Application of transcription factor ppbhlh14 in promoting chlorogenic acid biosynthesis in peach

Abstract

The application provides application of a transcription factor PpbHLH14 in promoting accumulation of chlorogenic acid in peach fruits. The PpbHLH14 directly combines with a promoter of a key enzyme gene PpHCT5 of chlorogenic acid biosynthesis, and promotes biosynthesis of chlorogenic acid in the peach fruits. In addition, a PpMYB308-PpbHLH14 complex enhances transcription activation of a downstream target gene PpHCT5 by the PpbHLH14, and further improves the content of the chlorogenic acid in the peach fruits.

Description

Technical Field

[0001] This invention relates to the application of transcription factor PpbHLH14 in promoting the biosynthesis of chlorogenic acid, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Chlorogenic acid (CGA) is an important phenylpropanoid secondary metabolite found in many plants. Studies have shown that CGA is a beneficial component with multiple functions. On the one hand, CGA possesses antibacterial, anti-inflammatory, antitumor, hypoglycemic, and hypolipidemic biological activities, and can be used to prevent and treat metabolic syndrome and related diseases. On the other hand, CGA not only has various physiological activities such as free radical scavenging, antioxidant, and preservation, preventing enzymatic browning in fruits and vegetables, but also has broad-spectrum antibacterial activity, inhibiting and killing various bacteria, fungi, yeasts, molds, and amoebas, showing significant disease resistance in susceptible fruits and vegetables such as peaches, apples, kiwifruit, and tomatoes. Furthermore, CGA plays an important role in stress resistance, defending against various biotic and abiotic stresses such as low temperature, drought, ultraviolet radiation, and pests by increasing its ability to eliminate reactive oxygen species and free radicals. Currently, CGA has become a hot topic in bioactive substance research.

[0003] Chlorogenic acid is the most abundant phenolic acid in peaches, playing a crucial role in enhancing their antioxidant capacity, disease resistance, and post-harvest preservation. It represents a significant entry point for improving the nutritional and resistant qualities of peaches. Studies have shown that chlorogenic acid and neochlorogenic acid significantly enhance the antioxidant capacity of peaches, surpassing the effects of other phenolic compounds. In immature peaches, high levels of chlorogenic acid and neochlorogenic acid can reduce fruit susceptibility to diseases or enhance resistance to brown rot by interfering with fungal melanin production. During storage, chlorogenic acid treatment can effectively reduce the diameter and rot index of lesions caused by Penicillium mold, and by activating the salicylic acid signaling pathway, it enhances the activity and expression of defense-related enzymes, thereby improving post-harvest resistance to Penicillium mold. Furthermore, treatment with chlorogenic acid-chitosan conjugates effectively maintained the firmness of peach flesh after harvest, delayed fruit softening, reduced fruit rot rate, and also delayed peel discoloration, increased soluble solids content, and decreased titratable acid content. The beneficial effects of chlorogenic acid make it possible to cultivate new 'medicinal, edible, and resistant' peach varieties. However, the regulatory mechanism of chlorogenic acid biosynthesis in peach fruit has not yet been reported.

[0004] Transcription factors play a crucial role in the regulation of chlorogenic acid metabolism in plants. These transcription factors can specifically bind to the promoter elements of one or more structural genes, thereby regulating chlorogenic acid biosynthesis by activating or inhibiting the expression levels of these structural genes. These include transcription factors such as MYB, bHLH, WRKY, and ERF. bHLH-type transcription factors regulate chlorogenic acid biosynthesis. In dandelion, TabHLH1 can regulate the expression of structural genes such as TaHQT2, Ta4CL, TaCHI, and TaF3′H. In particular, TabHLH1 can directly bind to the bHLH-binding elements in the promoter regions of TaHQT2 and Ta4CL. The chlorogenic acid and luteolin content was significantly increased in TabHLH1-OE overexpression lines, while it was significantly decreased in TabHLH1-RNAi lines. In *Salvia miltiorrhiza*, SmbHLH60 is a bHLH transcription factor with negative regulatory effects. On the one hand, SmbHLH60 negatively regulates the biosynthesis of phenolic acids and anthocyanins in *Salvia miltiorrhiza* by inhibiting the expression of target genes such as SmTAT1 and SmDFR; on the other hand, SmbHLH60 can also form a heterodimer with SmMYC2 to antagonize the regulation of phenolic acid and anthocyanin biosynthesis. In suspension-cultured carrot cells, DcMYB3 and DcMYB5 can bind to the cis-element box-L and activate the transcription of DcPAL1 and DcPAL3, promoting chlorogenic acid accumulation. In *Lonicera japonica*, LmMYB15 can bind to and activate the promoters of Lm4CL, LmMYB3, and LmMYB4, thereby promoting chlorogenic acid biosynthesis and phenylpropanoid metabolism. Increasingly, studies report that some transcription factors do not act alone but interact with other proteins to form transcriptional complexes, jointly regulating plant secondary metabolism. For example, MYB often forms the MYB-bHLH-WD40 (MBW) complex with bHLH and WD40 proteins, synergistically regulating plant secondary metabolism. However, research on the mechanisms by which MYB and bHLH regulate chlorogenic acid biosynthesis has not been reported. Therefore, identifying and utilizing regulatory genes of chlorogenic acid biosynthesis can help improve the chlorogenic acid content and nutritional value of plants, and promote the application of chlorogenic acid secondary metabolism engineering in plants. Summary of the Invention

