CN114085276B - Upstream regulatory factor IbERF10 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato - Google Patents
Upstream regulatory factor IbERF10 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato Download PDFInfo
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Abstract
The invention discloses an upstream regulatory factor IbERF10 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 IbERF10 of the IbbHLH2 gene through a yeast single hybrid library screening experiment. The interaction between the IbbHLH2 promoter and the upstream regulatory factor IbERF10 is proved by using a yeast single-hybridization rotation experiment and a dual-luciferase report system for detection. Subcellular localization results showed that IbERF10 localized to the nucleus. The results of the self-activating activity experiment show that IbERF10 has 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
Technical Field
The invention relates to the technical field of plant gene breeding, in particular to an upstream regulatory factor IbERF10 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potatoes.
Background
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.
In maize, the regulator genes, the leaf colour gene and the colour gene, which influence anthocyanin synthesis, act to regulate the expression of structural genes. Researches find that Lc genes have strong activation effects on DFR, F3'5' H, ANS and UF3GT and play an important role in anthocyanin synthesis pathways. After the Lc gene of corn is cloned, genes for encoding bHLH protein, such as DELILA gene for regulating the color of a flower crown of snapdragon, JAF13 and AN1 genes for regulating the color of petunia hybrida flowers, TT8 gene for regulating the color of arabidopsis seed coats and the synthesis of silique proanthocyanidins and the like, are found in sequence in other plants, and all the regulating genes regulate the expression of key enzyme genes in the downstream of the anthocyanin synthesis pathway. In blood oranges, the CsMYC2 protein was able to up-regulate the expression of UFGT in leaves, thereby darkening the color of the leaves. In petunia hybrida of Convolvulaceae, a transcription regulation gene IpIVS (IpbHLH2) can regulate and control the expression of related structural genes in an anthocyanin synthesis pathway, the regulation and control effects on DFR-B and ANS are most obvious, and the expression quantity of DFR-B and ANS is almost zero in a seed coat of a bHLH2 expression-deficient plant.
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 regulatory 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 DNApolymerase amplifies promoter DNA fragments of IbbHLH2 with ends with different enzyme cutting sites at two ends, a promoter IbbHLH2 is constructed into a pAbAi vector, and the specific sequence of a PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 is shown as follows:
PIbbHLH 2-F: 5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and
PIbbHLH2-R：5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-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 700 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 IbERF10 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 IbERF10 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 IbERF10, 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 IbERF10, wherein the specific sequence of the amplification primer is as follows:
IbERF 10-F: 5'-ATGGCTCCCAAGGAGAAGGG-3' and
IbERF10-R：5'-TTAGAGAGCGCCGGTTCCG-3'。
the sixth purpose of the invention is to provide the application of the upstream regulatory factor IbERF10 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 IbERF10 in promoting the biosynthesis of plant anthocyanin.
The eighth purpose of the invention is to provide the application of the upstream regulatory factor IbERF10 in the breeding of plant high anthocyanin varieties.
Further, the plant is purple sweet potato.
Further, in order to further verify that the selected upstream regulatory factor IbERF10 is combined with a promoter IbbHLH2, the IbERF10 is constructed into a pGADT7 yeast recombinant expression vector, and pGADT7-IbERF10 yeast recombinant expression vector plasmid and a pAbAi-PIbbHLH2 bait vector are jointly transformed into Y1HGold yeast to carry out a yeast single hybridization experiment, 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-IbERF10 was able to grow on SD/-Leu/AbA medium (FIG. 1), indicating that the IbERF10 protein could bind to the promoter IbbHLH 2.
The invention detects whether the upstream regulatory factor IbERF10 has self-activation activity again, constructs a pGBKT7-IbERF10 fusion expression vector, transfers the fusion expression vector into yeast Y2HGold after successful construction, evenly 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-IbERF10 can grow on the histidine defect culture medium and can enable X-alpha-Gal to show blue (figure 2). Indicating that the IbERF10 protein has self-activating activity.
