preparation of an AbA mother solution: 1mg of AbA was dissolved in 1mL of absolute ethanol to prepare a 1mg/mL AbA stock solution, which was stored at 4 ℃ in the dark.

(2) From Y1H [ pAbAi-prey]And Y1H [ p53AbAi]The culture dish of (1) picks larger monoclonal colony, uses 10 microliter 0.9% NaCl solution to resuspend the bacterial liquid, and dilutes the resuspended solution into 10^-1、10^-2And 10^-3A concentration gradient.

(3) Pipette 10. mu.L of resuspended suspension onto SD/-Ura, SD/-Ura/AbA (100 ng/mL-1000 ng/mL) medium.

(4) If colony Y1H [ pAbAi-prey ] does not grow at a certain concentration, but the control group Y1H [ p53AbAi ] grows normally, this concentration is the lowest AbA concentration that inhibits the recombinant yeast strain and can be used in subsequent experiments.

Note: the pAbAi-prey is pAbAi-PIbbHLH 2.

The minimum inhibitory concentration of the self-activating AbA of the pAbAi-PIbbHLH2 bait strain is 300 ng/mL.

Example 4: yeast single hybrid library screening

The Screening method of the Yeast single-Hybrid Library was performed according to the instructions of the Matchmaker Gold Yeast One-Hybrid Screening System of Clontech. The screening method of the yeast single hybrid library comprises the following steps:

(1) 25 μ L of Yeastmaker Carrier DNA was denatured in a water bath at 95 ℃ for 5min, quickly placed on ice for several minutes, and allowed to cool to 4 ℃ (repeated once).

(2) The following were added sequentially to a 10mL centrifuge tube which had been pre-cooled: 2.5mL PEG/LiAc, 25. mu.L denatured Yeastmaker Carrier DNA, 15. mu.g library plasmid (obtained in example 1), 600. mu. L Y1HGold competent cells (containing the bait expression vector pAbAi-PIbbHLH2), vortexed and mixed.

(3) Placing the centrifuge tube in 30 deg.C water bath for 45min, and gently mixing back and forth several times every 15 min.

(4) Add 160. mu.L DMSO, mix gently.

(5) The mixture was incubated in a 42 ℃ water bath for 20min, during which time it was gently mixed back and forth several times every 10 min.

(6) Centrifuging at 12000rpm for 30s, collecting bacterial liquid, discarding supernatant, adding 8mL of 0.9% NaCl solution, and resuspending the bacteria.

(7) 200. mu.L of the transformed yeast liquid was aspirated and uniformly spread on SD/-Leu and SD/-Leu/AbA plates, and the concentration of AbA was the lowest concentration for inhibiting self-activation (i.e., 300 ng/mL).

(8) And (3) carrying out inverted culture in an incubator at 30 ℃ for 48-96 h.

Selecting a single colony for colony PCR identification, wherein the identification method refers to example 2, selecting a universal primer pGADT7F/R for PCR detection of bacterial liquid, the sequence of the pGADT7F/R primer is shown in Table 1, selecting a sample which is brighter after electrophoresis and has a single band, sending the sample to Shanghai Biometrics Limited company for sequencing, and performing BLAST on the sequencing result in an NCBI database to analyze the sequencing result.

TABLE 1 general primers for Primary vectors

The upstream regulatory factor IbbHLH2 gene expression of the purple sweet potato is IbEBF2 obtained by screening through a yeast single hybridization method, the amino acid sequence of the upstream regulatory factor IbEBF2 is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2. And an amplification primer of an upstream regulatory factor IbEBF2 is designed to be IbEBF 2-F: 5'-ATGGCGTCCAGTGATCAAACG-3' and IbEBF 2-R: 5'-TTACAGGATCTGAGCAGCAGTGTT-3' are provided.

Example 5: verification of binding of upstream regulatory factor IbEBF2 and promoter IbbHLH2

IbEBF2 was constructed into pGADT7 yeast recombinant expression vector (Shanghai Linmai bioengineering Co., Ltd., product No. LM-1639), EcoRI and BamHI in the vector were selected as the restriction sites into which the target fragment was inserted, the synthetic primer sequences are shown in IbEBF2-ADF/IbEBF2-ADR in Table 2, and the construction method is referred to example 2. pGADT7-IbEBF2 yeast recombinant expression vector plasmid and pAbAi-PIbbHLH2 bait vector were co-transformed into Y1HGold single-hybrid yeast strain for yeast single-hybrid experiments.