[0005] The purpose of this invention is to provide the application of transcription factor PpbHLH14 in promoting chlorogenic acid biosynthesis.

[0006] The technical solution adopted in this invention is as follows:

[0007] The use of a transcription factor PpbHLH14 in promoting the biosynthesis of chlorogenic acid, wherein the nucleotide sequence of the PpbHLH14 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0008] Furthermore, transient transformation of peach fruits with the PpbHLH14 gene resulted in higher chlorogenic acid content in peach fruits overexpressing the PpbHLH14 gene compared to those injected with an empty vector.

[0009] Furthermore, the expression level of the PpHCT5 gene in peach fruits overexpressed with PpbHLH14 was significantly upregulated.

[0010] Furthermore, PpbHLH14 can bind to the promoter of the PpHCT5 gene and activate its expression.

[0011] Furthermore, the promoter sequence of the PpHCT5 gene is shown in SEQ ID NO.3.

[0012] This invention also provides an application of the PpMYB308-PpbHLH14 complex in synergistically promoting the biosynthesis of aucubin.

[0013] Furthermore, the nucleotide sequence of the PpMYB308 gene is shown in SEQ ID NO.4, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.5.

[0014] Furthermore, PpbHLH14 and PpMYB308 were co-converted.

[0015] Furthermore, the interaction between PpbHLH14 and PpMYB308 enhanced the transcriptional activation of the downstream target gene PpHCT5 by PpbHLH14, thereby further increasing the chlorogenic acid content in peach fruit.

[0016] The beneficial effects of this invention are:

[0017] This invention provides an application of the transcription factor PpbHLH14 in promoting chlorogenic acid accumulation in peach fruit. PpbHLH14 directly binds to the promoter of PpHCT5, a key enzyme gene in chlorogenic acid biosynthesis, thereby promoting chlorogenic acid biosynthesis in peach fruit. Furthermore, the PpMYB308-PpbHLH14 complex enhances the transcriptional activation of the downstream target gene PpHCT5 by PpbHLH14, further increasing the chlorogenic acid content in peach fruit. Attached Figure Description

[0018] Figure 1 Sequence alignment of peach PpbHLH14 with homologous proteins from other species. Yellow rectangles represent the conserved bHLH-MYC_N region and HLH domain, respectively.

[0019] Figure 2 Phylogenetic relationship analysis of peach PpbHLH14 with homologous proteins in other species.

[0020] Figure 3 Subcellular localization of PpbHLH14. GFP indicates green fluorescent protein localization, red fluorescence indicates nuclear localization signal, and the scale bar represents 20 μm.

[0021] Figure 4 Transient overexpression of PpbHLH14 in peach fruit. (A) Chlorogenic acid content in PpbHLH14-OE peach fruit. CGA, chlorogenic acid; NCGA, neochlorogenic acid; Total CGAs, total chlorogenic acid. (B) Expression level of PpHCT5 gene in PpbHLH14-OE peach fruit.

[0022] Figure 5 To analyze the activation effect of PpbHLH14 on the PpHCT5 promoter in a dual-luciferase reporter assay.

[0023] Figure 6 To verify the ability of PpbHLH14 to bind to the PpHCT5 promoter in yeast one-hybrid assays.

[0024] Figure 7 To verify the ability of PpbHLH14 to bind to the PpHCT5 promoter G-box element in yeast one-hybrid assays.

[0025] Figure 8 Gel migration analysis showed that PpbHLH14 directly binds to the G-box element of the PpHCT5 promoter.

[0026] Figure 9 Sequence alignment of PpMYB308 with homologous proteins from other species. Yellow rectangles represent the R2 MYB and R3 MYB domains, and red rectangles represent the conserved bHLH-binding motif.

