CN116063430B - Purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 and application thereof - Google Patents
Purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 and application thereof Download PDFInfo
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Abstract
The invention discloses a purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 and application thereof. According to the invention, a purple sweet potato strain A5 is used as an experimental material, a promoter sequence of IbbHLH2, ibMYB1 or IbWD40 is cloned, and an upstream regulatory factor IbPGP19 with regulatory effects on IbbHLH2, ibMYB1 and IbWD40 genes is obtained through a yeast single hybridization library screening experiment. The interaction between the IbbHLH2, ibMYB1 or IbWD40 promoter and the upstream regulatory factor IbPGP19 is confirmed by a yeast single hybridization rotation experiment and a double luciferase reporting system detection. The invention can enrich and deepen the basic theory of the molecular regulation of the biosynthesis of plant anthocyanin in theory, and is also expected to provide new ideas and clues for cultivation measures for improving the pigment content in purple sweet potato tuberous roots.
Description
Technical Field
The invention relates to the field of molecular mechanisms of plant genetics and mutation, in particular to a purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 and application thereof.
Background
Transcription factors regulating anthocyanin biosynthesis are mainly of three types: MYB, bHLH, WD40. The MYB transcription factor family refers to a transcription factor containing MYB structural domains, and is the largest family of plant transcription factors. The MYB transcription factor family, which is the most abundant class of transcription factors, plays an important role in the overall growth and development process of plants. It is involved not only in the signal transduction process in response to plant hormones and environmental factors (Carr and Kim, 2002), in cell differentiation and cell cycle regulation (Dubos et al, 2010), but also in important regulation of physiological processes such as plant secondary metabolism and anthocyanin formation (ilewellyn et al, 2008), coloration of the pericarp and pulp (Kobayashi et al, 2002), morphogenesis of organs such as petal leaves and root hairs (Zhang et al, 2009;Schellmann et al, 2002), and stress response (Kazuko et al, 1995;Ambawat et al, 2013). Among them, regulation of plant anthocyanin formation is one of its most important functions (Liu Zhongli et al 2012). Comprehensive analysis of the MYB transcription factor gene family has been performed in different plants to find 137R 2R 3-MYBs in arabidopsis thaliana (Arabidopsis thaliana); 95 of the rice (Oryza sativa) (fester et al 2011); 100 of the oranges (Citrus sinensis) (Liu et al, 2014).
WD40 repeat proteins (WDR) are a class of proteins with a beta propeller proteome structure, the core region of which consists of 40-60 amino acid residues; repeated WD40 motifs mediate interactions between proteins, which are immobilized upon protein interactions (michtra et al 2012), whereas ligand proteins that interact with WD40 proteins were found to be predominantly MYB and bHLH-type transcription factors using yeast two-hybrid experiments (somnornpalin et al 2002). WD40 protein is involved in a variety of physiological processes in plants, including mainly plant abiotic stress, growth and development, and flavonoid synthesis (Walker et al, 1999; huang et al, 2008;Miller et al, 2016). Studies have shown that the WD40 repeat protein of petunia is encoded by AN11 and comprises 5 WD40 repeat motifs that act upstream of AN2 to regulate petunia anthocyanin synthesis (de Vetten et al, 1997). TTG1 (TRANSPARENT TESTA GLABRA 1), which is highly homologous to petunia AN11 transcription factor in arabidopsis, can interact with GL3 and form a ternary complex with PAP1 to regulate the spatiotemporal expression of structural genes in the arabidopsis anthocyanin synthesis pathway (brueggamann et al, 2010). PAC1 of maize encodes WD40 protein, aleurone layer of seeds in PAC1 mutants is free of accumulation of anthocyanins (Carey et al 2004).
bHLH (basic helix-loop-helix) transcription factor is the second largest superfamily of transcription factors in plants, next to MYB transcription factors (Jin et al, 2014). The inventor of the present invention clones the gene sequence of transcription factor IbbHLH2 in Purple Sweet potato, and uses onion epidermis transient expression method to perform subcellular localization of the expression products of these transcription factor genes, through arabidopsis transformation experiment, it is proved that the expression quantity is located in the nucleus and is consistent with the variation trend of anthocyanin content (Fu Danwen; hui Yake; li Haihang; yang Shaohua; chen yanhui; gao Feng. Molecular Cloning and Functional Analysis of the Gene and Promoter of IbbHLH2 from pulse-Fleshed Sweet potato Potato. Hortichula Turaral SCIENCE and TECHNOLOGY,2021,40 (1)).
