Purple sweet potato anthocyanin synthesis regulatory factor IbACLA and application thereof

Abstract

The invention discloses a purple sweet potato anthocyanin synthesis regulatory factor IbACLA and application thereof. According to the invention, a purple sweet potato strain A5 is used as an experimental material, a promoter sequence of IbbHLH2 is cloned, and an upstream regulatory factor IbACLA of an IbbHLH2 gene is obtained through a yeast one-hybrid library screening experiment. A yeast single-hybridization rotary experiment and a dual-luciferase report system are used for detection to confirm that the IbbHLH2 promoter interacts with an upstream regulatory factor IbACLA. The basic theory of molecular regulation and control of plant anthocyanin biosynthesis can be enriched and deepened theoretically, and new thoughts and clues are expected to be provided for cultivation measures for improving the content of pigments in purple sweet potato tuberous roots.

Description

Technical Field

[0001] This invention relates to the field of molecular mechanisms of plant genetics and variation, specifically to IbACLA, a regulatory factor for anthocyanin synthesis in purple sweet potato, and its applications. Background Technology

[0002] Anthocyanins are a class of water-soluble natural pigments widely found in plants, belonging to the flavonoid family, which impart a rich variety of colors to plant tissues, including red, purple, and blue. Purple sweet potato (Ipomoea batatas L.) is rich in anthocyanins in its tubers, resulting in high nutritional value and health benefits, such as antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protective effects. Therefore, it has become an important crop for functional food development and health agriculture research. However, the synthesis of anthocyanins in purple sweet potato is influenced by various factors, including genetic background, environmental conditions, and regulatory networks, leading to significant differences in their content and composition among different varieties or under different growing conditions, thus limiting its stable production and commercial application.

[0003] There are three main types of transcription factors that regulate anthocyanin biosynthesis: MYB, bHLH, and WD40. Among them, bHLH (basic helix-loop-helix) transcription factors are the second largest transcription factor superfamily in plants, second only to MYB (Jin et al., 2014). The bHLH domain is highly conserved, containing about 60 amino acids and has two functionally distinct regions. A typical bHLH domain contains 13-17 basic amino acids at its N-terminus, called the basic region, which can recognize the cis-acting element E-Box (5'-CANNTG-3', where N represents any amino acid) on the promoter sequence of a target gene (Feller et al., 2011); the C-terminus contains about 40 amino acids, which is a helix-loop-helix (HLH) region composed of a variable-length loop region connecting two amphiphilic α-helices. bHLH transcription factors can form homo- or hetero-dimers depending on interactions between hydrophobic amino acids in the HLH region (Massari and Murre et al., 2000; Castilhos et al., 2014). Studies have shown that the highly conserved Leu-23 residue is structurally essential for the formation of bHLH dimers in human MAX and Arabidopsis PAR1 proteins, while the latter also requires the conserved Leu-52 residue (Brownlie et al., 1997; Carretero-Paulet et al., 2010). Furthermore, research indicates that the bHLH domains of certain proteins can also interact with non-bHLH proteins, and certain amino acids within these domains may determine the specificity of protein-protein interactions (Massari and Murre et al., 2000; Ciarapica et al., 2003). Further research is needed to fully and precisely understand the detailed mechanisms by which bHLH transcription factors form dimers.

[0004] The research team of the inventors cloned the gene sequence of transcription factor IbbHLH2 from purple-fleshed sweet potato and used the onion epidermal transient expression method to locate the subcellular products of these transcription factor genes. Through Arabidopsis transformation experiments, it was demonstrated that the gene is located in the cell nucleus and its expression level is consistent with the trend of anthocyanin content (Fu Danwen; Hui Yake; Li Haihang; Yang Shaohua; Chen YaHui; Gao Feng*. Molecular Cloning and Functional Analysis of the Gene and Promoter of IbbHLH2 from Purple-Fleshed Sweet Potato. HORTICULTURAL SCIENCE and TECHNOLOGY, 2021, 40(1).). Summary of the Invention

[0005] The technical problem to be solved by this invention is to screen an upstream regulatory factor that promotes the expression of the IbbHLH2 transcription factor in purple sweet potato.

[0006] To address the aforementioned technical issues, this invention first extracts RNA from sweet potato tubers using the Trizol method, then uses SMART technology to reverse transcribe and synthesize double-stranded cDNA, constructing a purple sweet potato yeast one-hybrid cDNA library.