[0027] Figure 10 Transcriptional activity analysis of PpbHLH14. SD / -Trp, tryptophan-deficient SD medium; SD / -Trp / -His / -Ade, tryptophan, histidine, and adenine-deficient SD medium; SD / -Trp / -His / -Ade / X-α-Gal, tryptophan, histidine, and adenine-deficient SD medium supplemented with X-α-Gal.

[0028] Figure 11 The yeast two-hybrid experiment showed the interaction between PpMYB308 and PpbHLH14.

[0029] Figure 12 The luciferase complementation assay demonstrated the interaction between PpMYB308 and PpbHLH14.

[0030] Figure 13 To investigate the synergistic regulation of chlorogenic acid biosynthesis by PpMYB308 and PpbHLH14. (A) DLR experiments show the transcriptional regulatory effects of PpMYB308 and PpbHLH14, alone or in synergy, on the PpHCT5 gene. (B) Expression levels of the PpHCT5 gene in peach fruits after transient injection of PpMYB308 and PpbHLH14. (C) Chlorogenic acid content in peach fruits after transient injection of PpMYB308 and PpbHLH14. Detailed Implementation

[0031] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0032] Example 1: Identification of the PpbHLH14 gene

[0033] 1. Cloning of the PpbHLH14 gene

[0034] Using peach cDNA as a template, specific primers were designed based on the sequence of the PpbHLH14 gene for PCR amplification. The specific primer sequences are as follows:

[0035] Upstream primer PpbHLH14-F: 5'-ATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 6)

[0036] Downstream primer PpbHLH14-R: 5'-GTTGTACCATCTTTTTATAATAGCG-3' (SEQ ID No. 7)

[0037] The PCR reaction program was as follows: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. The nucleotide sequence of the amplified PpbHLH14 gene is shown in SEQ ID NO.1.

[0038] Further analysis of the above sequence using DNAMAN software revealed that the coding region of the PpbHLH14 gene cloned in this application is 1479 bp long, encoding 492 amino acids. It contains a conserved basic DNA-binding region (bHLH-MYC_N) at amino acids 20–208 and a conserved basic helical-loop-helical (HLH) domain at amino acids 305–354. Figure 1 Its encoded protein amino acid sequence is shown in SEQ ID NO.2.

[0039] 2. Phylogenetic relationships of PpbHLH14

[0040] A phylogenetic tree was constructed using the neighbor-join (NJ) method in MEGA software, with the validation parameter set to Bootstrap = 1000. Phylogenetic analysis showed that peach PpbHLH14 and amygdala PdMYC2 proteins are most closely related. Figure 2 MYC2 protein is known to be an important regulator of the accumulation of plant secondary metabolites.

[0041] 3. Subcellular localization of PpbHLH14

[0042] Based on the cDNA sequence of the amplified target gene PpbHLH14, Nco I and Spe I restriction sites were selected, and homologous recombination was used to construct a pCAMBIA1302 vector containing a GFP tag. The recombinant plasmid was named PpbHLH14-GFP, and the primer sequences for PCR amplification are as follows:

[0043] PpbHLH14-GFP-F: 5'-GACTCTTGAccatggATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 8)

[0044] PpbHLH14-GFP-R: 5'-TTCTCCTTTactagtGTTGTACCATCTTTTTATAATAGCG-3' (SEQ IDNo.9)

[0045] The recombinant plasmid PpbHLH14-GFP was transformed into GV3101 Agrobacterium competent cells. Single colonies of Agrobacterium were collected and cultured in liquid medium containing kanamycin and rifampicin resistance, with Agrobacterium containing the empty vector pCAMBIA1302 as a control. The cells were cultured at 28°C until the OD600 reached 0.8–1.0. The cells were collected by centrifugation at 5000 rpm for 10 min and resuspended in infection solution (10 mM MgCl2, 10 mM MES, pH 5.7, 200 μM acetylsyleugenone) and incubated at 28°C in the dark for 2 h. One-month-old wild-type Nicotiana benthamiana with 4–5 leaves were selected for Agrobacterium infection. The subcellular localization of fluorescent proteins was observed and photographed using a laser scanning confocal microscope (Zeiss LSM880, Germany). Figure 3 As shown, PpbHLH14 is a nuclear localization protein.