Furthermore, in order to verify the interaction between the promoter IbbHLH2 and the upstream transcription factor IbERF10, the invention constructs the IbbERF 10 on an overexpression vector pGreenII 002962-SK, extracts plasmids from the recombinant bacterial liquid successfully sequenced, and then transfers the plasmids and the PIbbHLH2+ pGreenII 0800 LUC recombinant plasmids into an Arabidopsis protoplast. The results showed that IbERF10 can increase the activity of IbbHLH2 promoter (FIG. 3), indicating that IbERF10 can promote the expression of IbbHLH 2.
Furthermore, in order to clarify the function of the upstream regulatory factor IbERF10, the invention constructs a fusion protein of IbERF10 and Green Fluorescent Protein (GFP) and locates the action site of IbERF 10. The subcellular localization condition is observed by using a laser confocal microscope after an arabidopsis protoplast is transiently transformed by constructing a subcellular localization expression vector, and pCambia1300-GFP is used as a positive control. The GFP protein in the empty space can be expressed in each structure of the Arabidopsis protoplast, and the IbERF10 protein is expressed in the nucleus (FIG. 4), which shows that IbERF10 is a typical transcription factor.
The invention has the beneficial effects that: through a yeast single hybrid library screening experiment, the upstream regulatory factor IbERF10 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.
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FIG. 1 shows the results of gyration validation of the IbbHLH2 promoter and its upstream regulatory factor IbERF 10. The positive control is p53AbAi + AD-53, the negative control is PIbbHLH2-pAbAi + AD, the positive colony (PIbbHLH2-1-pAbAi + IbERF10-AD) indicates that the corresponding upstream regulatory factor protein (IbERF10) 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 IbERF 10.
FIG. 3 shows the interaction of the IbbHLH2 promoter with its upstream regulatory factor IbERF 10.
FIG. 4 shows the subcellular localization of the IbbHLH2 promoter upstream regulatory factor IbERF10 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 dH 2 And (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 ddH 2 And (4) dissolving out the O.
(4) pGADT7-SfiI vector (clontech, cat # 630490) was ligated with the appropriate post-column cDNA using DNA ligationKit. 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) Takes purple sweet potato root tuber DNA as a template and uses TaKaRa high fidelity enzyme
Max DNApolymerase amplifies promoter DNA fragments of IbbHLH2 with ends with different enzyme cutting sites at two ends, and the sequence of a PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 is PIbbHLH 2-F: 5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and PIbbHLH 2-R: 5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-3' is added. 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 TM Hind III and Quickcut TM Sma 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) 2 Method):
(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 OD 600 When the temperature is about 0.5, the mixture is placed 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) Aspirate 1mL of precooled 0.1M CaCl with a pipette 2 Suspending, 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 pipette 2 Suspending 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 -2 And 10 -3 A 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 700 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 with gentle mixing 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., 700 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 of IbbHLH2 gene expression obtained by screening through a yeast single hybridization method is IbERF10, the amino acid sequence of the upstream regulatory factor IbERF10 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 IbERF10 is designed to be IbERF 10-F: 5'-ATGGCTCCCAAGGAGAAGGG-3' and IbERF 10-R: 5'-TTAGAGAGCGCCGGTTCCG-3' are provided.
Example 5: verification of binding of upstream regulatory factor IbERF10 and promoter IbbHLH2
IbERF10 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 IbERF10-ADF/IbERF10-ADR in Table 2, and the construction method is referred to example 2. pGADT7-IbERF10 yeast recombinant expression vector plasmid and pAbAi-PIbbHLH2 bait vector were co-transformed into Y1HGold single hybrid yeast strain for yeast single hybrid experiments.
The Screening method of the Yeast single-Hybrid Library was carried out according to the protocol 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, 600. mu. L Y1HGold competent cells, 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., 700 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, 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-IbERF10 was able to grow on SD/-Leu/AbA medium (FIG. 1), indicating that the IbERF10 protein could bind to the promoter IbbHLH 2.