[0007] Furthermore, using purple sweet potato tuber DNA as a template, TaKaRa high-fidelity enzyme was used. Max DNAPolymerase amplified the IbbHLH2 promoter DNA fragment with different restriction sites at both ends, and constructed the IbbHLH2 promoter into the pAbAi vector. The specific sequences of the PCR primer pairs for amplifying the IbbHLH2 promoter DNA fragment are shown below:

[0008] PIbbHLH2-F: 5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and

[0009] PIbbHLH2-R: 5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-3'.

[0010] After self-activation detection, the minimum inhibitory concentration of AbA for self-activation of the constructed pAbAi-PIbbHLH2 decoy vector was determined to be 700 ng / mL.

[0011] The present invention further describes the preparation of Y1HGold competent cells from bait strains, the transfer of library plasmids into pAbAi-PIbbHLH2 competent cells, and the screening of binding proteins through yeast one-hybrid library screening. The upstream regulator of IbbHLH2 gene expression in purple sweet potato was identified as IbACLA.

[0012] Therefore, the first objective of this invention is to provide a purple sweet potato anthocyanin synthesis regulator IbACLA, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] A second objective of this invention is to provide a gene encoding the above-mentioned anthocyanin synthesis regulator IbACLA from purple sweet potato, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] A third objective of this invention is to provide a recombinant vector or recombinant bacterium containing the above-mentioned coding gene.

[0015] A fourth object of the present invention is to provide an expression cassette containing the above-described coding gene.

[0016] The fifth objective of this invention is to provide an amplification primer for the above-mentioned anthocyanin synthesis regulator IbACLA from purple sweet potato, the specific sequence of which is shown below:

[0017] IbACLA-F: 5'-ATGGCACGCAAGAAGATCAGA-3' and

[0018] IbACLA-R: 5'-TTATGCAGCTGCAGTGATGCA-3'.

[0019] The sixth objective of this invention is to provide the application of the above-mentioned purple sweet potato anthocyanin synthesis regulator IbACLA in promoting the expression of sweet potato IbbHLH2 transcription factor.

[0020] The seventh objective of this invention is to provide the application of the above-mentioned purple sweet potato anthocyanin synthesis regulator IbACLA in promoting sweet potato anthocyanin biosynthesis.

[0021] The eighth objective of this invention is to provide the application of the above-mentioned purple sweet potato anthocyanin synthesis regulator IbACLA in the breeding of sweet potato varieties with high anthocyanin content.

[0022] The ninth objective of this invention is to provide a method for promoting the synthesis of sweet potato anthocyanins by overexpressing the aforementioned regulatory factor IbACLA in sweet potato plants.

[0023] Furthermore, to further verify the binding of the screened regulatory factor IbACLA to the promoter IbbHLH2, IbACLA was constructed into the pGADT7 yeast recombinant expression vector. EcoRI and BamHI in the vector were selected as the restriction enzyme sites for the insertion of the target fragment. The synthesized primer sequences are shown in Table 2. The pGADT7-IbACLA yeast recombinant expression vector plasmid and the pAbAi-PIbbHLH2 bait vector were co-transformed into Y1HGold yeast for yeast one-hybrid experiments. The results showed that the positive control strain transformed with p53AbAi+AD-53 could grow on SD / -Leu / AbA medium, while the negative control strain transformed with the empty vector PIbbHLH2-1-pAbAi+pGADT7 could not grow on SD / -Leu / AbA medium. This indicates that the yeast one-hybrid experiment can effectively detect whether the protein binds to the promoter. The PIbbHLH2-1-pAbAi+IbACLA-pGADT7 strain could grow on SD / -Leu / AbA medium. Figure 1 This indicates that the IbACLA protein can bind to the promoter IbbHLH2.

[0024] Furthermore, to verify the interaction between the promoter IbbHLH2 and its regulatory factor IbACLA, IbACLA was constructed into the overexpression vector pGreenII 0029 62-SK. PIbbHLH2 was inserted into the front end of the luciferase vector pGreenII0800-LUC as a reporter plasmid. Sac I and Xho I in the pGreenII 0029 62-SK vector and Kpn I and Nco I in the pGreenII 0800-LUC vector were selected as restriction enzyme sites for the inserted target fragment. The primer sequences for the constructed vector are shown in Table 2. After extracting the plasmid from the successfully sequenced recombinant bacterial culture, it was co-transformed into Arabidopsis protoplasts with the PIbbHLH2+pGreenII0800LUC recombinant plasmid. The results showed that IbACLA could enhance the activity of the IbbHLH2 promoter (…). Figure 2 This indicates that IbACLA can promote the expression of IbbHLH2.