(2) The following were added sequentially to a 10mL centrifuge tube which had been pre-cooled: 2.5mL PEG/LiAc, 25. mu.L denatured Yeastmaker Carrier DNA, 15. mu.g library plasmid, 600. mu. L Y1HGold competent cells, vortexed and mixed.

(4) Add 160. mu.L DMSO, mix gently.

Selecting a single colony for colony PCR identification, selecting a universal primer pGADT7F/R (the sequence is shown in table 1), selecting a sample which is brighter after electrophoresis and has a single band, sending the sample to Shanghai Biometrics Limited company for sequencing, and performing BLAST on the sequencing result in an NCBI database to analyze the sequencing result.

As a result, it was found that: the positive control p53AbAi + AD-53 (i.e., inserting the positive control 53 gene sequence into the pAbAi vector to obtain p53AbAi, inserting the positive control 53 gene sequence into the pGADT7 vector to obtain AD-53, the construction method refers to example 2) transformed strain can grow on SD/-Leu/AbA culture medium; while the negative control pAbAi-PIbbHLH2+ pGADT7 no-load transformed strain was able to grow on SD/-Leu/AbA medium; thus, the yeast single-hybridization experiment can effectively detect whether the protein is combined on the promoter. pAbAi-PIbbHLH2+ pGADT7-IbEBF2 was able to grow on SD/-Leu/AbA medium (FIG. 1), indicating that the IbEBF2 protein could bind to the promoter IbbHLH 2.

Example 6: detecting the self-activating activity of an upstream regulatory factor IbEBF2

IbEBF2 was introduced into pGBKT7 plasmid (Shanghai Linmai bioengineering Co., Ltd., cat No. LM-8123), pGBKT7-IbEBF2 fusion expression vector was constructed, EcoR I and BamH I in the vector were selected as the enzyme cleavage sites into which the objective fragment was inserted, synthetic primer sequences were shown in IbEBF2-BDF/IbEBF2-BDR in Table 2, and the construction method was referred to in example 2. After the construction is successful, the fusion expression vector is transferred into a Y2HGold yeast strain, the transformed bacterial liquid is uniformly coated on a tryptophan-deficient culture medium (Takara Cat #630413) to grow, a positive single cloning point is selected to be cultured on a histidine-deficient culture medium (namely SD/-His/ABA/X-alpha-Gal plus culture medium, Takara Cat #630415), and the result shows that the yeast strain transformed by pGBKT7-IbEBF2 can grow on the histidine-deficient culture medium and can enable X-alpha-Gal to show blue (figure 2). The above results indicate that the IbEBF2 protein has self-activating activity.

Example 7: construction of Dual-luciferase reporter vectors

The gene sequence of an upstream regulatory factor IbEBF2 is constructed on an overexpression vector pGreenII 002962-SK (Shanghai Qincheng Biotech Co., Ltd., product number QCP0465), which is called an effector plasmid, and PIbbHLH2 is inserted into the front end of a vector pGreenII 0800-LUC (Kohlei Biotech Co., Ltd., product number kl-zl-0808) luciferase to be used as a reporter plasmid. Sac I and Xho I in pGreenII 002962-SK vector and Kpn I and Nco I in pGreenII 0800-LUC vector were selected as restriction sites for inserting the objective fragment, vector primer sequences for construction were IbPb2-0800F/IbPb2-0800R and IbEBF2-62-SKF/IbEBF2-62-SKR in Table 2, and the construction method was as in reference example 2.

Example 8: preparation and transformation of Arabidopsis protoplasts

1. The preparation steps of the arabidopsis protoplast are as follows:

(1) preparing enzymolysis liquid, and preheating in a 55 ℃ water bath kettle.

(2) Selecting wild type Arabidopsis leaves before bolting after four weeks, tearing off the lower epidermis of the leaves, and quickly putting the leaves into the enzymolysis solution.

(3) Carrying out enzymolysis for 50min at 25 deg.C under 50rpm in dark condition, observing the form of protoplast under microscope, and obtaining better cell state when the cells are round and bright.