Disclosure of Invention
The invention aims to solve the technical problem of screening an upstream regulatory factor for promoting expression of transcription factors of IbbHLH2, ibMYB1 and/or IbWD40 of purple sweet potato.
In order to solve the technical problems, the invention firstly uses Trizol method to extract RNA from sweet potato tubers, uses SMART technology to reverse transcribe and synthesize double-chain cDNA, and constructs a purple sweet potato yeast single hybrid cDNA library.
Furthermore, the purple sweet potato tuber DNA is used as a template, and TaKaRa high-fidelity enzyme is usedMax DNA Polymerase A promoter DNA fragment of IbbHLH2, ibMYB1 or IbWD40 with different restriction enzyme sites at both ends is amplified, and the promoter IbbHLH2, ibMYB1 or IbWD40 is constructed into a pAbAi vector, and the specific sequence of a PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 is as follows:
PIbbHLH2-F:5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and is provided with
PIbbHLH2-R：5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-3'。
The specific sequences of the PCR primer pairs for amplifying the promoter DNA fragment of IbMYB1 are as follows:
PIbMYB1-F:5'-TTATTACATCAAGCTAAATAAATACGATTTG-3' and is provided with
PIbMYB1-R：5'-TATATATATTGAAGGGTGTCGGAAATTC-3'。
The specific sequences of the PCR primer pairs for amplifying the promoter DNA fragment of IbWD40 are as follows:
PIbWD40-F:5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and is provided with
PIbWD40-R：5'-TCTCATACCACCACACCCTAGTGG-3'。
After self-activation detection, the constructed pAbAi-PIbbHLH2 bait carrier determines that the lowest inhibition concentration of the self-activated AbA is 700ng/mL.
After self-activation detection, the constructed pAbAi-PIbMYB1 bait carrier is determined to have the lowest inhibition concentration of self-activation AbA of 400ng/mL.
After self-activation detection, the constructed pAbAi-PIbWD40 bait carrier determines that the lowest inhibition concentration of the self-activated AbA is 300ng/mL.
The invention prepares the bait strain into Y1HGold competent cells, transfers library plasmids into pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40 competent cells, screens binding proteins through a yeast single hybridization screen library, and screens to obtain the upstream regulatory factor expressed by genes IbbPGP 19 of IbbHLH2, ibMYB1 and IbWD40 of the purple sweet potato.
Therefore, a first object of the present invention is to provide a purple sweet potato anthocyanin synthesis regulatory factor IbPGP19, 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 purple sweet potato anthocyanin synthesis regulatory factor IbPGP19, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The third object of the present invention is to provide a recombinant vector or recombinant bacterium containing the above-mentioned coding gene.
The fourth object of the present invention is to provide an expression cassette containing the above-mentioned coding gene.
The fifth object of the present invention is to provide an amplification primer of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19, wherein the specific sequence of the amplification primer is as follows:
IbPGP19-F:5'-ATGGCGAGCACAAGTTATACGA-3' and is provided with
IbPGP19-R：5'-CTAAAATACCGTGACACATGAGCC-3'。
The sixth object of the present invention is to provide an application of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 in promoting expression of sweet potato IbbHLH2, ibMYB1 and/or IbWD40 transcription factors.
The seventh object of the present invention is to provide an application of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 in promoting biosynthesis of sweet potato anthocyanin.
The eighth object of the invention is to provide the application of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 in the breeding of sweet potato high anthocyanin varieties.
The ninth object of the present invention is to provide a method for promoting synthesis of anthocyanin in sweet potato, which is to overexpress the regulatory factor IbPGP19 in sweet potato plants.
Further, to further verify that the screened regulatory factor IbPGP19 binds to the promoters IbbHLH2, ibMYB1 or IbWD40, ibPGP19 was constructed into pGADT7 yeast recombinant expression vector, ecori and bamhi were selected as cleavage sites for insertion of the desired fragment in the vector, and the synthetic primer sequences are shown in table 2. Yeast single hybridization experiments were performed on pGADT7-IbPGP19 yeast recombinant expression vector plasmids and pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40 bait vectors transformed into Y1HGold yeast, and found that: the positive control p53Abai+AD-53 transformed strain was able to grow on SD/-Leu/AbA medium, whereas the negative control PIbbHLH2-1-pAbai+pGADT7, PIbMYB1-4-pAbai+pGADT7 or PIbWD40-1-pAbai+pGADT7 empty transformed strain was unable to grow on SD/-Leu/AbA medium. The yeast single hybridization experiment can effectively detect whether the protein is bound on the promoter. Whereas PIbbHLH2-1-pAbAi+IbPGP19-pGADT7, PIbMYB1-4-pAbAi+IbPGP19-pGADT7 or PIbWD40-1-pAbAi+IbPGP19-pGADT7 was able to grow on SD/-Leu/AbA medium (FIGS. 1-3), indicating that IbPGP19 protein was able to bind to the promoter IbbHLH2, ibMYB1 or IbWD40.