[0046] Example 2 Functional verification of the PpbHLH14 gene

[0047] 1. Construction of PpbHLH14 overexpression vector

[0048] First, based on the cDNA sequence of the target gene PpbHLH14 obtained by amplification in Example 1, BamHI and HindIII restriction sites were selected, and homologous recombination was used to construct it in the pGreen II 0029 62-SK vector. The recombinant plasmid was named 35S-PpbHLH14, and the primer sequences for PCR amplification are as follows:

[0049] 35S-PpbHLH14-F:5'-TAGAACTAGTggatccATGGAAGAAATTATGTCCTCTTGTT-3'(SEQ IDNo.10)

[0050] 35S-PpbHLH14-R:5'-GGTATCGATaagcttCTAGTTGTACCATCTTTTTATAATAGCG-3'(SEQID No.11)

[0051] 2. Transient transformation of PpbHLH14 peach fruit

[0052] The recombinant plasmid 35S-PpbHLH14 was transformed into GV3101 (pSoup) Agrobacterium competent cells. Single colonies of Agrobacterium were collected and cultured in liquid medium containing kanamycin and rifampicin resistance, with Agrobacterium containing the empty vector pGreen II 002962-SK as a control. The cells were cultured at 28°C until the OD600 reached 0.8–1.0. After centrifugation at 5000 rpm for 10 min, the cells were collected and resuspended in the infection solution (10 mM MgCl2, 10 mM MES, pH = 5.7, 200 μM acetylsyl syringone) and incubated at 28°C in the dark for 2 h.

[0053] 'Xiahui 5' peaches at their second expansion stage (70 DAFB) were selected as experimental material. The above suspensions were injected into the peach fruits using a 1 ml syringe. Due to individual differences in the experimental fruits and variations in Agrobacterium injection efficiency, symmetrical portions of the same peach fruit were used as the experimental and control groups, with three biological replicates, each containing eight peaches. Five days later, the injected peaches were immediately frozen in liquid nitrogen and stored at -80°C.

[0054] 3. Analysis of chlorogenic acid content and gene expression in peach fruits after transient injection of PpbHLH14

[0055] The extraction method of chlorogenic acid is as follows: First, grind the plant sample with liquid nitrogen, accurately weigh 1g of sample, and add 7mL of methanol (0.1% H3PO4) extractant; then, extract the mixed solution with ultrasound in the dark for 30min, and centrifuge at 10000r / min for 10min at 4℃; finally, take the supernatant, filter it through a 0.22μm organic filter, and then enter the chromatographic column.

[0056] The determination method for chlorogenic acid is as follows: Chlorogenic acid was determined using an Agilent 1260 Infinity HPLC system with an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) and a DAD detector. Mobile phase A was methanol (0.1% H3PO4), and mobile phase B was water (0.1% H3PO4). The injection volume was 5 μL, the flow rate was 1.0 mL / min, and the column temperature was 30 °C. Gradient elution was used under the following conditions: 0–22 min, 5%–71% A; 22–30 min, 71%–5% A; 30–35 min, 5% A. The detection wavelength for chlorogenic acid was 320 nm, and its content was calculated based on the peak area.

[0057] qRT-PCR is used to detect gene expression levels, according to... qPCR The reaction was performed according to the instructions for the Green Master Mix kit, and the 20 μL reaction volume used was as follows: qPCR The reagents included 10 μL Green Master Mix, 1 μL cDNA, 0.8 μL each of upstream and downstream primers, and 7.4 μL ddH2O. The ABI 7500 real-time PCR instrument (Applied Biosystems, USA) was used with the following reaction program: 95°C denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 30 s, for a total of 40 cycles, followed by a 72°C extension for 30 s. Peach PpTEF2 was used as the internal control gene. The real-time primer sequences are as follows:

[0058] q-PpHCT5-F: 5'-GCAGATGGCCTTTCTGGTCT-3' (SEQ ID No. 12)

[0059] q-PpHCT5-R: 5'-TGTGATCGAATGCAGGCTGT-3' (SEQ ID No. 13)

[0060] Results of instantaneous injection of peach fruit Figure 4 As shown in the figure. HPLC analysis results showed that, compared with peach fruits injected with the empty vector, overexpression of PpbHLH14 significantly induced the accumulation of chlorogenic acid and neochlorogenic acid in peach fruits, ultimately leading to a significant increase in the total chlorogenic acid content. Figure 4 A). Furthermore, using symmetrical parts of the same peach fruit as the experimental and control groups, three biological replicates were set up, each containing eight peaches, to detect the expression level of the PpHCT5 gene in the peach fruit. It was observed that the expression of the PpHCT5 gene was significantly upregulated in PpbHLH14-OE peach fruit. Figure 4 B).

[0061] Example 3: PpbHLH14 binds to the G-box element on the promoter to activate PpHCT5 expression.

[0062] 1. Dual-luciferase reporter assay to analyze the activation effect of PpbHLH14 on the PpHCT5 promoter.