(4) Diluting the enzyme solution with an equal volume of W5 solution, gently mixing, washing with clear water to remove a 75 μm nylon mesh, soaking with W5 solution, and filtering to obtain protoplast.

Preparation of W5 solution (100 mL):

(5) centrifuge at 800rpm for 2min, aspirate the supernatant as much as possible, and resuspend the protoplasts with 1mL of W5 solution (repeat this step three times).

(6) The protoplasts were resuspended in 1mL of W5 solution and then placed on ice for 30 min.

2. The transformation procedure for arabidopsis protoplasts was as follows:

(1) 10-20. mu.g of the target plasmid (IbEBF 2-pGreenII 002962-SK recombinant plasmid and PIbbHLH2-pGreenII 0800-LUC recombinant plasmid constructed in example 7) was added to a 2mL EP tube, 100. mu.L of Arabidopsis protoplast was added thereto, the mixture was gently mixed, and the mixture was immediately put on ice after the addition.

(2) Add 110. mu.L PEG/CaCl₂Flicking the centrifugal tubeMix well and incubate at room temperature for 10 min.

(3) Add 220. mu. L W5 solution to ice, invert the tube and mix well, and leave on ice for 1 min.

(4) The 440. mu. L W5 solution was added to the tube again, turned upside down, and placed on ice for 1 min.

(5) Finally, 880. mu. L W5 solution was added to the tube, the mixture was inverted and placed on ice for 1 min.

(6) Centrifuge at 800rpm for 3min at 4 ℃ and aspirate the supernatant.

(7) The protoplast is resuspended in 500 mu L W5 solution and cultured for 16-20 h at 22 ℃ in the dark.

Example 9: detection of Dual-luciferase reporter System

Use Dual-

Reporter Assay (Promega) detects the activity of two luciferases LUC and REN, and comprises the following steps:

(1) preparation of 100. mu.L of 1 XPLB lysate: 20 μ L of 5 XPassive lysine Buffer was added to 100 μ L of water. Preparation of 10mL LAR II: 10mL of ice-thawed Luciferase Assay Buffer II was pipetted into Luciferase Assay Substrate and gently shaken to dissolve it (-20 ℃ for one month, -70 ℃ for one year). 100 μ L Stop&

Preparation of Reagent: aspirate 100. mu.L of Stop&

Buffer, add 2. mu.L of 50 × Stop&

Substrate, which was mixed by vortexing slightly (15 d at-20 ℃).

(2) The transformed Arabidopsis protoplast solution of example 8 was centrifuged at 13200rpm at 4 ℃ for 90 seconds to remove the W5 solution.

(3) Add 100. mu.L of 1 XPLB lysate, gently blow and mix well, transfer to 24-well plate, place in horizontal shaking table, shake 15min at room temperature and low speed.

(4) And (3) collecting the lysate, transferring the lysate to a 1.5mL centrifuge tube, centrifuging the lysate for 10min at the temperature of 4 ℃ and the rpm of 13200, and taking 60 mu L of supernatant to place in ice to obtain luciferase to be detected.

(5) And under the condition of keeping out of the sun, adding 100 mu L of LAR II into a black 96-hole enzyme label plate, then adding 20 mu L of cell lysate, and gently mixing the mixture for 2-3 times by using a gun head to avoid generating bubbles.

(6) The LUC was placed in a microplate reader to detect the enzyme activity and the data was recorded.

(7) The 96-well plate was removed and 100. mu.L of Stop was added to the same well&

Reagent, mixing gently for 2-3 times by using a gun head, and avoiding strong light irradiation in the whole operation process.

(8) The enzyme activity of REN was detected in a microplate reader and the data recorded.

(9) The experiment is repeated for 3 times, the average value is taken, and the activation effect of the transcription factor on the promoter is detected by comparing the ratio of LUC/REN of different samples.

The results show that: IbEBF2 was able to increase the activity of the IbbHLH2 promoter (FIG. 3), indicating that IbEBF2 was able to promote expression of IbbHLH 2.