Further, in order to verify the interaction of the promoters IbbHLH2, ibMYB1 or IbWD40 and the regulatory factor IbPGP19, the invention constructs IbPGP19 on an over-expression vector pGreenII 002962-SK, inserts the PIbbHLH2, the PIbMYB1 or the PIbWD40 into the front end of the vector pGreenII 0800-LUC luciferase to serve as a reporter plasmid, selects Sac I and Xho I in the pGreenII 002962-SK vector and Kpn I and Nco I in the pGreenII 0800-LUC vector to serve as enzyme cleavage sites for inserting target fragments, and constructs a vector primer sequence shown in Table 2. And (3) extracting plasmids from the recombinant bacterial liquid which is successfully sequenced, and then transferring the plasmids and the PIbbHLH2+pGreenII0800LUC, PIbMYB1+pGreenII0800LUC or PIbWD40+pGreenII0800LUC recombinant plasmids into arabidopsis protoplast. The results show that IbPGP19 is able to increase the activity of IbbHLH2, ibMYB1 or IbWD40 promoters (fig. 4-6), demonstrating that IbPGP19 is able to promote expression of IbbHLH2, ibMYB1 or IbWD40.
Furthermore, in order to clarify the function of the upstream regulatory factor IbPGP19, the invention constructs a fusion protein of IbPGP19 and Green Fluorescent Protein (GFP) to locate the action site of IbPGP19. After transient transformation of Arabidopsis protoplast by constructing subcellular localization expression vector, subcellular localization was observed with laser confocal microscope, and pCambia1300-GFP was used as positive control. The GFP protein in the empty vector was expressed in the various structures of Arabidopsis protoplasts and the IbPGP19 protein was expressed in the nucleus (FIG. 7), indicating that IbPGP19 is a typical transcription factor.
The invention has the beneficial effects that: through a yeast single hybrid library screening experiment, the factors IbPGP19 with the regulation function on all 3 promoters are successfully obtained in the screening of upstream regulation factors of the promoters IbbHLH2, ibMYB1 and IbWD40. The result of the invention can theoretically enrich and deepen the basic theory of the molecular regulation of the biosynthesis of plant anthocyanin; the novel genetic marker can be provided for purple sweet potato high anthocyanin variety breeding in application, suitable operating elements or reconstruction targets can be selected for molecular breeding, and novel ideas and clues can be provided for cultivation measures for improving pigment content in purple sweet potato tuberous roots.
Drawings
FIG. 1 shows the results of the rotation verification of the IbbHLH2 promoter and its upstream regulator IbPGP19. The positive control was p53AbAi+AD-53, the negative control was PIbbHLH2-1-pAbAi+AD, and the positive colony (PIbbHLH 2-1-pAbAi+IbPGP 19-AD) indicated that the corresponding upstream regulatory factor protein (IbPGP 19) was able to bind to the IbbbHLH 2 promoter, AD: pGADT7.
FIG. 2 shows the results of the rotation verification of the IbMYB1 promoter and its upstream regulator IbPGP19. The positive control was p53AbAi+AD-53, the negative control was PIbMYB1-4-pAbAi+AD, and the positive colonies (PIbMYB 1-4-pAbAi+IbPGP 19-AD) indicated that the corresponding upstream regulatory factor protein (IbPGP 19) was able to bind to the IbMYB1 promoter, AD: pGADT7.
FIG. 3 shows the results of the regulatory factor IbPGP19, and the IbWD40 promoter. The positive control was p53AbAi+AD-53, the negative control was PIbWD40-1-pAbAi+AD, and the positive colonies (PIbWD 40-1-pAbAi+IbPGP 19-AD) indicated that the corresponding upstream regulatory factor protein (IbPGP 19) was able to bind to the IbWD40 promoter, AD: pGADT7.
FIG. 4 is a graph showing the interaction of the IbbHLH2 promoter with its upstream regulator IbPGP19.
FIG. 5 is a graph showing the interaction of the IbMYB1 promoter with its upstream regulator IbPGP19.
FIG. 6 is a graph showing the interaction of the IbWD40 promoter with its upstream regulator IbPGP19.
