Vdbahd-at-dcr, a vaccinium dunifolium 6'-o-coffeate arbutin synthase gene, and application thereof

Abstract

This invention discloses a gene for synthase of 6′-O-caffeoyl arbutin from *Vaccinium camphora*. VdBAHD-AT-DCR Its applications. The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. This protein belongs to the BAHD acyltransferase family, contains the conserved motifs HXXXD and DFGWG, and can catalyze the production of 6′-O-caffeoylarbutin (CA) using caffeoyl-CoA as the acyl donor and β-arbutin as the acyl acceptor. In in vitro enzymatic reactions, the CA yield of 0.175 mg / mL of enzyme protein was 5.66 ng, with a transformation efficiency of 0.65%. After constructing a plant overexpression vector and transforming Arabidopsis thaliana, the CA content of the transgenic plants increased by 24.22%–32.42%, and the resistance to the pathogenic tomato strain DC3000 of *Pseudomonas syringae* was significantly enhanced; the optimal inhibitory concentration of CA against this pathogen was 50 μg / mL, with an inhibition rate of 96.27%. This invention provides a key gene resource for the green biomanufacturing of CA and plant disease-resistant breeding, and has important application value in the fields of medicine, cosmetics, and agriculture.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and enzyme engineering technology, specifically relating to a BAHD acyltransferase gene isolated from Vaccinium dunalianum. VdBAHD-AT-DCR The gene and its encoded protein, as well as its applications in catalyzing the biosynthesis of 6′-O-caffeoylarbutin and improving plant disease resistance. Background Technology

[0002] 6′-O-Caffeoylarbutin (CA) is a caffeoyl derivative of β-arbutin (β-Arb), possessing a variety of important pharmacological activities, including skin whitening, antioxidant, neuroprotective, hepatoprotective, and anti-COVID-19 effects. Studies have shown that CA's skin whitening activity is twice that of β-arbutin, while its cytotoxicity is only half that of β-arbutin, indicating broad application prospects in the cosmetics and pharmaceutical fields.

[0003] Blueberry (Vaccinium bracteatum) is a specialty tea tree species in southwestern China. Its leaf buds contain up to 31.76% (dry weight) of carboxylic acid (CA), making it the plant resource with the highest known CA content. However, CA is mainly found in the tender leaves and buds of Blueberry, and due to factors such as the long plant growth cycle and low extraction rate, large-scale extraction of CA from natural plants is difficult to meet market demand.

[0004] Currently, the biosynthetic mechanism of acetic acid (CA) remains unclear. In previous research, the applicant performed transcriptome sequencing on leaves of *Vaccinium bracteatum* at different ages (NCBI accession number: PRJNA1037676) and used weighted gene co-expression network analysis (WGCNA) to correlate differentially expressed genes with CA content. The study found a significant positive correlation between the expression level of a BAHD acyltransferase family gene and CA content (r=0.96, p<0.01). Based on the chemical structural relationship between β-Arb and CA, CA may be generated by acyltransferase-catalyzed caffeylation at the 6′-O-position of β-Arb. However, the key enzyme and its gene catalyzing this reaction have not yet been reported. Therefore, identifying and characterizing the key acyltransferase gene catalyzing this reaction is of great value for achieving large-scale in vitro biosynthesis of CA through enzyme engineering and fermentation engineering, while simultaneously reducing over-harvesting of *Vaccinium bracteatum* to protect wild resources. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides a 6′-O-caffeoylarbutin synthase gene VdBAHD-AT-DCR and its encoded protein screened and cloned from blueberry leaves. This gene encodes an acyltransferase that catalyzes the reaction of caffeoyl coenzyme A with β-arbutin to produce CA.

[0006] Another aspect of this invention provides the application of this gene in the in vitro enzymatic synthesis of CA, increasing the CA content in plants, and enhancing plant disease resistance.

[0007] The technical solution of the present invention is as follows: A 6′-O-caffeoylarbutin synthase gene VdBAHD-AT-DCR of *Vaccinium camphora*, wherein the nucleotide sequence of the gene is the sequence shown in SEQ ID NO.1, or the nucleotide sequence having at least 95% identity with SEQ ID NO.1 and encoding a protein having 6′-O-caffeoylarbutin synthase activity.