[0025] The beneficial effects of this invention are as follows: Through yeast one-hybrid library screening experiments, the upstream regulator of the promoter IbbHLH2, IbACLA, was successfully obtained. Theoretically, this result can enrich and deepen the fundamental theory of molecular regulation of anthocyanin biosynthesis in plants; in application, it can provide new genetic markers for breeding high-anthocyanin varieties of purple sweet potato, enabling the screening of suitable operational elements or modification targets for molecular breeding. Furthermore, it is expected to provide new ideas and clues for cultivation measures to increase the pigment content in purple sweet potato tubers. Attached Figure Description

[0026] Figure 1 This is the result of the IbbHLH2 promoter and its upstream regulator IbACLA rotation validation. The positive control was p53AbAi+AD-53, and the negative control was PIbbHLH2-1-pAbAi+AD. The positive colony (PIbbHLH2-1-pAbAi+IbACLA-AD) indicates that the corresponding upstream regulator protein (IbACLA) can bind to the IbbHLH2 promoter. AD: pGADT7.

[0027] Figure 2 This refers to the interaction between the IbbHLH2 promoter and its upstream regulator IbACLA. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional experimental methods. Unless otherwise specified, the experimental reagents and consumables mentioned in the following embodiments are all from conventional biochemical reagent companies.

[0029] Example 1: Construction of a one-hybrid cDNA library of *Polygonum cuspidatum*

[0030] (1) RNA was extracted from the tubers of purple sweet potato (strain A5) using the Trizol method, and double-stranded cDNA was synthesized by reverse transcription using SMART technology.

[0031] (2) The amplified cDNA should be purified using the TaKaRa MiniBEST DNA Fragment Purification Kit to dissolve dH2O.

[0032] (3) The cDNA digested with restriction endonuclease SfiI was subjected to column treatment, followed by PCI / CI purification treatment, and finally ddH2O was used to dissolve it.

[0033] (4) The pGADT7-SfiI vector (clontech, catalog number 630490) was ligated with an appropriate amount of column-passed cDNA using a DNA ligation kit. The ligation buffer was purified to obtain a primary cDNA library.

[0034] (5) A small amount of primary library ligation solution was transferred into competent E.coli HST08 cells by electroporation. After identification, an appropriate amount of bacterial solution was spread on LB plates containing Amp resistance and cultured at 37°C for 12 h. The volume of the primary library was calculated based on the number of colonies grown on the plate.

[0035] (6) The amplified colonies were cultured overnight, and then plasmids were extracted to obtain library plasmids.

[0036] Example 2: Construction of pAbAi-PIbbHLH2 decoy carrier

[0037] (1) Using purple sweet potato tuber DNA as a template, TaKaRa high-fidelity enzyme was used. Max DNA Polymerase amplifies the IbbHLH2 promoter DNA fragment with different restriction sites at both ends. The PCR primer pairs for amplifying the IbbHLH2 promoter DNA fragment are PIbbHLH2-F: 5'-GCATAACTTATAATCTTAAGTATGATGATCATAT-3' and PIbbHLH2-R: 5'-CTACCTAAGAATTTCTAGTAGAGGTAAATTGTA-3'. The reaction system (20 μL) is as follows:

[0038]

[0039]

[0040] The PCR reaction conditions are as follows:

[0041]

[0042] (2) The promoter IbbHLH2 (whose sequence has been disclosed in patent CN114085276A) was constructed into the pAbAi vector (Kelei Biotechnology Co., Ltd., catalog number kl-zl-0879). The steps were as follows: using the TaKaRa restriction endonuclease QuickCut TM Hind III and QuickCut TM The pAbAi plasmid was double-digested with SmaⅠ. The primer sequences were synthesized as shown in Table 2 (Pb1-1F / Pb1-1R). The reaction conditions were 37℃ and digestion time was at least 3 hours. The reaction system is as follows:

[0043]

[0044] Enzyme digestion products that are correctly identified by electrophoresis are then recovered by gel extraction.