FIG. 7 shows subcellular localization of the regulatory factor IbPGP19 in Arabidopsis protoplasts (scale 20 μm). A: green fluorescence map; b: chloroplast autofluorescence; c: a bright field map; d: and (5) superposing the diagrams.
Detailed Description
The present invention will be further described with reference to examples, wherein the test methods are conventional test methods unless otherwise specified, and wherein the test reagents and consumables described in the examples are from conventional Biochemical reagent company unless otherwise specified.
Example 1: construction of a purple sweet potato Yeast Single hybrid cDNA library
(1) RNA was extracted from purple sweet potato (line A5) tubers by Trizol method, and double-stranded cDNA was synthesized by reverse transcription using SMART technology.
(2) Amplified cDNA is purified by TaKaRa MiniBEST DNA Fragment Purification Kit to give dH 2 O dissolves out.
(3) The cDNA cut by the restriction enzyme SfiI is subjected to column treatment and PCI/CI purification treatment, and finally ddH is obtained 2 O dissolves out.
(4) pGADT7-SfiI vector (clontech, cat. No. 630490) was ligated with the appropriate amount of post-column cDNA using the DNA ligation Kit. Purifying the purified ligation mixture to obtain a primary cDNA library.
(5) Transferring a small amount of primary library connecting solution into competent cells E.coli HST08 by an electrotransformation method; after identification, a proper amount of bacterial liquid is coated on an LB plate containing Amp resistance, and the culture is carried out for 12 hours at 37 ℃; the primary library capacity was calculated from the number of colonies grown on the plates.
(6) Amplified colonies were cultured overnight and then subjected to plasmid extraction to obtain library plasmids.
Example 2: construction of pAbAi-PIbbHLH2, pAbAi-PIbMYB1, pAbAi-PIbWD40 bait vector
(1) Using purple sweet potato tuber DNA as template and TaKaRa high-fidelity enzymeMax DNA Polymerase promoter DNA fragments of IbbHLH2, ibMYB1 or IbWD40 with different restriction site ends at both ends were amplified. Wherein, the liquid crystal display device comprises a liquid crystal display device,
specific sequences of the PCR primer pair for amplifying the promoter DNA fragment of IbbHLH2 are as follows:
PIbbHLH2-F:5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and is provided with
PIbbHLH2-R：5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-3'。
The specific sequences of the PCR primer pairs for amplifying the promoter DNA fragment of IbMYB1 are as follows:
PIbMYB1-F:5'-TTATTACATCAAGCTAAATAAATACGATTTG-3' and is provided with
PIbMYB1-R：5'-TATATATATTGAAGGGTGTCGGAAATTC-3'。
The specific sequences of the PCR primer pairs for amplifying the promoter DNA fragment of IbWD40 are as follows:
PIbWD40-F:5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and is provided with
PIbWD40-R：5'-TCTCATACCACCACACCCTAGTGG-3'。
The reaction system (20. Mu.L) was as follows:
the PCR reaction conditions were:
(2) The promoters IbbHLH2, ibMYB1, or IbWD40 (IbbHLH 2 sequence has been disclosed in patent CN114085276a, ibMYB1 sequence has been disclosed in patent CN 113881688A, ibWD40 sequence has been disclosed in patent CN113929759 a) were constructed into the pAbAi vector (Ke Lei biotechnology limited, cat No. kl-zl-0879) by: using TaKaRa restriction endonuclease QuickCut TM HindIII and QuickCut TM SmaI carries out double enzyme digestion on pAbAi plasmid, the synthetic primer sequences are shown in Pb2-1F/Pb2-1R, PM1-4F/PM1-4R, PW1-1F/PW1-1R in Table 2, the reaction conditions are 37 ℃, the enzyme digestion is carried out for more than 3 hours, and the reaction system is as follows:
and (5) detecting the correct enzyme digestion product through electrophoresis, and performing gel digestion recovery.
The fragment of interest and the expression vector were ligated using Clon Express II One Step Cloning Kit kit (Vazyme) at 37℃for 30min. The reaction system is as follows:
(3) The ligation product was used to subsequently transform E.coli DH 5. Alpha. Competent cells.
Preparation of E.coli DH 5. Alpha. Competent cells (CaCl) 2 Method):
(1) Coli DH 5. Alpha. To 5mL of LB liquid medium was inoculated, and cultured overnight at 37℃with shaking at 220 rpm.
(2) Transferring 2mL of the bacterial liquid cultured overnight into 100mL of LB liquid medium, and continuously shaking culture until OD 600 About 0.5, and standing on ice for 30min.
(3) 1mL of the bacterial liquid was taken into a new 1.5mL centrifuge tube, centrifuged at 4000rpm at 4℃for 10min, and the supernatant was aspirated with a pipette.