[0063] The overexpression vector constructed in Example 2 was used as the effector vector 35S-PpbHLH14; a 2000bp fragment of the PpHCT5 promoter region was cloned and constructed into the pGreen II 0800-LUC vector as the reporter vector proPpHCT5-LUC. The primers for vector construction are shown below:

[0064] proPpHCT5-LUC-F: 5'-GACGGTATCGATaagcttGATTTTTTTTTTCTTCTATCA-3' (SEQ IDNo.14)

[0065] proPpHCT5-LUC-R: 5'-TCTAGAACTAGTggatccCTTTTTGCTGGGCCTCAC-3' (SEQ IDNo.15)

[0066] The promoter sequence of the PpHCT5 gene is shown in SEQ ID NO.3.

[0067] The recombinant plasmids were transformed into Agrobacterium GV3101 (pSoup). Agrobacterium injection solutions containing the reporter gene and effector gene were mixed at a ratio of 10:1 and injected into the underside of Tobacco Benzovia leaves using a sterile syringe. The empty vector pGreen II0029 was also used.

[0068] A mixed injection of 62-SK and proPpHCT5-LUC served as a negative control. Three days after injection, samples were taken from tobacco leaves using a 6mm diameter punch for dual-luciferase assay. The Reporter Assay System (Vazyme, Nanjing, China) was used to measure the luminescence signals of luciferase LUC from *Fireflya spp.* and luciferase REN from *Reniformis spp.* Figure 5 As shown, PpbHLH14 significantly induced PpHCT5 transcription.

[0069] 2. Yeast one-hybrid experiments were used to verify the ability of PpbHLH14 to bind to the PpHCT5 promoter G-box element.

[0070] (1) Construction of bait carriers and prey carriers

[0071] The full-length promoter P0 (-1 to -2000 bp) of PpHCT5 (approximately 2000 bp) was inserted into the pAbAi vector via Sac I and Xho I restriction sites to generate the bait plasmid pAbAi-proPpHCT5; the full-length CDS sequence of the transcription factor was inserted into the pGADT7 vector via EcoRI and BamHI restriction sites to generate the prey plasmid AD-PpbHLH14. The primers used for vector construction are shown below:

[0072] pAbAi-P0-F: 5'-AAGCTTGAATTCgagctcGATTTTTTTTTTCTTCTATCA-3' (SEQ ID No. 16)

[0073] pAbAi-P0-R: 5'-ATACAGAGCACATGCctcgagCTTTTTGCTGGGCCTCAC-3' (SEQ ID No. 17)

[0074] AD-PpbHLH14-F: 5'-GAGGCCAGTgaattcATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 18)

[0075] AD-PpbHLH14-R:5'-GAGCTCGATggatccCTAGTTGTACCATCTTTTTATAATAGCG-3'(SEQID No.19)

[0076] (2) Bait plasmid transformation of yeast competent cells

[0077] The pAbAi-proPpHCT5 vector was linearized and recovered using BbsI or BstB I, and then transformed into the Y1HGold yeast strain using the PEG / LiAc method. The transformed cells were plated on SD / -Ura medium and cultured at 30°C for 3–5 days. Single colonies were then picked and PCR colony reactions were performed using the MatchmakerInsertCheckPCRMix 1 kit. If a fragment of approximately 1.35kb+X was amplified, it indicated that the bait plasmid had been successfully integrated into the yeast Y1H Gold genome, thus identifying the bait strain.

[0078] (3) Screening for the lowest inhibitory concentration of AbA

[0079] After identifying the positive strain, select a single clone and dilute it to OD using 0.9% NaCl solution. 600 The concentration was set at 0.2. Then, 10 μL of bacterial culture was diluted 1, 10, and 100 times and spotted onto SD / -Ura plates containing a certain concentration gradient of AbA (adenosine A). The plates were incubated at 30°C, and the growth of yeast was monitored to determine the minimum inhibitory concentration of AbA.

[0080] (5) Preparation and interaction verification of yeast competent cells

[0081] Select a single clone of the successfully identified bait strain and incubate it overnight at 30°C in 2 mL of YPDA liquid medium. Add 100 μL of the bacterial culture to 50 mL of YPDA liquid medium and continue incubation at 30°C until OD (Organic Degradation) is achieved. 600 Once the pH reaches 0.4–0.5, centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend in 10 mL of sterile water. Centrifuge again, discard the supernatant, and resuspend in 2 mL of TE / LiAc solution. Finally, aliquot 100 μL into 1.5 mL sterile centrifuge tubes, ready for transformation.

[0082] (6) Verification of yeast heterozygosity-yeast interaction

[0083] The constructed AD-TF recombinant plasmid was transformed into bait yeast competent cells using the PEG / LiAc method. The cells were then plated on SD / -Leu / AbA plates and cultured at 30℃ for 3–5 days. Yeast growth was observed, with the empty vector pGADT7 used as a negative control. Figure 6 As shown, PpbHLH14 can bind to the promoter of PpHCT5.