[0008] The protein encoded by the gene described in this invention has the amino acid sequence shown in SEQ ID NO.2, or is a protein that has been substituted, deleted, or added with conserved amino acids in SEQ ID NO.2 and still has 6′-O-caffeoylarbutin synthase activity.

[0009] The present invention provides a recombinant expression vector containing the aforementioned gene.

[0010] The present invention provides recombinant host cells containing the recombinant expression vector.

[0011] This invention provides the application of the gene or protein in the in vitro preparation of 6′-O-caffeoylarbutin, using caffeoyl coenzyme A as an acyl donor and β-arbutin as an acyl acceptor, and carrying out an acyl transfer reaction under the catalysis of the protein.

[0012] This invention provides a method for preparing 6′-O-caffeoylarbutin, comprising: using caffeoyl-CoA and β-arbutin as substrates, and reacting for 30-180 minutes at 25-37°C and pH 6.0-7.5 under the catalysis of the protein encoded by the gene described in claim 1 or the protein expressed by the recombinant vector described in claim 3, to generate 6′-O-caffeoylarbutin.

[0013] Furthermore, the reaction conditions were 30°C, pH 6.8, reaction time 120 minutes, and substrate concentration of 0.5 mM.

[0014] A method for increasing the content of 6′-O-caffeoylarbutin in plants, comprising introducing the gene into the plant genome and overexpressing it, wherein the plant is Arabidopsis thaliana or Vaccinium bracteatum.

[0015] This invention provides the application of the gene or protein in the cultivation of transgenic plants resistant to the pathogenic tomato strain DC3000 of Pseudomonas syringae.

[0016] This invention provides a method for cultivating transgenic plants with high 6′-O-caffeoylarbutin content and enhanced disease resistance, wherein the plants are Arabidopsis thaliana or Vaccinium bracteatum, comprising the following steps: (1) Construct a recombinant plant expression vector containing the gene described in claim 1; (2) Transform the recombinant plant expression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Transform plant cells or tissues using the recombinant Agrobacterium; (4) Transgenic plants with increased 6′-O-caffeoyl arbutin content and enhanced resistance to Pst DC3000 were screened and cultivated.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention marks the first cloning of the key acyltransferase gene VdBAHD-AT-DCR, catalyzing the synthesis of cyanobacteria (CA), from *Vaccinium bracteatum*, filling a gap in the gene pool for key enzymes in CA biosynthesis. By constructing a prokaryotic expression system, a recombinant protein with catalytic activity was successfully obtained, providing a novel biocatalyst for the in vitro enzymatic synthesis of CA. Through the construction of a plant overexpression vector and its transformation into *Arabidopsis thaliana*, it was demonstrated that this gene significantly increases CA content in plants (by 24.22%–32.42% in selected lines) and significantly enhances the transgenic plants' resistance to bacterial diseases (such as Pst DC3000). This provides a new gene resource for plant disease-resistant breeding, and this invention lays an important foundation for the green biomanufacturing of CA and the genetic improvement of plant disease resistance traits. Attached Figure Description