(4) 1mL of pre-chilled 0.1M CaCl was aspirated with a pipette 2 Suspending and precipitating, mixing by light blowing, and standing on ice for 30min.
(5) Centrifuging at 4000rpm at 4deg.C for 10min, pipetting the supernatant with a pipette, pipetting 0.2mL of pre-chilled 0.1M CaCl with a pipette 2 Suspending and precipitating, and standing on ice for 5h for transformation.
Ligation product transformation E.coli DH 5. Alpha. Competent cells:
(1) Taking 100 mu L of the prepared escherichia coli DH5 alpha competent cells to a new 1.5mL centrifuge tube, adding 10 mu L of DNA connection product into a super clean bench, flicking, uniformly mixing, and standing on ice for 30min.
(2) The conversion product was heat shocked in a 42℃water bath for 90s and immediately removed and placed on ice for 5min.
(3) 1mL of LB liquid medium without resistance is added, and shaking culture is carried out at 180rpm at 37 ℃ for 60-90 min.
(4) Centrifuge at 5000rpm for 4min at room temperature, pipette 900. Mu.L of supernatant under sterile conditions, gently blow to resuspend the remaining 200. Mu.L of liquid.
(5) Uniformly coating the bacterial liquid in LB solid medium containing Amp, standing for 30min and airing.
(6) The incubator is inverted and cultured for 12 to 16 hours at 37 ℃.
Screening and sequencing identification of positive clones:
a single colony with resistance is picked from a culture dish by a sterile small gun head and cultured for 4 hours at 37 ℃ under shaking at 220rpm, and 2 mu L of the bacterial liquid is taken as a template for colony PCR detection. And (3) 200 mu L of bacterial liquid of a positive strain with the same size as the target fragment obtained by amplification in the PCR reaction is sent to Shanghai biological limited company for sequencing. 20% sterilized glycerol is added into bacterial liquid with correct sequencing, the bacterial liquid is stored in a centrifuge tube with the concentration of 1.5mL, and pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40 bait plasmids are extracted by a refrigerator with the temperature of minus 80 ℃.
Example 3: self-activation detection of pAbAi-PIbbHLH2, pAbAi-PIbMYB1, pAbAi-PIbWD40 bait strains
The Y1H yeast is transformed by pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40 bait plasmid to obtain bait strain. The lowest AbA concentration of the inhibition bait strain is tested by adopting a self-activation test, the growth condition of the inhibition bait strain on SD/-Ura solid medium is observed, and the self-activation detection of the bait strain and the determination method of the lowest AbA concentration are as follows:
(1) Preparation of AbA mother liquor: 1mL of absolute ethanol was used to dissolve 1mg of AbA to prepare 1mg/mL of AbA mother liquor, which was stored at 4℃in the absence of light.
(2) From Y1H [ pAbAi-prey ]]And Y1H [ p53AbAi ]]Larger monoclonal colonies were picked up in dishes, the bacterial suspension was resuspended in 10. Mu.L of 0.9% NaCl solution, and the resuspension solution was diluted to 10 -1 、10 -2 And 10 -3 Concentration gradient.
(3) 10. Mu.L of the resuspension bacteria were pipetted 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 control group Y1H [ p53AbAi ] grows normally, this concentration is the lowest AbA concentration inhibiting the recombinant yeast strain and can be used in subsequent experiments.
Note that: pAbAi-prey is pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40.
The detection shows that the lowest inhibition concentration of the self-activated AbA of the pAbAi-PIbbHLH2 bait strain is 700ng/mL, the lowest inhibition concentration of the self-activated AbA of the pAbAi-PIbMYB1 bait strain is 400ng/mL, and the lowest inhibition concentration of the self-activated AbA of the pAbAi-PIbWD40 bait strain is 300ng/mL.
Example 4: yeast single hybrid library screening
Yeast Single hybrid library screening was performed according to Clontech Matchmaker Gold Yeast One-Hybrid Library Screening System. The screening method of the yeast single hybridization library comprises the following steps:
(1) Denaturation was effected by taking 25 mu L Yeastmaker Carrier DNA in a 95℃water bath for 5min, rapidly placing on ice for several minutes, and repeating once until the temperature was reduced to 4 ℃.
(2) Sequentially adding into a pre-cooled 10mL centrifuge tube: 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 bait expression vectors pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD 40) were vortexed.
(3) The centrifuge tube was placed in a water bath at 30℃for 45min with gentle mixing several times every 15min.
(4) 160. Mu.L of DMSO was added and gently mixed.