Example 6: detecting the self-activating activity of an upstream regulatory factor IbERF10
IbERF10 was introduced into pGBKT7 plasmid (Shanghai Linmai bioengineering Co., Ltd., cat No. LM-8123), pGBKT7-IbERF10 fusion expression vector was constructed, EcoR I and BamH I in the vector were selected as the enzyme cleavage sites into which the target fragment was inserted, synthetic primer sequences were shown in IbERF10-BDF/IbERF10-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-IbERF10 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 IbERF10 protein has self-activating activity.
Example 7: construction of Dual-luciferase reporter vectors
The gene sequence of an upstream regulatory factor IbERF10 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 are selected as the restriction enzyme cutting sites of the inserted target fragment, the sequences of the primers for constructing the vector are shown in IbPb2-0800F/IbPb2-0800R and IbERF10-62-SKF/IbERF10-62-SKR in Table 2, and the construction method is referred to 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) Vibrating and performing enzymolysis at 25 deg.C and 50rpm in dark for 50min until mesophyll cells are completely enzymolyzed, observing the form of protoplast under microscope, and obtaining better 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 (IbERF 10-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, the mixture was gently mixed, and the mixture was immediately placed on ice after the addition.
(2) Add 110. mu.L PEG/CaCl 2 Flick the tube and mix it evenly, incubate it for 10min at room temperature.
(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 of
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: mu.L of 5 XPassive lysine Buffer was added to 100. mu.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: IbERF10 was able to increase the activity of the IbbHLH2 promoter (FIG. 3), suggesting that IbERF10 could promote the expression of IbbHLH 2.
Example 10: the function of an upstream regulatory factor IbERF10 is clarified
Constructs the fusion protein of IbERF10 and Green Fluorescent Protein (GFP) and locates the action site of IbERF 10. The gene sequence of the upstream regulatory factor IbERF10 is constructed on a subcellular localization expression vector pCambia1300 (Shanghai Lianmai bioengineering Co., Ltd., product No. LM1375), BamHI and HindII in the pCambia1300 vector are selected as enzyme cutting sites for inserting a target fragment, a specific primer containing an initiation codon and not containing a stop codon is designed, the sequence of the synthesized primer is shown in IbERF10-1300F/IbERF10-1300R in Table 2, and the construction method is referred to example 2. After the Arabidopsis protoplast was transiently transformed with IbERF10-pCambia1300, subcellular localization was observed using confocal laser microscopy, and pCambia1300-GFP was used as a positive control. The GFP protein in the empty space can be expressed in each structure of the Arabidopsis protoplast, and the IbERF10 protein is expressed in the nucleus (FIG. 4), which shows that IbERF10 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 IbERF10 and application thereof in regulation and control of IbbHLH2 expression of purple sweet potato
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 260
<212> PRT
<213> sweet potato (Ipomoea batatas)
<400> 1
Met Ala Pro Lys Glu Lys Gly Thr Ala Val Ala Val Ala Ala Ala Lys
1 5 10 15