[0018] Figure 1 For the present invention VdBAHD-AT-DCR Gene PCR amplification agarose gel electrophoresis image; Figure 2 For the present invention VdBAHD-AT-DCR Phylogenetic tree analysis of the protein with BAHD acyltransferases from other species; Figure 3 For the present invention VdBAHD-AT-DCR Analysis diagram of conserved domains of the protein and conserved motifs characteristic of the BAHD family; Where, a represents the search results for conserved protein domains; b represents the multiple sequence alignment verification of conserved functional motifs; Figure 4 For the present invention VdBAHD-AT-DC Predicted secondary and tertiary structures of proteins; Figure 5 A graph showing the correlation between the expression level of the VdBAHD-AT-DCR gene and the CA content in different tissues of *Vaccinium bracteatum* leaves. Among them, 5a shows the differences in the natural contents of β-Arb and CA in different tissues (LB, TL, ML, FB, F) of *Vaccinium bracteatum* leaves; 5b shows the dynamic changes in the contents of β-Arb and CA in the leaves after treatment with five exogenous signaling substances (CK, MT, SA, ABA, MeJA) for 0 / 6 / 12 / 24 / 48 h; different lowercase letters on the column indicate significant differences between groups (P<0.05). Figure 6 Images of recombinant VdBAHD-AT-DCR-His protein detected by SDS-PAGE and Western blot. Figure 7 The graph shows the results of VdBAHD-AT-DCR acyltransferase activity assay. Figure 8 This is the UHPLC-MS / MS MRM chromatogram of the gradient CA standards of this invention; Figure 9 Subcellular localization results of VdBAHD-AT-DCR protein; Figure 10 This is a quantitative gene expression level diagram of Arabidopsis VdBAHD-AT-DCR-His in Example 8; Figure 11 Example 8: Transgenic Arabidopsis thaliana VdBAHD-AT-DCR Gene expression level detection graph; Figure 12 This is a chromatogram of HPLC peak elution times for β-Arb and CA in Example 9; Figure 13 The graph shows the contents of β-Arb(a) and CA(b) in Arabidopsis thaliana at different times in Example 9; Figure 14 This is a diagram showing the effect of CA on the root length of Arabidopsis thaliana Col-0 and VdBAHD-AT-DCR-His in Example 9; Figure 15 The effect of CA on the root length of Arabidopsis thaliana Col-0 (a) and VdBAHD-AT-DCR-His (b) under short light conditions is shown in Example 9. Figure 16 The effect of CA on the root length of Arabidopsis thaliana Col-0 (a) and VdBAHD-AT-DCR-His (b) under long light conditions in Example 9 is shown in Figure 9. Figure 17 This is a fluorescence detection image of Pst DC3000 cultured for 36 h under different concentrations of CA and β-Arb treatment in Example 10; Figure 18 This is a fluorescence intensity detection graph of P st DC3000 cultured for 36 h under different concentrations of CA and β-Arb treatment in Example 10; Figure 19 The infection status of Arabidopsis thaliana Col-0 and VdBAHD-AT-DCR-His Pst DC3000 in Example 11; Figure 20 The graph shows the antibacterial effect of different concentrations of CA on Pst DC3000. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the embodiments.

[0020] Information on biomaterials involved in the following examples: The camphor leaf blueberry (Vaccinium dunalianum) was collected from Lizhai, Tuanjie Township, Xishan District, Kunming City, Yunnan Province (102°34'20.291"; 25°05'1.80").

[0021] The vector pET-28a(+) was purchased from Sangon Biotech Co., Ltd.

[0022] The Escherichia coli Trelief™ 5α competent cells were purchased from Qingke Biotechnology Co., Ltd.

[0023] Arabidopsis thaliana Col-0 wild type.

[0024] Example 1 VdBAHD-AT-DCR Gene cloning 1. Plant materials The *Vaccinium camphora* plant material was collected on October 7, 2024, from Lizhai Orchard, Tuanjie Township, Xishan District, Kunming City, Yunnan Province (102°34'20.291"; 25°05'1.80"). Several two-year-old potted *Vaccinium camphora* seedlings were brought back to the laboratory greenhouse for cultivation, and leaf buds, young leaves, and mature leaf tissues were collected. The pET-28a(+) vector was purchased from Sangon Biotech Co., Ltd.; *E. coli* Trelief™ 5α competent cells were purchased from Qingke Biotechnology Co., Ltd.

[0025] 2. Total RNA extraction and cDNA synthesis Total RNA was extracted from *Vaccinium bracteatum* leaves using the OminiPlant RNA Kit (Dnase I). RNA integrity and concentration / purity (OD) were assessed using 1.2% agarose gel electrophoresis and a micro-ultraviolet spectrophotometer. 260 / OD 280 The ratio is between 1.8 and 2.0.

[0026] First-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser, with 1 μg of total RNA as a template. Reaction conditions: 37℃ for 15 min, 85℃ for 5 s. The resulting cDNA was stored at -20℃ for subsequent gene cloning and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis.