(5) The mixture was incubated in a 42℃water bath for 20min, during which time the mixture was gently mixed several times every 10min.
(6) The cells were collected by centrifugation at 12000rpm for 30 seconds, the supernatant was discarded, and 8mL of 0.9% NaCl solution was added to resuspend the cells.
(7) 200 mu L of the transformed saccharomycete liquid is absorbed and uniformly coated on SD/-Leu and SD/-Leu/AbA culture dishes, and the AbA concentration is the lowest concentration for inhibiting self-activation.
(8) Culturing in an incubator at 30 ℃ for 48-96 hours in an inverted mode.
Single colonies are selected for colony PCR identification, the identification method refers to example 2, universal primers pGADT7F/R are selected for PCR detection of bacterial liquid, the sequences of the pGADT7F/R primers are shown in table 1, a sample which is brighter after electrophoresis and has a single strip is selected for sequencing by Shanghai biological limited company, BLAST is carried out on sequencing results in NCBI database, and sequencing results are analyzed.
TABLE 1 general primers for major vectors
The factors with regulation and control effects on the expression of the purple sweet potato IbbHLH2, ibMYB1 and IbWD40 genes are screened by a yeast single hybridization method to obtain IbPGP19, and the amino acid sequence of the regulation and control factor IbPGP19 is shown as SEQ ID NO.1, and specifically comprises the following steps: MASTSYTKYNSPQIGHLNEKQLVKRLDINEKVEWQIISIQKKKKKLPPPPSFCKPPFLEHTVPCSLLQSVSCKRGSCVTVF.
The nucleotide sequence of the regulatory factor IbPGP19 is shown as SEQ ID NO.2, and specifically comprises the following steps: ATGGCGAGCACAAGTTATACGAAGTACAATAGCCCACAGATAGGACACCTAAATGAGAAGCAATTAGTGAAAAGGTTGGACATTAATGAAAAGGTAGAATGGCAGATTATTTCAATACAAAAAAAAAAAAAAAAGCTTCCTCCTCCTCCATCTTTCTGTAAACCTCCATTTCTTGAACATACTGTTCCTTGTTCTTTGCTGCAATCTGTATCATGCAAAAGAGGCTCATGTGTCACGGTATTTTAG. And the amplification primer of the upstream regulatory factor IbPGP19 is designed as IbPGP19-F:5'-ATGGCGAGCACAAGTTATACGA-3' and IbPGP19-R:5'-CTAAAATACCGTGACACATGAGCC-3'.
Example 5: verification of binding of upstream regulatory factor IbPGP19 to promoters IbbHLH2, ibMYB1, ibWD40
IbPGP19 was constructed into pGADT7 yeast recombinant expression vector (Shanghai-Haimai bioengineering Co., ltd., cat. No. LM-1639), ecoRI and BamHI were selected as cleavage sites for insertion of the desired fragment in the vector, and the sequence of the synthetic primer was shown in Table 2 as IbPGP19-ADF/IbPGP19-ADR, and the construction method was described in example 2. Yeast single hybridization experiments were performed by co-transforming pGADT7-IbPGP19 yeast recombinant expression vector plasmids with pAbAi-PIbbHLH2, pAbAi-PIbMYB1 or pAbAi-PIbWD40 bait vectors in Y1HGold single hybrid yeast strains.
Yeast Single hybrid library screening was performed according to Clontech Matchmaker Gold Yeast One-Hybrid Library Screening System. The screening method of the yeast single hybridization library comprises the following steps:
(1) Denaturation was effected by taking 25 mu L Yeastmaker Carrier DNA in a 95℃water bath for 5min, rapidly placing on ice for several minutes, and repeating once until the temperature was reduced to 4 ℃.
(2) Sequentially adding into a pre-cooled 10mL centrifuge tube: 2.5mL PEG/LiAc, 25. Mu.L denatured Yeastmaker Carrier DNA, 15. Mu.g library plasmid, 600. Mu. L Y1HGold competent cells, vortex well.
(3) The centrifuge tube was placed in a water bath at 30℃for 45min with gentle mixing several times every 15min.
(4) 160. Mu.L of DMSO was added and gently mixed.
(5) The mixture was incubated in a 42℃water bath for 20min, during which time the mixture was gently mixed several times every 10min.
(6) The cells were collected by centrifugation at 12000rpm for 30 seconds, the supernatant was discarded, and 8mL of 0.9% NaCl solution was added to resuspend the cells.
(7) 200 mu L of the transformed saccharomycete liquid is absorbed and uniformly coated on SD/-Leu and SD/-Leu/AbA culture dishes, and the AbA concentration is the lowest concentration for inhibiting self-activation.