Example 10: the function of an upstream regulatory factor IbEBF2 is clarified

A fusion protein of IbEBF2 and Green Fluorescent Protein (GFP) is constructed, and the action site of IbEBF2 is located. The gene sequence of the upstream regulatory factor IbEBF2 is constructed on a subcellular localization expression vector pCambia1300 (Shanghai Linmai bioengineering Co., Ltd., product No. LM1375), BamHI and HindII in the pCambia1300 vector are selected as enzyme cutting sites for inserting a target fragment, specific primers containing an initiation codon and no stop codon are designed, the sequence of the synthesized primers is shown in IbEBF2-1300F/IbEBF2-1300R in Table 2, and the construction method is referred to example 2. Then IbEBF2-pCambia1300 was transiently transformed into Arabidopsis protoplasts, and subcellular localization was observed using confocal laser microscopy, using pCambia1300-GFP as a positive control. The GFP protein in the empty vector was expressed in the respective structures of Arabidopsis protoplasts, and the IbEBF2 protein was expressed in the nucleus (FIG. 4), indicating that IbEBF2 is a typical transcription factor.

TABLE 2 construction of vectors Using primer sequences (restriction sites underlined)

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Sequence listing

<110> research institute of south seed reproduction of academy of sciences of Guangdong province

<120> upstream regulatory factor IbEBF2 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 286

<212> PRT

<213> sweet potato (Ipomoea batatas)

<400> 1

Met Ala Ser Ser Asp Gln Thr Val Leu Gln Ile Ser Ser Pro Ser Ser

1 5 10 15

Thr Thr Leu Ser Ala Arg Val His Pro Leu Val Ile Phe Asn Ile Cys

20 25 30

Asp Cys Phe Val Arg Arg Pro Asp Gln Ala Glu Arg Val Ile Gly Thr

35 40 45

Leu Leu Gly Ser Val Leu Pro Asp Gly Thr Val Asp Ile Arg Asn Ser

50 55 60

Tyr Ala Val Pro His Asn Glu Ser Gln Asp Gln Val Ala Leu Asp Ile

65 70 75 80

Asp Tyr His His Asn Met Leu Ala Ser His Gln Lys Val Asn Pro Lys

85 90 95

Glu Val Ile Val Gly Trp Phe Ser Thr Gly Phe Gly Val Ser Gly Gly

100 105 110

Ser Ala Leu Ile His Asp Phe Tyr Thr Arg Glu Val Thr Asn Pro Ile

115 120 125

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

130 135 140

Ala Phe Ile Ser Val Asn Leu Ser Leu Gly Asp Gln Pro Leu Ala Ala

145 150 155 160

Gln Phe Gln Glu Ile Pro Leu Asp Leu Arg Met Ile Glu Ala Glu Arg

165 170 175

Val Gly Phe Asp Met Leu Lys Thr Thr Val Val Asp Lys Leu Pro Asn

180 185 190

Asp Leu Glu Gly Met Glu Ala Ser Met Glu Arg Leu Leu Ala Leu Ile

195 200 205

Asn Asp Val His Lys His Val Asp Asp Val Val Glu Gly Arg Val Pro

210 215 220

Ala Asp Asn Asn Leu Gly Arg Leu Ile Ser Glu Thr Val Asn Ser Ile

225 230 235 240

Pro Lys Leu Ser Pro Gln Glu Phe Asp Lys Leu Val Asn Asp Ser Leu

245 250 255

Gln Asp Gln Leu Leu Leu Leu Tyr Leu Ser Ser Ile Thr Arg Thr Gln

260 265 270

Leu Ser Leu Ala Glu Lys Leu Asn Thr Ala Ala Gln Ile Leu

275 280 285

<210> 2

<211> 861

<212> DNA

<213> sweet potato (Ipomoea batatas)