[0027] PCR amplification of target gene Candidate VdBAHD-AT-DCR gene sequences were obtained through screening using the Vaccinium bracteatum third-generation transcriptome database (NCBI accession number: PRJNA1037676). Specific primers were designed. F1: 5′-ATGGCAGCTGAAGTTG-3′; R1: 5′-TCAAGCCACCACTTCTTCCA-3′; PCR amplification was performed using cDNA from *Vaccinium bracteatum* leaves as a template. The PCR reaction system (50 μL) consisted of: 25 μL of 2×PrimeSTAR Max DNA Polymerase, 2 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, and ddH2O to a final volume of 50 μL. The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1.5 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0028] The PCR products were detected by 1.2% agarose gel electrophoresis, and the target band of approximately 1400 bp was recovered. Figure 1

[0029] Example 2 VdBAHD-AT-DCR Bioinformatics analysis of proteins Analysis using NCBI ORF Finder showed that SEQ ID NO:1 contains a 1344 bp open reading frame encoding 447 amino acids, as shown in SEQ ID NO:2. MAAEVAKEEEKVVGVKIQSKAHVKPNKPIGRRECQLITFDLPYIAFYYNQKLLIYKGGVSDFEGIVGRLKDGLGVVLEEYYQLAGKLGKYEDGVFKVEYDDDMDGVEVAVA AAEGIAIADLAVEEGTDKLKELVPYNGVLNVEGLRQPLLAVQITKLKDGGLALGCAFNHAILDGTATWHFMSSWAEICNGSRSISVPPFLDRTKARNTRVTLDISLPSSNGDV The protein TTDPASPPLREKVFKFPESAIDKIKSRVNSTPNPSGKPFSTFQALSSHVWRAITRARNLKPEDITVYCVFADCRKRVDPPMPESYFGNLIQAVFTGTAAGLLAAHPAEFAAGTIQKAIEAHDAKAIEARNSEWEANPKIFQWKDAGVNCVAVGSSPRFKVYEVDFGFGKPEIVRSGFNNRFDGMVYLYQGKSGGRSIDVEISLEGGAMENLEKDEEFLMEEVVA has a predicted molecular weight of 49.04 kDa, a theoretical isoelectric point (pI) of 5.36, and an instability index of 32.60 (<40), indicating that the protein is relatively stable. Its overall average hydrophilicity is -0.198, classifying it as a hydrophilic protein.

[0030] NCBI CD-Search conserved domain analysis indicated that VdBAHD-AT-DCR belongs to the transferase superfamily (pfam02458). Figure 3 As shown in a, the conserved HXXXD and DGWG motifs of the BAHD acyltransferase family are as follows: Figure 3 As shown in b, it has high homology with spermidine hydroxycinnamoyltransferase (SHT) and hydroxycinnamoyl-CoA: shikimic acid / quinic acid hydroxycinnamoyltransferase (HCT).

[0031] SignalP 5.0 prediction showed that the protein had no signal peptide at the N-terminus; TMHMM 2.0 predicted no transmembrane domain; NetPhos 3.1 predicted 23 high-confidence phosphorylation sites, including 16 serine sites (69.6%), 5 threonine sites (21.7%), and 2 tyrosine sites (8.7%).

[0032] like Figure 2 The phylogenetic tree analysis shown indicates that VdBAHD-AT-DCR is most closely related to the BAHD acyltransferase DCR protein of Ericaceae plants such as evergreen blueberry (KAH7853961.1), rhododendron (KAI8564856.1), and azalea (KAF7148805.1), and has high homology with flavonoid acyltransferases.

[0033] like Figure 4 As shown in Figure a, SOPMA secondary structure prediction reveals that this protein is predominantly composed of α-helices, β-sheets, and random coils. SWISS-MODEL homology modeling yielded the three-dimensional structure as shown in Figure a. Figure 4 As shown in b, the GMQE value is 0.91, sequence consistency is 0.53, and coverage is 0.99, indicating high model quality. Considering the conserved structural domains, phylogenetic relationships, and three-dimensional structure prediction results, it is believed that... VdBAHD-AT-DCR It encodes a typical BAHD acyltransferase family protein with the potential to catalyze acyl transfer reactions.