(8) Culturing in an incubator at 30 ℃ for 48-96 hours in an inverted mode.
And (3) 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 single band, sending the sample to Shanghai biological limited company for sequencing, performing BLAST on a sequencing result in an NCBI database, and analyzing the sequencing result.
The result shows that: positive control p53abai+ad-53 (i.e. inserting a positive control 53 gene sequence into the pAbAi vector to obtain p53AbAi, inserting a positive control 53 gene sequence into the pGADT7 vector to obtain AD-53, construction method reference example 2) the transformed strain was able to grow on SD/-Leu/AbA medium; whereas the negative control PIbbHLH2-1-pAbAi+pGADT7, PIbMYB1-4-pAbAi+pGADT7 or PIbWD40-1-pAbAi+pGADT7 empty transformed strain could not grow on SD/-Leu/AbA medium; the yeast single hybridization experiment can effectively detect whether the protein is bound on the promoter. PIbbHLH2-1-pAbAi+IbPGP19-pGADT7, PIbMYB1-4-pAbAi+IbPGP19-pGADT7 or PIbWD40-1-pAbAi+IbPGP19-pGADT7 can be grown on SD/-Leu/AbA medium (FIGS. 1-3), indicating that IbPGP19 protein can bind to the promoter IbbHLH2, ibMYB1 or IbWD40.
Example 6: construction of double luciferase reporting system carrier
The gene sequence of the upstream regulatory factor IbPGP19 was constructed on an over-expression vector pGreenII 002962-SK (Shanghai Qing City Biotechnology Co., ltd., cat. No. QCP 0465), called an effector plasmid, and PIbbHLH2, PIbMYB1 or PIbWD40 was inserted into the front end of the vector pGreenII 0800-LUC (Ke Lei Biotechnology Co., ltd., cat. No. kl-zl-0808) luciferase 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 cleavage sites for insertion of the desired fragment, and vector primer sequences were constructed as shown in Table 2, ibPb2-1 0800F/IbPb2-1 0800R, ibPM1-4 0800F/IbPM1-4 0800R, ibPW1 0800F/IbPW1 0800R and IbPGP19-62-SKF/IbPGP19-62-SKR, and the construction method was as described in example 2.
Example 7: 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 water bath kettle at 55 ℃.
(2) Selecting wild arabidopsis leaves after four weeks and before bolting, tearing off the epidermis under the leaves, and rapidly putting the leaves into enzymolysis liquid.
(3) The enzymolysis is carried out for 50min at 25 ℃ under 50rpm in a dark place until mesophyll cells are completely hydrolyzed, the form of protoplast can be observed under a microscope, and the state is better when the cells are round and transparent.
(4) Diluting the enzyme solution with an equal volume of W5 solution, gently mixing, washing the nylon mesh cloth with 75 mu m by using clean water, soaking the nylon mesh cloth with the W5 solution, and filtering the protoplast.
Preparation of W5 solution (100 mL):
(5) Centrifugation at 800rpm for 2min, the supernatant was aspirated as much as possible, and protoplasts were resuspended with 1mL of W5 solution (this procedure was repeated three times).
(6) The protoplasts were resuspended in 1mL of W5 solution and then kept on ice for 30min.
2. The transformation procedure for Arabidopsis protoplasts was as follows:
(1) 10-20 mug of target plasmid (IbPGP 19-pGreenII 002962-SK recombinant plasmid and PIbbHLH2-pGreenII 0800-LUC recombinant plasmid constructed in example 6, PIbMYB1-pGreenII 0800-LUC recombinant plasmid or PIbWD40-pGreenII 0800-LUC recombinant plasmid) was added into a 2mL EP tube, 100 uL of Arabidopsis protoplast was added, gently mixed, and immediately placed on ice after the addition.
(2) 110. Mu.L PEG/CaCl was added 2 The centrifuge tube was flicked to mix them evenly and incubated for 10min at room temperature.
(3) The 220 mu L W solution was added to ice, the centrifuge tube was inverted and mixed well and left on ice for 1min.
(4) The tube was again filled with 440 μ L W solution, gently inverted and placed on ice for 1min.
(5) Finally, 880 mu L W of solution is added into the centrifuge tube, the mixture is inverted and mixed evenly, and the mixture is placed on ice for 1min.
(6) The supernatant was aspirated by centrifugation at 800rpm at 4℃for 3 min.
(7) Protoplasts were resuspended in 500. Mu. L W5 solution and incubated at 22℃for 16-20 h in the dark.