[0084] Preferably, the truncated promoter fragments P1 (-1 to -500 bp), P2 (-501 to -968 bp), P3 (-1515 to -1893 bp), and P4 (-1894 to -2000 bp) of PpHCT5 are inserted into the pAbAi vector via Sac I and Xho I restriction sites to generate a bait plasmid. The above experiment is then repeated. The primers used for vector construction are shown below:

[0085] pAbAi-P1-F: 5'-AAGCTTGAATTCgagctcCGATATCTACCTATCACATCACGT-3' (SEQ ID No. 20)

[0086] pAbAi-P1-R: 5'-ATACAGAGCACATGCctcgagCTTTTTGCTGGGCCTCAC-3' (SEQ ID No. 21)

[0087] pAbAi-P2-F: 5'-AAGCTTGAATTCgagctcGTTATTGGTTCCAAGCATAAGTT-3' (SEQ ID No. 22)

[0088] pAbAi-P2-R: 5'-ATACAGAGCACATGCctcgagTATTCATTCTCACCCCCTGA-3' (SEQ ID No. 23)

[0089] pAbAi-P3-F: 5'-AAGCTTGAATTCgagctcGAGAAGCACGATAATGGGC-3' (SEQ ID No. 24)

[0090] pAbAi-P3-R: 5'-ATACAGAGCACATGCctcgagTCATTCTAACTCGAGGGTCTTG-3' (SEQ ID No. 25)

[0091] pAbAi-P4-F: 5'-AAGCTTGAATTCgagctcGATTTTTTTTTTCTTCTATCA-3' (SEQ ID No. 26)

[0092] pAbAi-P4-R: 5'-ATACAGAGCACATGCctcgagGCCCATTATCGTGCTTCTC-3' (SEQ ID No. 27)

[0093] like Figure 7 As shown, PpbHLH14 binds directly to the PpHCT5 promoter P1 fragment, which contains a potential binding site G-box element associated with bHLH.

[0094] 3. Gel migration analysis showed that PpbHLH14 directly binds to the G-box element of the PpHCT5 promoter.

[0095] The CDS sequence of PpbHLH14 was cloned and constructed into the pET-28a vector to generate the His-PpbHLH14 recombinant plasmid. This plasmid was transformed into BL21(DE3) cells, and the protein was induced with 0.5 mM IPTG and cultured at 16°C for 20 h. The fusion protein was purified using the Ni-agarose His-Tagged Purification Kit (CWbiotech, Beijing, China) for gel migration analysis. The primers used for vector construction are shown below:

[0096] His-PpbHLH14-F: 5'-ATGGGTCGCggatccATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 28)

[0097] His-PpbHLH14-R: 5'-TGCGGCCGCaagcttCTAGTTGTACCATCTTTTTATAATAGCG-3' (SEQID No. 29)

[0098] Based on the positional information of the cis-acting element in the PpHCT5 promoter sequence, a 30bp sequence containing the G-box element was biotin-labeled as a labeled probe, the unlabeled sequence was used as a cold probe, and the biotin-labeled mutant sequence was used as a competing probe. All probes were labeled and synthesized by Shanghai Sangon Biotech Co., Ltd. Furthermore, gel migration analysis was performed using the LightShift Chemiluminescent EMSA Kit (Beyotime, Shanghai, China). Figure 8 As shown, His-PpbHLH14 can bind to biotin probes containing G-box elements to form a translocation band, indicating that PpbHLH14 activates PpHCT5 expression by directly binding to G-box elements.

[0099] Example 4: PpMYB308 and PpbHLH14 interact to form a complex.

[0100] 1. Cloning of the PpMYB308 gene

[0101] Following the method described in Example 1, using peach cDNA as a template, specific primers were designed based on the sequence of the PpMYB308 gene for PCR amplification. The specific primer sequences are as follows:

[0102] Upstream primer PpMYB308-F: 5'-ATGGGAAGGGCTCCTTGT-3' (SEQ ID No. 30)

[0103] Downstream primer PpMYB308-R: 5'-TATCAGCAGTGACTCAGCAA-3' (SEQ ID No. 31)

[0104] The nucleotide sequence of the amplified PpMYB308 gene is shown in SEQ ID NO.4.

[0105] Further analysis of the above sequence using DNAMAN software revealed that the coding region of the cloned PpMYB308 gene is 879 bp long, encoding 292 amino acids. Its R3 repeat region contains a typical bHLH-interacting motif [(D / E)LX2(R / K)X3LX6LX3R], which can interact with the bHLH transcription factor. Figure 9 Its encoded protein amino acid sequence is shown in SEQ ID NO.5.