[0034] Example 3 VdBAHD-AT-DCR Tissue-specific gene expression qRT-PCR detection VdBAHD-AT-DCR Gene expression levels in different tissues of *Vaccinium bracteatum* leaves. Vd60S-2 The primer sequence for the internal reference gene is as follows: qVdBAHD-AT-DCR-F2: 5′-GTTCGGGAAGCCGGAGATAG-3′; qVdBAHD-AT-DCR-R2: 5′-CCCCCCACTCTTCCCCTGATA-3′; The results are as follows Figure 5 As shown in a, VdBAHD-AT-DCR Gene expression levels in different tissues showed the following distribution: leaf bud > young leaf > stem > mature leaf > fruit, consistent with the distribution trend of CA content, and the two were significantly positively correlated (r=0.96, p<0.01).

[0035] like Figure 5 As shown in b, after treating camphor blueberry leaves with 100 μM salicylic acid (SA) for 24 h, the amount of... VdBAHD-AT-DCRThe gene expression level was significantly increased, and the CA content increased by 15.29% simultaneously, further confirming the association between this gene and CA synthesis.

[0036] Example 4 Prokaryotic expression and purification of recombinant VdBAHD-AT-DCR protein

[0037] 1. Construction of prokaryotic expression vectors Using pET-28a(+) as the vector backbone, homologous recombination primers were designed: pET28a-VdBAHD-AT-DCR-F: 5′-GGGTCGCGGATCCATGGCAGCTGAAGTTGC-3′, pET28a-VdBAHD-AT-DCR-R: 5′-GCTTGTCGACGGAGCTCAGCCACCACTTCTTC-3′. The pET-28a(+) vector was double-digested with BamHI and SacI. The VdBAHD-AT-DCR gene ORF fragment was cloned into pET-28a(+) via homologous recombination to obtain the recombinant plasmid pET-28a-VdBAHD-AT-DCR (containing a 6×His tag). Plasmid extraction was performed using the PurePlasmid Mini Kit according to the manufacturer's instructions. Sequencing was performed by Qingke Biotechnology Co., Ltd. Plasmids with correct sequences were stored at -20℃ or -80℃ for later use.

[0038] 2. Protein induction expression and purification The recombinant plasmid was transformed into Escherichia coli BL21(DE3). Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD600≈0.6. IPTG was added to a final concentration of 0.9 mM and expression was induced at 16°C for 12 h.

[0039] Collect bacterial cells, resuspend them in lysis buffer containing 10 mM imidazole (50 mM NaH2PO4, 300 mM NaCl, pH 7.4), add lysozyme to a concentration of 1 mg / mL, and incubate on ice for 30 minutes. Sonicate the cells in an ice-water bath (2 seconds on, 4 seconds off, total 20 minutes), centrifuge at 14000 rpm for 30 minutes at 4°C, and collect the supernatant.

[0040] The recombinant protein was purified using His-tag Ni-NTA affinity chromatography. Washing and elution were performed with buffers containing 10 mM, 25 mM, and 500 mM imidazole, respectively. SDS-PAGE analysis showed the following results. Figure 6 As shown in a, a single target band (VdBAHD-AT-DCR-His fusion protein) was obtained at approximately 55 kDa, with a purified protein concentration of approximately 1.0–1.5 mg / mL. Figure 6As shown in b, Western blot further confirmed the expression of the recombinant protein.

[0041] Example 5: Detection of acyltransferase activity of VdBAHD-AT-DCR protein

[0042] The acyltransferase activity of VdBAHD-AT-DCR was determined using an acyltransferase activity assay kit. Principle: Acetyl-CoA reacts with butanol under the catalysis of acyltransferase to produce acetylbutyl ester and free CoA. The sulfhydryl group of free CoA reacts with DTNB to generate TNB, which has a characteristic absorption peak at 412 nm.