Val His Glu Ile Arg Lys Glu Gln Val His Phe Arg Gly Val Arg Lys
20 25 30
Arg Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Gly Lys Lys
35 40 45
Ser Arg Val Trp Leu Gly Thr Phe Asp Thr Ala Glu Glu Ala Ala Arg
50 55 60
Ala Tyr Asp Lys Ala Ala Arg Glu Phe Arg Gly Ser Lys Ala Lys Thr
65 70 75 80
Asn Phe Pro Val Pro Glu Asp Leu Leu Pro Leu Asn Phe Ala Ala Gln
85 90 95
Leu His Val Lys Asn Ser Val Cys Glu Lys Asn Ala Gly Gly Ser Pro
100 105 110
Ser Pro Ser Ser Thr Val Glu Ser Ser Ser Gly Gly Gly Gly Gly Gly
115 120 125
Cys Gly Arg Ile Ser Pro Ala Val Met Ala Asp Ser Ser Pro Leu Asp
130 135 140
Leu Ser Leu Gly Gly Arg Ala Pro Val Met Phe Pro Ile Gln Asn His
145 150 155 160
Pro Gln Arg Val Val His Pro Met Val Pro Gly Ala Tyr Pro Ala Gly
165 170 175
Val Tyr Pro Val Ser Gln Ile Val Tyr Leu Asn Ala Leu Ala Arg Ala
180 185 190
Val Ala Val Asn Pro Pro Leu Asn Arg Gln Thr Glu Thr Leu Asn Phe
195 200 205
Phe Ala Pro Asn Ala Asn Cys Gly Asp His Ser Asp Ser Asp Ser Ser
210 215 220
Ser Val Ile Asp Leu Asn Ala Gly Tyr Val Glu Pro Arg Lys Gly Gly
225 230 235 240
Pro Ile Asn Asn Phe Asp Leu Asn His Pro Pro Pro Glu Ser Leu Gly
245 250 255
Thr Gly Ala Leu
260
<210> 2
<211> 783
<212> DNA
<213> sweet potato (Ipomoea batatas)
<400> 2
atggctccca aggagaaggg gacggcggtg gcggttgcgg cggcgaaagt acatgagatt 60
cggaaggagc aggtgcattt taggggcgtg aggaagaggc cgtgggggag atatgcggcg 120
gagattaggg atcccgggaa gaagagccgc gtttggctcg ggacgtttga tacggcggag 180
gaggcggcta gggcgtacga taaggcggcg agggagtttc ggggttccaa ggcgaagact 240
aattttccgg tgccggagga tctccttccg ctcaatttcg ccgcgcaatt gcacgtgaag 300
aatagcgtgt gtgagaagaa cgccggcggg agtccgagcc cgagcagtac tgttgagtcg 360
tcgagcggcg gcggcggcgg aggctgtggt cgtatctcgc cggcggtgat ggcggactcg 420
tcgcctctgg atctaagcct aggcggccgt gcgccggtga tgtttccgat acagaatcac 480
ccccagcgcg tggttcatcc catggttccc ggcgcgtacc cggccggggt ctatcccgtt 540
agccagattg tttatctcaa cgcgctcgcc cgtgccgtgg cggtgaaccc tccgctcaac 600
cggcaaaccg agacgctcaa tttcttcgcc cccaacgcca actgtgggga ccacagcgac 660
tccgactctt catcggtcat tgacttgaac gccggctatg tcgaaccgcg aaagggtggg 720
cccattaaca atttcgatct caaccaccca ccgccagaga gtctcggaac cggcgctctc 780
taa 783
<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 (7)
1. An upstream regulatory factor IbERF10 of IbbHLH2 transcription factor of purple sweet potato, the amino acid sequence of which is shown in SEQ ID NO. 1.
2. A gene encoding the upstream regulatory factor IbERF10 of the transcription factor IbbHLH2 of purple sweet potato 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. An expression cassette comprising the coding gene of claim 2 or 3.
6. The amplification primer of the upstream regulatory factor IbHLH 2 transcription factor IbERF10 of the purple sweet potato IbbHLH2, which is characterized in that the amplification primer is IbERF 10-F: 5'-ATGGCTCCCAAGGAGAAGGG-3'
And IbERF 10-R: 5'-TTAGAGAGCGCCGGTTCCG-3' is added.
7. The use of the upstream regulatory factor IbERF10 in promoting the expression of IbbHLH2 transcription factor of purple sweet potato.
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Application publication date: 20220225 Assignee: Guangzhou Chengfeng Ecological Farm Co.,Ltd. Assignor: Nanfan Seed Industry Research Institute Guangdong Academy of Sciences Contract record no.: X2023980035449 Denomination of invention: Upstream regulatory factor IbERF10 and its application in regulating the expression of IbbHLH2 in purple heart sweet potatoes Granted publication date: 20220916 License type: Common License Record date: 20230511 |