[0106] 2. Yeast two-hybrid experiments showed the interaction between PpMYB308 and PpbHLH14.

[0107] The CDS sequences of PpMYB308 and PpbHLH14 were cloned and constructed into the pGADT7 and pGBKT7 vectors, respectively. The primers for vector construction are as follows:

[0108] AD-PpMYB308-F: 5'-ATGGGTCGCggatccATGGGAAGGGCTCCTTGT-3' (SEQ ID No. 32)

[0109] AD-PpMYB308-R: 5'-GAGCTCGATggatccTATCAGCAGTGACTCAGCAA-3' (SEQ ID No. 33)

[0110] BD-PpbHLH14-F: 5'-GCCATGGAGGCCgaattcATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 34)

[0111] BD-PpbHLH14-R:5'-CAGGTCGACggatccCTAGTTGTACCATCTTTTTATAATAGCG-3'(SEQID No.35)

[0112] BD-PpbHLH14N-F: 5'-GCCATGGAGGCCgaattcATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 36)

[0113] BD-PpbHLH14N-R: 5'-CAGGTCGACggatccTTCCTCAACCTTTGCTTTGAG-3' (SEQ ID No. 37)

[0114] BD-PpbHLH14C-F: 5'-GCCATGGAGGCCgaattcTTGGAGGCTAAAATCCAACAA-3' (SEQ ID No. 38)

[0115] BD-PpbHLH14C-R: 5'-CAGGTCGACggatccCTAGTTGTACCATCTTTTTATAATAGCG-3' (SEQID No. 39)

[0116] The two vector plasmids were co-transformed into the Y2HGold yeast strain, plated on SD / -Leu / -Trp (DDO) medium, and cultured at 30°C. The resulting single colonies were cultured in YPDA liquid medium and then plated on DDO, SD / -Ade / -His / -Leu / -Trp (QDO), and SD / -Leu / -Trp / -His / -Ade / AbA / X-α-Gal (QDO / AbA / X-α-Gal) media, and cultured at 30°C for 3–5 days. Yeast growth was observed, with pGBKT7-p53+pGADT7-T serving as a positive control and pGBKT7-Lam+pGADT7-T as a negative control.

[0117] like Figure 10 As shown, the cutoff test indicates that the C-terminus of PpbHLH14 is self-activated, while the N-terminus is not. Figure 11 As shown, AD-PpMYB308 and BD-PpbHLH14 NThe co-transformed yeast cells were able to grow normally on QDO medium and turn blue on QDO / AbA / X-α-Gal medium, indicating that pMYB308 can interact with PpbHLH14.

[0118] 3. Luciferase complementation assays showed the interaction between PpMYB308 and PpbHLH14.

[0119] The CDS sequences of PpMYB308 and PpbHLH14 were cloned and constructed into the pCAMBIA1300-cLUC and pCAMBIA1300-nLUC vectors, respectively (the stop codon was removed from the nLUC vector). The primers for vector construction are as follows:

[0120] cLUC-PpMYB308-F: 5'-TCCCGGGGCggtaccATGGGAAGGGCTCCTTGT-3' (SEQ ID No. 40)

[0121] cLUC-PpMYB308-P: 5'-GCTCTGCAGgtcgacTATCAGCAGTGACTCAGCAA-3' (SEQ IDNo. ​​41)

[0122] cLUC-PpbHLH14-F: 5'-TCCCGGGGCggtaccATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 42)

[0123] cLUC-PpbHLH14-R:5'-GCTCTGCAGgtcgacCTAGTTGTACCATCTTTTTATAATAGCG-3'(SEQID No.43)

[0124] nLUC-PpMYB308-F: 5'-GACGAGCTCggtaccATGGGAAGGGCTCCTTGT-3' (SEQ ID No. 44)

[0125] nLUC-PpMYB308-R: 5'-CGAGATCTGgtcgacTATCAGCAGTGACTCAGCAA-3' (SEQ ID No. 45)

[0126] nLUC-PpbHLH14-F: 5'-GACGAGCTCggtaccATGGAAGAAATTATGTCCTCTTGTT-3' (SEQ ID No. 46)

[0127] nLUC-PpbHLH14-R: 5'-CGAGATCTGgtcgacGTTGTACCATCTTTTTATAATAGC-3' (SEQ ID No. 47)

[0128] The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells. Agrobacterium buffer was mixed at a 1:1 ratio and injected into the underside of tobacco leaves. Two days after injection, the underside of the tobacco leaves was sprayed with 1 mM D-luciferin fluorescein substrate reaction solution and reacted in the dark for 15 min. Finally, the reaction was performed using a plant in vivo imaging system (ChemiDoc). TM MP (Bio-Rad) was used to detect luminescence. The results are as follows: Figure 12 As shown, co-injection of PpMYB308 and PpbHLH14 into tobacco leaves produces a strong fluorescence signal, while PpMYB308 or PpbHLH14 alone, along with other negative controls, fails to produce a fluorescence signal, indicating that PpMYB308 and PpbHLH14 interact in vivo.