[0043] The reaction system contained acetyl-CoA, butanol, DTNB, and an appropriate amount of purified protein. The reaction was carried out at 30℃ for 120 min, and the absorbance change at 412 nm was detected. The results are shown in Figure 7. The OD412 value continuously increased with increasing reaction time, and the reaction rate slowed down and stabilized around 102 min. At 120 min, the net difference in OD412 between the experimental group and the control group was 0.1104, while the absorbance of the control group was close to 0. Based on the calculation formula for the 96-well plate provided with the kit, the acyltransferase activity of the recombinant protein was 22080 U / mg (1 U is defined as: 0.0005 units of catalytic absorbance change per minute per gram of sample per mL of reaction system at 37℃). The OD412 of the control group (with an equal volume of deionized water added) showed almost no fluctuation, confirming that VdBAHD−AT−DCR possesses acyltransferase catalytic activity.

[0044] Example 6: In vitro catalytic synthesis of CA using VdBAHD-AT-DCR

[0045] An in vitro enzymatic reaction system (total volume 400 μL) was constructed as follows: 100 mM Tris-HCl buffer (pH 6.8), 0.5 mM caffeoyl-CoA, 0.5 mM β-arbutin, and purified VdBAHD-AT-DCR protein at concentrations of 0, 0.0875, 0.175, and 0.35 mg / mL, respectively. After incubation at 30°C for 120 min, samples were taken (based on the enzyme activity curve in Example 5, 120 min was within the linear growth range of the enzymatic reaction, suitable for product quantification). The reaction was terminated by adding 2 volumes of ice-cold methanol. The mixture was centrifuged at 15000 rpm for 3 min to remove the precipitated protein. The supernatant was concentrated and reconstituted with 200 μL of methanol.

[0046] like Figure 8As shown in Table 1, UHPLC-MS / MS was used to quantify the product in the supernatant of the in vitro enzymatic reaction. Before detection, a quantitative standard curve was established using gradient CA standards: Y = 189.08X (R² = 0.999). (UHPLC-MS / MS detection of the target compound requires a known standard for that compound; pure CA extracted and purified from *Vaccinium bracteatum* was used as the standard in the pre-established assay system). The results are shown in Table 1. In the reaction system with 0.175 mg / mL enzyme protein, the CA yield was 5.66 ng (13.0 fmol), with a conversion efficiency of 0.65%. In contrast, the control group without enzyme showed only trace amounts of CA (0.3 ng / mL) remaining in the substrate, confirming that VdBAHD-AT-DCR can catalyze the synthesis of CA from caffeoyl-CoA and β-arbutin.

[0047] Table 1 Results of CA in vitro catalytic synthesis

[0048] Note: The conversion rate is obtained by dividing the molar amount of CA produced by the molar amount of substrate (0.2 µmol).

[0049] Example 7 Subcellular localization of VdBAHD-AT-DCR protein

[0050] 1. Construction of subcellular localization vectors Using pCAMBIA1300-GFP as the vector backbone, homologous recombination primers were designed: 1300-GFP-VdBAHD-AT-DCR-F: 5′-CAAGAGACAGGATCCGAATTCATGGCAGCTGAAGTTGC-3′; 1300-GFP-VdBAHD-AT-DCR-R: 5′-CGGTGCACTAGTGTCGACTCAAGCCACCACTTCTTC-3′; After linearization of pCAMBIA1300-GFP by double digestion with EcoRI and SalI, pCAMBIA1300-GFP was constructed via homologous recombination. VdBAHD-AT-DCR Recombinant vector.

[0051] 2. Instantaneous transformation of tobacco The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101. Positive single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin, and cultured at 28°C with shaking until OD600≈0.6. The cells were collected by centrifugation at 5000 rpm for 10 min, resuspended in osmotic medium (containing 10 mM MES, 150 μM acetylsyl syringone, pH 5.6) and diluted to OD600≈0.8.

[0052] Injected onto the underside of *Nicotiana benthamiana* leaves, the cells were incubated in the dark at 22°C for 24 hours, then transferred to a 16-hour light / 8-hour dark cycle for 2 days. GFP fluorescence signal was observed using a ZEISS fluorescence microscope. Results showed... Figure 9 As shown, green fluorescence is distributed in both the cytoplasm and the nucleus.