[0045] The target fragment and expression vector were ligated using the Clon Express II One Step Cloning Kit (Vazyme) at 37°C for 30 min. The reaction system is as follows:

[0046]

[0047]

[0048] (3) The ligation product is used for subsequent transformation of Escherichia coli DH5α competent cells.

[0049] Preparation of Escherichia coli DH5α competent cells (CaCl2 method):

[0050] (1) Inoculate Escherichia coli DH5α into 5 mL of LB liquid medium and culture overnight at 37°C with shaking at 220 rpm.

[0051] (2) Transfer 2 mL of the overnight culture to 100 mL of LB liquid medium and continue to culture with shaking until OD. 600 Set the temperature to around 0.5 and place it on ice for 30 minutes.

[0052] (3) Take 1 mL of bacterial culture into a new 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 10 min at 4℃, and use a pipette to remove the supernatant.

[0053] (4) Use a pipette to take 1 mL of pre-cooled 0.1 M CaCl2 suspension precipitate, gently blow to mix, and place on ice for 30 min.

[0054] (5) Centrifuge at 4000 rpm for 10 min at 4℃, remove the supernatant with a pipette, and use a pipette to remove 0.2 mL of pre-cooled 0.1 M CaCl2 suspension precipitate. Place on ice for 5 h before conversion.

[0055] Transformation of the ligation product into E. coli DH5α competent cells:

[0056] (1) Take 100 μL of the prepared Escherichia coli DH5α competent cells into a new 1.5 mL centrifuge tube, add 10 μL of DNA ligation product in a clean bench, gently mix and place on ice for 30 min.

[0057] (2) Heat shock the conversion product in a 42°C water bath for 90 seconds, then immediately place it on ice for 5 minutes.

[0058] (3) Add 1 mL of LB liquid medium without resistance and incubate at 37°C and 180 rpm for 60-90 min with shaking.

[0059] (4) Centrifuge at 5000 rpm for 4 min at room temperature. Under aseptic conditions, use a pipette to remove 900 μL of supernatant and gently resuspend the remaining 200 μL of liquid.

[0060] (5) Spread the bacterial solution evenly on LB solid medium containing Amp and let it dry for 30 minutes.

[0061] (6) Incubate in an inverted incubator at 37℃ for 12-16 hours.

[0062] Screening and sequencing identification of positive clones:

[0063] Single resistant colonies were picked from the culture dish using a sterile pipette tip and placed in LB broth containing the resistant culture medium. The culture was incubated at 37°C with shaking at 220 rpm for 4 hours. 2 μL of the incubation was used as a template for colony PCR detection. 200 μL of the positive bacterial culture, which amplified to the size of the target fragment, was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. 20% sterile glycerol was added to the correctly sequenced bacterial culture in a 1.5 mL centrifuge tube and stored at -80°C. The pAbAi-PIbbHLH2 bait plasmid was then extracted.

[0064] Example 3: Detection of self-activation of pAbAi-PIbbHLH2 bait strain

[0065] The pAbAi-PIbbHLH2 bait plasmid was transformed into Y1H yeast to obtain the bait strain. A self-activation assay was used to determine the minimum AbA concentration that inhibited the bait strain, and their growth on SD / -Ura solid medium was observed. The methods for detecting the self-activation of the bait strain and determining the minimum AbA concentration are as follows:

[0066] (1) Preparation of AbA stock solution: Dissolve 1 mg AbA in 1 mL of anhydrous ethanol to prepare a 1 mg / mL AbA stock solution, and store it at 4 °C in the dark.

[0067] (2) Pick larger single colonies from the culture dishes of Y1H[pAbAi-prey] and Y1H[p53AbAi], resuspend the bacterial culture in 10 μL of 0.9% NaCl solution, and dilute the resuspended solution to 10 μL. -1 10 -2 and 10 -3 Concentration gradient.

[0068] (3) Take 10 μL of resuspended bacterial solution and spot it onto SD / -Ura, SD / -Ura / AbA (100 ng / mL~1000 ng / mL) medium.

[0069] (4) If colony Y1H[pAbAi-prey] does not grow at a certain concentration, while the control group Y1H[p53AbAi] grows normally, then this concentration is the lowest AbA concentration that inhibits the recombinant yeast strain and can be used for subsequent experiments.