[0129] Example 5: PpMYB308 and PpbHLH14 synergistically regulate the biosynthesis of chlorogenic acid.

[0130] 1. Construction of PpMYB308 overexpression vector

[0131] Following the method for constructing the overexpression vector in Example 2, the CDS sequence of PpMYB308 was constructed into the pGreen II 0029 62-SK vector using homologous recombination. The recombinant plasmid was named 35S-PpMYB308, and the primer sequences for PCR amplification are as follows:

[0132] 35S-PpMYB308-F: 5'-TAGAACTAGTggatccATGGGAAGGGCTCCTTGT-3' (SEQ ID No. 48)

[0133] 35S-PpMYB308-R: 5'-GGTATCGATaagcttTTATATCAGCAGTGACTCAGCAA-3' (SEQ ID No. 49)

[0134] 2. Synergistic effect of PpMYB308 and PpbHLH14 on the transcriptional regulation of the PpHCT5 gene

[0135] Following the dual-luciferase reporter assay method in Example 3, Agrobacterium injection solutions containing the aforementioned reporter gene (proPpHCT5-LUC) and effector genes (35S-PpMYB308 and 35S-PpbHLH14) were mixed at a ratio of 10:1 and injected together into tobacco epidermal cells, and the results were detected. Figure 13 As shown in Figure A, co-expression of PpMYB308 and PpbHLH14 significantly enhanced the transcriptional activation effect on the PpHCT5 promoter compared with expression of PpMYB308 or PpbHLH14 alone.

[0136] 3. Analysis of chlorogenic acid content and gene expression in peach fruits after transient injection of PpMYB308 and PpbHLH14

[0137] Following the transient injection method for peach fruits in Example 2, *Agrobacterium EV* (empty vector), *PpMYB308-OE* (35S-PpMYB308), *PpbHLH14-OE* (35S-PpbHLH14), and *PpMYB308-OE+PpbHLH14-OE* were injected into peach fruits, with *PpMYB308-OE+PpbHLH14-OE* being co-injected with *PpMYB308* and *PpbHLH14* in equal proportions. Chlorogenic acid content and *PpHCT5* gene expression were analyzed. The transient injection experiment showed that, compared with peach fruits that transiently overexpressed *PpMYB308* or *PpbHLH14* alone, simultaneous injection of *PpMYB308* and *PpbHLH14* significantly increased the expression level of the *PpHCT5* gene. Figure 13 B). More importantly, co-injection of PpMYB308 and PpbHLH14 leads to a significant increase in the content of chlorogenic acid in peach fruit. Figure 13 C). In summary, the above results indicate that PpMYB308 and PpbHLH14 interact to form a transcriptional complex, which synergistically activates the expression of the PpHCT5 gene, thereby promoting the biosynthesis of chlorogenic acid in peach fruit.

Claims

1. The application of a transcription factor PpbHLH14 in promoting chlorogenic acid biosynthesis, characterized in that, The nucleotide sequence of the PpbHLH14 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Transiently transform peach fruits with the PpbHLH14 gene.

3. The application according to claim 2, characterized in that, PpbHLH14 overexpression results in higher chlorogenic acid content in peaches.

4. The application according to claim 2, characterized in that, The expression level of the PpHCT5 gene was significantly upregulated in peach fruits overexpressed with PpbHLH14.

5. The application according to claim 4, characterized in that, PpbHLH14 binds to the promoter of the PpHCT5 gene and activates the expression of the PpHCT5 gene.

6. The application according to claim 5, characterized in that, The promoter sequence of the PpHCT5 gene is shown in SEQ ID NO.

3.

7. An application of the PpMYB308-PpbHLH14 complex in synergistically promoting the biosynthesis of chlorogenic acid, wherein the nucleotide sequence of the PpMYB308 gene is shown in SEQ ID NO. 4, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO. 5; the nucleotide sequence of the PpbHLH14 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

8. The application according to claim 7, characterized in that, The interaction between PpbHLH14 and PpMYB308 enhances the transcriptional activation of the downstream target gene PpHCT5 by PpbHLH14, further increasing the chlorogenic acid content in peach fruit.

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