[0053] Example 8: Obtaining and Identifying Transgenic Arabidopsis

[0054] 1. Inflorescence immersion method for transformation Arabidopsis thaliana Columbia ecotype (Col-0) was transformed using an Agrobacterium-mediated inflorescence immersion method. GV3101 bacterial suspension containing pCAMBIA1300-GFP-VdBAHD-AT-DCR was resuspended in 5% sucrose solution (containing 0.02% Silwet L-77), OD600≈0.8. Arabidopsis inflorescences were immersed in the bacterial suspension for 30 s, incubated in the dark for 24 h, and then cultured normally. T0 generation seeds were harvested after seed maturity.

[0055] 2. Screening and identification of transgenic plants After disinfecting T1 generation seeds with 75% ethanol for 2 min and 5% sodium hypochlorite for 5 min, they were sown on 1 / 2 MS medium containing 50 μg / mL hygromycin to screen for resistant seedlings. Genomic DNA was extracted from the resistant seedlings for PCR identification, and positive plants were obtained. The T2 generation was further screened until the T3 generation to obtain homozygous lines.

[0056] 3. Detection of expression levels in transgenic plants Total RNA was extracted from leaves of T3 generation transgenic Arabidopsis thaliana and reverse transcribed into cDNA. The expression level of the VdBAHD-AT-DCR gene was detected by qRT-PCR (primers as in Example 3), and high-expression lines were screened.

[0057] like Figure 10 As shown, for transgenic Arabidopsis thaliana VdBAHD-AT-DCR-His-1 , 2 , 3 , 6 , 8 , 12 , 18 , 19 , 20 and 21 Quantitative analysis was performed on the strains. The strain with the lowest expression level was selected. VdBAHD-AT-DCR-His-6 The expression level was used as the standard value "1", and strain 8 showed the highest expression level. For example... Figure 11 As shown, Western blot analysis further confirmed that the expression level of VdBAHD-AT-DCR-His protein was the highest in strain 8, and this strain was selected for subsequent experiments.

[0058] Example 9: Effect of CA on Arabidopsis thaliana root length

[0059] Weigh out the transgenic Arabidopsis thaliana from Example 8 above. VdBAHD-AT-DCR-His-8 0.1 g each of wild-type Col-0 leaves were ground with liquid nitrogen, and then 1 mL of methanol was added for ultrasonic extraction for 30 min. The mixture was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was filtered through a 0.22 μm filter membrane for HPLC detection.

[0060] like Figure 12 As shown, the HPLC conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-0.5% acetic acid (35:65, v / v), flow rate: 1.0 mL / min, detection wavelength: 280 nm, column temperature: 30 °C. The β-arbutin standard curve was Y = 4066.6X - 1.572 (R² = 1.00), and the CA standard curve was Y = 12078X - 38.128 (R² = 0.999).

[0061] The results are as follows Figure 13 As shown, the β-arbutin content in transgenic Arabidopsis thaliana decreased by up to 80.28% over time compared to the wild type. Figure 13 a), CA content increased by a maximum of 32.42% ( Figure 13 (b) This confirms that VdBAHD-AT-DCR can efficiently catalyze the conversion of β-arbutin to CA in plants. Figure 14-16 This further demonstrates the effect of CA on root length.

[0062] Example 10: Detection of in vitro antibacterial activity of CA

[0063] 1. Strains culture The tomato pathogenic strain DC3000 of Pseudomonas syringae (containing a luciferase reporter gene) was inoculated into KB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 28°C with shaking.

[0064] 2. Antibacterial test Prepare KB liquid medium containing different concentrations of CA or β-arbutin (0, 0.5, 1, 2.5, 5, 10, 25, 50, 100 μg / mL), inoculate with an equal volume of Pst DC3000 bacterial suspension, and incubate at 28℃ for 24 h. Then, dilute the bacterial suspension 100-fold and spread it onto KB solid medium containing kanamycin. After 36 h, detect the colony fluorescence intensity as shown in the figure. Figure 17 As shown in the image.

[0065] The results show that... Figure 18As shown in Table 2, the inhibition rate of CA reached 96.27% at a concentration of 50 μg / mL and 98.99% at 100 μg / mL; while the inhibition rate of β-arbutin was only 54.07% at 100 μg / mL. This indicates that CA has a strong inhibitory effect on Pst DC3000, which is significantly better than that of β-arbutin.