[0070] Note: pAbAi-prey is the same as pAbAi-PIbbHLH2.

[0071] Testing revealed that the minimum inhibitory concentration of AbA for self-activation of the pAbAi-PIbbHLH2 bait strain was 700 ng / mL.

[0072] Example 4: Screening of yeast one-hybrid libraries

[0073] The yeast one-hybrid library screening method was performed according to the Clontech Matchmaker Gold Yeast One-Hybrid Library Screening System manual. The yeast one-hybrid library screening method is as follows:

[0074] (1) Take 25 μL of Yeastmaker Carrier DNA and incubate it in a water bath at 95°C for 5 min to denature it. Then quickly place it on ice for a few minutes until the temperature drops to 4°C (repeat once).

[0075] (2) Add the following to a pre-cooled 10 mL centrifuge tube in sequence: 2.5 mL PEG / LiAc, 25 μL denatured Yeastmaker Carrier DNA, 15 μg library plasmid (obtained in Example 1), and 600 μL Y1HGold competent cells (containing bait expression vector pAbAi-PIbbHLH2), and vortex to mix.

[0076] (3) Place the centrifuge tubes in a 30°C water bath for 45 minutes, and gently mix them several times every 15 minutes.

[0077] (4) Add 160 μL of DMSO and mix gently.

[0078] (5) Warm bath in a 42℃ water bath for 20 minutes, gently stirring several times every 10 minutes during the process.

[0079] (6) Centrifuge at 12000 rpm for 30 s, collect the bacterial culture, discard the supernatant, add 8 mL of 0.9% NaCl solution, and resuspend the bacterial cells.

[0080] (7) Take 200 μL of the transformed yeast culture and spread it evenly on SD / -Leu and SD / -Leu / AbA culture dishes. The AbA concentration is the minimum concentration to inhibit self-activation (i.e., 700 ng / mL).

[0081] (8) Incubate upside down in a 30℃ incubator for 48–96 h.

[0082] Single colonies were selected for colony PCR identification, following the method described in Example 2. Universal primers pGADT7F / R were used for PCR detection of the bacterial culture. The pGADT7F / R primer sequences are shown in Table 1. Samples with bright and single bands after electrophoresis were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were BLASTed in the NCBI database and analyzed.

[0083] Table 1. Common primers for main vectors

[0084]

[0085] The upstream regulator of the IbbHLH2 gene expression in purple sweet potato was identified as IbACLA using a yeast one-hybrid assay. The amino acid sequence of IbACLA is shown in SEQ ID NO.1, specifically: MARKKIREYDSKRLLKEHFKRLGGYDLAIKSAQVTESTDLNELVDKEPWLSSTKLVVKPDMLFGKRGKSGLVALKLDLAEVAVFVKERLGKEVEMGGCKGPITTFIVEPFVPHNEEFYL NIVSERLGCSISFSECGGIEIEENWDKVKTIFVPTGVPLTSDLCAPLVATLPLEIKTVIEDFIKVVYALFLDLDFSFLEMNPFTLVDGKPYPLDMRGELDDTAAFKNFKKWGDVEFPLPFGRVMSATESFIHGLDEKTSASLKFTVLNPKGR IWTMVAGGGASVIYADTVGDLGYASELGNYAEYSGAPNEEEVLQYARVVIDCATADPDGRKRALVIGGGIANFTDVAATFNGIIRALKESKLKAARMSLFVRRGGPNYQRGLAKMRALAEEIGIPIEVYGPEATMTGICKQAIECITAAA*