<400> 2

atggcgtcca gtgatcaaac ggtgctccag atttcgtctc cttcttcaac aaccctctcc 60

gctagggttc acccgctggt gattttcaac atctgcgact gctttgtccg gcgacccgac 120

caagccgagc gcgtcattgg tacgcttctc ggatccgtct tacccgacgg caccgtcgat 180

attcgcaact cctatgccgt tcctcacaac gagtcccaag atcaggttgc tttggatatt 240

gattatcatc ataacatgtt ggcatcccat cagaaagtga atcctaagga agtcattgtt 300

ggatggtttt ccactgggtt tggagtttca ggcggtagcg ctctaatcca tgatttttac 360

actagagaag ttacaaatcc tatccatttg actgttgaca ctggattcac aaatggggag 420

gctaccatca aagcttttat ttctgtgaat ttgtcacttg gggatcaacc tcttgctgca 480

cagttccaag aaattccatt ggacttgcga atgattgaag ctgagcgggt tggatttgat 540

atgctgaaga caacagtggt tgacaaactt ccaaatgacc tagaaggaat ggaggcatca 600

atggagagat tacttgctct gatcaatgat gttcacaaac atgttgatga tgttgtggaa 660

ggtcgtgttc cagcagacaa taaccttgga agacttatat ctgagaccgt aaactctatt 720

ccaaaactat caccacaaga atttgataag cttgtgaatg acagtcttca ggatcaattg 780

ctcctactat atttgtcgag catcacaaga acacaactca gcttggctga aaagttgaac 840

actgctgctc agatcctgta a 861

<210> 3

<211> 1021

<212> DNA

<213> sweet potato (Ipomoea batatas)

<400> 3

cggccgcttt attggtgaag ggtaagacaa gaatttgtag ccacgcataa cttataatct 60

taagtatgat gatcatattt tattcattaa gctactcttt ttggagtaac catatcatgt 120

ttgtctaatt tttcatttaa tagctagaga gaatatttaa agatcataaa tttgattttt 180

gtcaaaattt tcttagtcga tagctgcaca aaattttctt agtgtagttt acctctgcag 240

tgtggtctgc aagttgttac atattaataa aatttattca aataaactct cagataataa 300

aatggggttt cttataaaaa aaataaaaat taaaaataaa ctataggaac agaaaatagg 360

catactcttg ataaataaga gatgatgaag aattgaagac ctcttgtgcg cagagtgcac 420

aagtaagcga tgaaaacttg aagacagata aggcaactta caatttacct ctactagaaa 480

ttcttaggta gtatgtggtt ctgaacgttt agaattaatt agagttgtcg catggtagct 540

tgatcaattg gtcacatgtg tgaagtttgg aagaagaaat caaggatcga gtctcatcag 600

tggcaatgta ggagcaaccc cttaaagtga gggggtcctt gtgcctggtt tagtccactg 660

aggctcaaat ccacccccat ccaccccata tgaaggtgaa accgggtgtc actaaatcac 720

aagtctttga cagaagaatt aaatagagct gaaaaaccta actaaattta taataaacaa 780

aaaaaaaaaa gtgacactag tatagtaata tgatacacgt gggacacata aaaggcaagg 840

acaaaaacct aatcttgaat tctcctattt tgtcgtctct ttcccagtcc caatacccga 900

ccggttgaca ccaaccagtc aaaatccaac tccccgacaa acaaataaat tcaaccttaa 960

cccctctccg gtctgcaact cttaatttca tatgtaaacc actctgtctc acatctttcc 1020

c 1021

Claims

1. An upstream regulatory factor IbEBF2 of IbbHLH2 transcription factor of Ibby sweetpotato, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. A gene encoding the upstream regulatory factor IbEBF2 of the IbbHLH2 transcription factor of Ibber sweetpotato of claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

4. A recombinant vector or a recombinant bacterium comprising the coding gene according to claim 2 or 3.

5. A transgenic cell line or expression cassette comprising the encoding gene of claim 2 or 3.

6. The amplification primer of the upstream regulatory factor IbEBF2 of the purple sweet potato IbbHLH2 transcription factor of claim 1, which is characterized in that the amplification primer is IbEBF 2-F: 5'-ATGGCGTCCAGTGATCAAACG-3' and IbEBF 2-R: 5'-TTACAGGATCTGAGCAGCAGTGTT-3' are provided.

7. The use of the upstream regulatory factor IbEBF2 of claim 1 for promoting the expression of IbbHLH2 transcription factor of Ipomoea batatas.

8. The use of the upstream regulatory factor IbEBF2 of claim 1 for promoting plant anthocyanin biosynthesis.

9. The use of the upstream regulatory factor IbEBF2 in the breeding of plant high anthocyanin varieties.

10. The use according to claim 8 or 9, wherein the plant is sweetpotato purple.