[0066] Table 2 Antibacterial rates of different concentrations of CA and β-Arb

[0067] Example 11 Disease resistance identification of transgenic Arabidopsis thaliana Four-week-old transgenic Arabidopsis thaliana VdBAHD-AT-DCR-His-8 and wild-type Col-0 plants were sprayed with Pst DC3000 bacterial solution (OD600≈0.01), and the disease symptoms were observed after 3 days.

[0068] The fluorescence imaging results of the pathogen infection phenotype are shown in Figure 19: at 0 days of infection, wild-type Col-0 and... VdBAHD-AT-DCR-His Transgenic Arabidopsis leaves showed no lesions or pathogen fluorescence signals. Three days after infection, wild-type Col-0 leaves developed numerous yellow lesions with strong pathogen fluorescence signals and dense lesion distribution. In contrast, transgenic lines showed significantly fewer and smaller lesion areas and lower overall pathogen fluorescence intensity. Further analysis of infected leaf tissues, including grinding and gradient dilution plating, revealed pathogen gradient fluorescence imaging results (Figure 20). At the same dilution gradient, the number of viable Pst·DC3000 bacteria recovered from transgenic line leaves was significantly lower than that of wild-type, while the blank BC control group showed no obvious colony signals. These phenotypic and in vivo pathogen proliferation quantification results collectively confirm that overexpression... VdBAHD-AT-DCR The gene can enhance Arabidopsis' resistance to Pst·DC3000 by promoting CA accumulation.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A gene for synthase synthesis of 6′-O-caffeoylarbutin from *Vaccinium camphora*. VdBAHD-AT-DCR Its characteristics are, The nucleotide sequence of the gene is the sequence shown in SEQ ID NO.1, or the nucleotide sequence that has at least 95% identity with SEQ ID NO.1 and encodes a protein with 6′-O-caffeoylarbutin synthase activity.

2. The protein encoded by the gene according to claim 1, characterized in that, Its amino acid sequence is the sequence shown in SEQ ID NO.2, or a protein that has been substituted, deleted or added with conserved amino acids in SEQ ID NO.2 and still has 6′-O-caffeoylarbutin synthase activity.

3. A recombinant expression vector containing the gene of claim 1.

4. A recombinant host cell containing the recombinant expression vector of claim 3.

5. The use of the gene of claim 1 or the protein of claim 2 in the in vitro preparation of 6′-O-caffeoylarbutin, characterized in that, An acyl transfer reaction was carried out under the catalysis of the protein, using caffeoyl-CoA as the acyl donor and β-arbutin as the acyl acceptor.

6. A method for preparing 6′-O-caffeoylarbutin, characterized in that, include: Using caffeoyl-CoA and β-arbutin as substrates, 6′-O-caffeoylarbutin is generated by reacting at 25–37°C and pH 6.0–7.5 for 30–180 minutes under the catalysis of the protein encoded by the gene described in claim 1 or the protein expressed by the recombinant vector described in claim 3.

7. The method as described in claim 6, characterized in that, The reaction conditions were 30°C, pH 6.8, and 120 minutes, with a substrate concentration of 0.5 mM.

8. A method for increasing the content of 6′-O-caffeoylarbutin in plants, characterized in that, This includes introducing the gene of claim 1 into the genome of a plant and overexpressing it, wherein the plant is Arabidopsis thaliana or Vaccinium bracteatum.

9. The use of the gene of claim 1 or the protein of claim 2 in the cultivation of transgenic plants resistant to the pathogenic tomato strain DC3000 of Pseudomonas syringae.

10. A method for cultivating transgenic plants with high 6′-O-caffeoylarbutin content and enhanced disease resistance, characterized in that, The plant is Arabidopsis thaliana or Vaccinium camphora, and the steps include: (1) Construct a recombinant plant expression vector containing the gene described in claim 1; (2) Transform the recombinant plant expression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Transform plant cells or tissues using the recombinant Agrobacterium; (4) Transgenic plants with increased 6′-O-caffeoyl arbutin content and enhanced resistance to Pst DC3000 were screened and cultivated.

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