[0086] The nucleotide sequence of the upstream regulatory factor IbACLA is shown in SEQ ID NO.2, specifically: ATGGCACGCAAGAAGATCAGAGAGTATGATTCCAAGAGGCTGTTGAAGGAGCATTTCAAGAGGCTCGGTGGGTATGATTTGGCCATCAAATCTGCTCAAGTTACAGAATCGACTGATCTCAATGAGCTGGTCGACAAAGAGCCTTGGCTCTCCTCAACAAAATTGGTTGTCAAGCCTGATATGCTCTTCGGCAAGCGTGGGAAAAGCGGTCTGGTTGCCTTGAAGCTAGATCTGGCTGAAGTCGCTGTGTTTGTGAAGGAAAGGCTTGGCAAGGAGGTTGAGATGGGTGGATGCAAAGGTCCCATCACAACTTTCATTGTTGAGCCCTTTGTCCCACACAACGAGGAGTTTTACCTTAACATCGTCTCTGAGAGGCTTGGATGTAGCATAAGCTTTTCGGAATGTGGAGGAATTGAAATCGAAGAGAACTGGGACAAGGTTAAGACCATTTTTGTTCCAACGGGGGTGCCTTTAACCTCAGATCTGTGTGCTCCACTTGTTGCAACACTTCCGCTTGAGATCAAGACTGTGATTGAGGACTTCATCAAAGTTGTTTATGCTCTATTTTTAGATTTGGATTTCAGTTTCCTTGAGATGAATCCTTTCACATTGGTTGACGGAAAGCCTTATCCGCTGGACATGAGGGGGGAACTAGATGACACTGCTGCTTTTAAGAACTTTAAGAAGTGGGGAGATGTTGAATTCCCGTTGCCATTTGGAAGGGTCATGAGCGCTACAGAAAGCTTTATCCATGGGCTGGATGAGAAGACAAGTGCATCTTTGAAGTTCACCGTCTTGAACCCAAAGGGACGAATCTGGACCATGGTGGCTGGTGGAGGTGCCAGTGTCATCTATGCCGATACTGTTGGAGACCTTGGATACGCA TCTGAACTCGGGAACTATGCTGAATATAGCGGGGCTCCTAATGAAGAAGAGGTCCTGCAGTATGCCAGAGTCGTAATTGATTGTGCAACTGCAGACCCTGATGGTCGTAAAAGAGCCCTCGTGATTGGT GGTGGTATAGCAAACTTTACTGACGTTGCTGCTACATTTAATGGCATAATCCGAGCTTTGAAGGAGAAGGAATCGAAGCTCAAGGCTGCTAGAATGAGTCTCTTTGTAAGGAGAGGCGGTCCGAACTAC CAAAGGGGTCTTGCTAAGATGAGAGCTCTTGCAGAGGAAATCGGCATCCCCATTGAGGTCTACGGCCCGGAGGCAACCATGACAGGCATTTGCAAACAGGCAATTGAATGCATCACTGCAGCTGCATAA.

[0087] The amplification primers for the upstream regulator IbACLA were designed as IbACLA-F: 5'-ATGGCACGCAAGAAGATCAGA-3' and IbACLA-R: 5'-TTATGCAGCTGCAGTGATGCA-3'.

[0088] Example 5: Verification of the binding of the upstream regulatory factor IbACLA to the promoter IbbHLH2

[0089] IbACLA was constructed into the pGADT7 yeast recombinant expression vector (Shanghai Lianmai Biotechnology Co., Ltd., catalog number LM-1639). EcoRI and BamHI in the vector were selected as the restriction enzyme sites for the insertion of the target fragment. The primer sequences are shown in Table 2 (IbACLA-ADF / IbACLA-ADR). The construction method is as described in Example 2. The pGADT7-IbACLA yeast recombinant expression vector plasmid and the pAbAi-PIbbHLH2 bait vector were co-transformed into Y1HGold yeast strain for yeast one-hybrid experiments.

[0090] The yeast one-hybrid library screening method was performed according to the Clontech Matchmaker Gold Yeast One-Hybrid Library Screening System manual. The yeast one-hybrid library screening method is as follows:

[0091] (1) Take 25 μL of Yeastmaker Carrier DNA and incubate it in a 95°C water bath for 5 min to denature it. Then quickly place it on ice for a few minutes until the temperature drops to 4°C (repeat once).

[0092] (2) Add the following to a pre-cooled 10mL centrifuge tube in sequence: 2.5mL PEG / LiAc, 25μL denatured Yeastmaker Carrier DNA, 15μg library plasmid, and 600μL Y1HGold competent cells, and vortex to mix.

[0093] (3) Place the centrifuge tubes in a 30°C water bath for 45 minutes, and gently mix them several times every 15 minutes.

[0094] (4) Add 160 μL of DMSO and mix gently.

[0095] (5) Warm bath in a 42℃ water bath for 20 minutes, gently stirring several times every 10 minutes during the process.