Example 8: detection of dual luciferase reporter systems
UsingThe report Assay (Promega) detects the activity of two luciferases, LUC and REN, and comprises the following specific steps:
(1) Preparation of 100. Mu.L of 1 XPLB lysate: 20. Mu.L of 5X Passive Lysis Buffer water was added to 100. Mu.L. Preparation of 10mL LAR II: 10mL of ice thawed Luciferase Assay Buffer II was pipetted into Luciferase Assay Substrate and gently shaken to dissolve (-20℃for one month, -70℃for one year). 100 mu L Stop&Preparation of Reagent: 100 mu L Stop is sucked&/>Buffer, add 2. Mu.L of 50 XSTOP&/>The Substrate was slightly vortexed to mix it (15 d at-20 ℃).
(2) The solution of the transformed Arabidopsis thaliana protoplast of example 7 was centrifuged at 13200rpm at 4℃for 90s to remove the W5 solution.
(3) 100. Mu.L of 1 XPLB lysate was added, gently swirled and mixed, transferred to a 24-well plate, and placed on a horizontal shaker and shaken at low speed for 15min at room temperature.
(4) The lysate was collected, transferred to a 1.5mL centrifuge tube, centrifuged at 13200rpm at 4℃for 10min, and 60. Mu.L of the supernatant was placed on ice for the luciferase to be assayed.
(5) Under the dark condition, 100 mu L of LAR II is added into a black 96-well ELISA plate, then 20 mu L of cell lysate is added, and the mixture is gently mixed for 2 to 3 times by a gun head, so that bubbles are avoided.
(6) The enzyme activity of the LUC was measured in an enzyme-labeled instrument and the data were recorded.
(7) The 96-well plate was removed and 100. Mu.L Stop was added to the same well&And (3) the Reagent is gently mixed for 2-3 times by using a gun head, and strong light irradiation is avoided in the whole operation process.
(8) The REN enzyme activity was detected in an microplate reader and the data were recorded.
(9) Experiments were repeated 3 times, averaged, and the activation of promoters by transcription factors was examined by comparing the LUC/REN ratios of the different samples.
The results show that: ibPGP19 was able to increase the activity of IbbHLH2, ibMYB1, or IbWD40 promoters (fig. 4-6), demonstrating that IbPGP19 was able to promote expression of IbbHLH2, ibMYB1, or IbWD40.
Example 9: defining the function of the upstream regulatory factor IbPGP19
Fusion proteins of IbPGP19 and Green Fluorescent Protein (GFP) were constructed to localize the site of action of IbPGP19. The gene sequence of the upstream regulatory factor IbPGP19 is constructed on a subcellular localization expression vector pCambia1300 (Shanghai-associated biological engineering Co., ltd., cat.No. LM 1375), bamHI and HindII in the vector pCambia1300 are selected as cleavage sites for insertion of the target fragment, a specific primer containing the initiation codon but not the termination codon is designed, and the sequence of the synthetic primer is shown in IbPGP19-1300F/IbPGP19-1300R in Table 2, and the construction method is described in example 2. Then, after the IbPGP19-pCambia1300 was transiently transformed into Arabidopsis protoplast, subcellular localization was observed by using a laser confocal microscope, and pCambia1300-GFP (p 1300 GFP) was used as a positive control. The GFP protein in the empty vector was expressed in the various structures of Arabidopsis protoplasts and the IbPGP19 protein was expressed in the nucleus (FIG. 7), indicating that IbPGP19 is a typical transcription factor.
TABLE 2 construction of vectors Using primer sequences (underlined as cleavage sites)
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The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended 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 such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. The amino acid sequence of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 is shown as SEQ ID NO. 1.
2. A gene encoding the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.
4. A recombinant vector or 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. An amplification primer of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 according to claim 1, wherein the amplification primer is IbPGP19-F:5'-ATGGCGAGCACAAGTTATACGA-3' and IbPGP19-R:5'-CTAAAATACCGTGACACATGAGCC-3'.
7. Use of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 of claim 1 for promoting expression of sweet potato IbbHLH2, ibMYB1 and/or IbWD40 transcription factors.
8. Use of the purple sweet potato anthocyanin synthesis regulator IbPGP19 of claim 1 for promoting sweet potato anthocyanin biosynthesis.
9. The use of the purple sweet potato anthocyanin synthesis regulatory factor IbPGP19 according to claim 1 in breeding of sweet potato high anthocyanin varieties.
10. A method for promoting the synthesis of sweet potato anthocyanin, wherein the regulatory factor IbPGP19 of claim 1 is overexpressed in sweet potato plants.
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