[0096] (6) Centrifuge at 12000 rpm for 30 s, collect the bacterial culture, discard the supernatant, add 8 mL of 0.9% NaCl solution, and resuspend the bacterial cells.

[0097] (7) Take 200 μL of the transformed yeast culture and spread it evenly on SD / -Leu and SD / -Leu / AbA culture dishes. The AbA concentration is the minimum concentration to inhibit self-activation (i.e., 700 ng / mL).

[0098] (8) Incubate upside down in a 30℃ incubator for 48–96 h.

[0099] Single colonies were selected for colony PCR identification using universal primers pGADT7F / R (sequences shown in Table 1). Samples with bright and single bands after electrophoresis were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were BLASTed in the NCBI database and analyzed.

[0100] The results showed that the strain transformed with the positive control p53AbAi+AD-53 (i.e., p53AbAi obtained by inserting the positive control 53 gene sequence into the pAbAi vector, and AD-53 obtained by inserting the positive control 53 gene sequence into the pGADT7 vector, constructed as described in Example 2) could grow on SD / -Leu / AbA medium; while the strain transformed with the negative control PIbbHLH2-1-pAbAi+pGADT7 empty vector could not grow on SD / -Leu / AbA medium. This indicates that the yeast one-hybrid assay can effectively detect whether a protein is bound to the promoter. PIbbHLH2-1-pAbAi+IbACLA-pGADT7 can grow on SD / -Leu / AbA medium. Figure 1 This indicates that the IbACLA protein can bind to the promoter IbbHLH2.

[0101] Example 6: Construction of a dual-luciferase reporter system vector

[0102] The gene sequence of the upstream regulatory factor IbACLA was constructed into the overexpression vector pGreenII 0029 62-SK (Shanghai Qincheng Biotechnology Co., Ltd., catalog number QCP0465), referred to as the effector plasmid. PIbbHLH2 was inserted into the front end of the luciferase in the vector pGreenII 0800-LUC (Kelei Biotechnology Co., Ltd., catalog number kl-zl-0808) as the reporter plasmid. Sac I and Xho I in the pGreenII 0029 62-SK vector and KpnI and Nco I in the pGreenII 0800-LUC vector were selected as restriction enzyme sites for the inserted target fragment. The primer sequences for constructing the vector are shown in Table 2: IbPb1-1 0800F / IbPb1-1 0800R and IbACLA-62-SKF / IbACLA-62-SKR. The construction method is as described in Example 2.

[0103] Example 7: Preparation and transformation of Arabidopsis protoplasts

[0104] 1. The preparation steps of Arabidopsis thaliana protoplasts are as follows:

[0105] (1) Prepare the enzyme hydrolysate and preheat it in a water bath at 55°C.

[0106] (2) Select wild-type Arabidopsis leaves four weeks after bolting, peel off the lower epidermis of the leaves and quickly put them into the enzymatic hydrolysate.

[0107] (3) At 25℃, shake at 50 rpm in the dark for 50 min until the mesophyll cells are completely digested. The morphology of the protoplasts can be observed under a microscope. When the cells are round and translucent, the condition is better.

[0108] (4) Dilute the enzyme solution with an equal volume of W5 solution, mix gently, wash the 75μm nylon mesh with water, and then filter the protoplasts after wetting it with W5 solution.

[0109] Preparation of W5 solution (100 mL):

[0110]

[0111]

[0112] (5) Centrifuge at 800 rpm for 2 min, remove as much supernatant as possible, and resuspend the protoplasts in 1 mL of W5 solution (repeat this step three times).

[0113] (6) Resuspend the protoplast in 1 mL of W5 solution and place it on ice for 30 min for later use.

[0114] 2. The transformation steps of Arabidopsis protoplasts are as follows:

[0115] (1) Add 10-20 μg of the target plasmid (IbACLA-pGreenII002962-SK recombinant plasmid and PIbbHLH2-pGreenII 0800-LUC recombinant plasmid constructed in Example 6) to a 2 mL EP tube, add 100 μL of Arabidopsis protoplasts, mix gently, and place on ice immediately after addition.

[0116] (2) Add 110 μL PEG / CaCl2, gently tap the centrifuge tube to mix, and incubate at room temperature for 10 min.

[0117] (3) Add 220 μL of W5 solution to ice, invert the centrifuge tube to mix it, and place it on ice for 1 min.

[0118] (4) Add 440 μL of W5 solution to the centrifuge tube again, gently invert it, and place it on ice for 1 min.

[0119] (5) Finally, add 880 μL of W5 solution to the centrifuge tube, invert to mix, and place on ice for 1 min.

[0120] (6) Centrifuge at 800 rpm for 3 min at 4℃ and remove the supernatant.

[0121] (7) The protoplasts were resuspended in 500 μL of W5 solution and cultured in the dark at 22°C for 16–20 h.

[0122] Example 8: Detection using a dual-luciferase reporter system

[0123] use The Reporter Assay (Promega) detects the activities of two luciferases, LUC and REN, using the following steps:

[0124] (1) Preparation of 100 μL 1×PLB lysis buffer: Add 20 μL 5×Passive Lysis Buffer to water to a final volume of 100 μL. Preparation of 10 mL LAR II: Add 10 mL of Luciferase Assay Buffer II (thawed on ice) to Luciferase Assay Substrate and gently shake to dissolve (can be stored at -20℃ for one month, or at -70℃ for one year). 100 μL Stop& Preparation of Reagent: Take 100 μL of Stop& Add 2μL of 50×Stop& buffer Substrate can be vortexed slightly to mix it (it can be left to stand at -20°C for 15 days).

[0125] (2) Take the Arabidopsis protoplast solution transformed in Example 7, centrifuge at 13200 rpm for 90 s at 4°C, and remove the W5 solution.

[0126] (3) Add 100 μL of 1×PLB lysis buffer, gently mix by blowing and transfer to a 24-well plate, place on a horizontal shaker, and shake at low speed for 15 min at room temperature.

[0127] (4) Collect the lysis buffer, transfer it to a 1.5 mL centrifuge tube, centrifuge at 13200 rpm for 10 min at 4 °C, and take 60 μL of the supernatant and place it on ice to test for luciferase.

[0128] (5) Under light-protected conditions, add 100 μL of LAR II to a black 96-well microplate, then add 20 μL of cell lysis buffer, and gently mix with a pipette tip 2-3 times to avoid generating bubbles.

[0129] (6) Place it in an enzyme reader to detect the enzyme activity of LUC and record the data.

[0130] (7) Remove the 96-well plate and add 100 μL of Stop& to the same well. Use the spray nozzle to gently mix the Reagent 2-3 times. Avoid strong light exposure during the entire process.

[0131] (8) Place it in an enzyme reader to detect the enzyme activity of REN and record the data.

[0132] (9) The experiment was repeated 3 times and the average value was taken. The activation effect of transcription factors on promoters was detected by comparing the LUC / REN ratio of different samples.

[0133] The results showed that IbACLA could enhance the activity of the IbbHLH2 promoter. Figure 2 This indicates that IbACLA can promote the expression of IbbHLH2.

[0134] Table 2 shows the primer sequences used for constructing the vectors (underlined lines indicate restriction enzyme sites).

[0135]

[0136]

[0137] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A purple sweet potato anthocyanin synthesis regulator IbACLA, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A gene encoding IbACLA, the anthocyanin synthesis regulator of purple sweet potato as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. A recombinant vector or recombinant bacteria containing the encoding gene as described in claim 2 or 3.

5. An expression cassette containing the encoding gene as described in claim 2 or 3.

6. An amplification primer for the anthocyanin synthesis regulator IbACLA of purple sweet potato as described in claim 1, characterized in that, The amplification primers are IbACLA-F: 5'-ATGGCACGCAAGAAGATCAGA-3' and IbACLA-R: 5'-TTATGCAGCTGCAGTGATGCA-3'.

7. The application of the purple sweet potato anthocyanin synthesis regulator IbACLA as described in claim 1 in promoting the expression of sweet potato IbbHLH2 transcription factor.

8. The application of the purple sweet potato anthocyanin synthesis regulator IbACLA as described in claim 1 in promoting sweet potato anthocyanin biosynthesis.

9. The application of the purple sweet potato anthocyanin synthesis regulator IbACLA as described in claim 1 in the breeding of sweet potato varieties with high anthocyanin content.

10. A method for promoting the synthesis of sweet potato anthocyanins, characterized in that, Overexpression of the regulatory factor IbACLA as described in claim 1 in sweet potato plants.

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