Flavonoid glycosyl transferase gene and application thereof

Abstract

The invention discloses a flavonoid glycosyl transferase gene and an application thereof. The gene sequence of the flavonoid glycosyl transferase is a nucleotide sequence for coding an amino acid sequence as shown in SEQ ID NO.2, or a sequence complementarily paired with the nucleotide sequence for coding the amino acid sequence as shown in SEQ ID NO.2. The flavonoid glycosyl transferase can be used for catalyzing myricetin to form myricetin 3-O-glucoside. The fusarium wilt resistance of a transgenic hairy root plant over-expressed by the flavonoid glycosyl transferase obtained by a transgenic means is obviously improved. The flavonoid glycoside transferase provided by the invention provides a candidate gene for biosynthesis of flavonoid glycoside (myricetin 3-O-glucoside), and can be applied to creation of new germplasm of high-resistance plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering and enzyme engineering, in particular, it relates to a flavonoid glycosyltransferase gene and application thereof. BACKGROUND

[0002] Flavonoids (UDP) play a key role in plants, such as attracting pollinating insects, resisting microbial infection and other environmental challenges. Importantly, flavonoids are a component of the plant immune response, which can enhance the ability of plants to resist diseases. In addition, it is believed that they help the ecological interactions between plants and their surroundings - including biotic and abiotic interactions. By using genetic, biochemical and molecular biology methods, the biosynthesis of flavonoids has become a widely characterized subject in many plant lineages. The biosynthesis of flavonoids is regulated both spatially and temporally during plant development. This regulation is influenced by a variety of environmental and endogenous stimuli, including pathogen attack, various stresses and plant growth regulators. Therefore, the research on the regulation of plant flavonoid biosynthesis and metabolism has gradually become a hot topic.

[0003] Vernicia, a genus in the family Euphorbiaceae, is an important source of industrial oil trees, especially known for its ability to extract tung oil from fruits. Among the species in this genus, Vernicia fordii (Chinese tung tree, V. fordii) is the main cultivated species due to its oil production. The oil extracted from Chinese tung tree seeds is widely used in various industrial applications, playing a key role in the manufacture of paints and coatings, inks, lubricants, synthetic rubber, and biodiesel. However, tung wilt caused by Fusarium. oxysporum f. sp. Fordii (Fof-1) seriously affects the growth and development of Chinese tung tree. F. oxysporum (Fusarium oxysporum) ranks among the top ten fungal plant diseases, which can cause vascular wilt in more than 120 plants including cotton, tomatoes, and bananas, causing huge economic losses. However, the specific resistance mechanisms of Chinese tung tree to Fof-1 are still largely unknown, and there is currently no effective strategy to completely control its outbreak and spread, which hinders the prevention and control of Chinese tung wilt and the advancement of Chinese tung wilt-resistant breeding. Therefore, it is of great significance to effectively inhibit the Chinese tung wilt fungus and further cultivate high-quality and high-resistance cultivated varieties.

[0004] Plant molecular breeding provides a new way for the directional breeding and genetic improvement of oil camphor. Mining and functional verification of key genes regulating disease resistance are the basis for plant molecular breeding. Flavonoids and their glycosylated derivatives are ubiquitous secondary metabolites in various plant species. These compounds are essential for the overall health and survival of plants. For example, they play a crucial role in plant defense mechanisms, helping to prevent the growth of pathogens and manage interactions between plants and their symbiotic partners. In addition, the glycosylation process has a variety of important functions in addition to plant defense functions. Glycosylation of anthocyanins at the 3-OH site is crucial to ensure their stability, thereby affecting the color characteristics of plants. UDP-dependent glycosyltransferases (UGTs) transfer activated sugar molecules to various substrates in plants during the final stage of flavonoid biosynthesis. The glycosylation process of flavonoids is usually catalyzed by stereospecific and site-specific UDP-glycosyltransferases, which play a key role in the resistance interactions between plants and their environment, including biological and abiotic stress. The function of UGTs is essential to promote the stability of metabolites.

[0005] UGTs have been extensively studied for their key role in disease resistance through the production of secondary metabolites such as flavonoids and flavonol glycosides. To date, UGTs from model and non-model plants have been the subject of research, and their functions have been characterized in terms of their regulation of several metabolic pathways. For example, TOGT1 is a specific UDP-glucosyltransferase (UGT) found in tobacco plants. This enzyme plays an important role in enhancing plant resistance to viral infection. It achieves this goal by altering the content of key antiviral compounds, namely scopoletin and its glucoside derivatives. Through this biochemical process, TOGT1 can help plants resist viruses. In Arabidopsis thaliana, UGT73C6 promotes the transfer of UDP-glucose to the 3-O-rhamnose and quercetin 3-O-rhamnose 7-hydroxyl positions of kaempferol and quercetin. In addition, soybean GmUGT79A6 and UGT91Q2 in tea have been identified as catalysts for flavonol glycoside and nerolidol glycoside biosynthesis, respectively, which in turn affects the cold resistance of plants. Given that different UGTs have different substrates and form plant-specific metabolites through glycosylation, different UGTs have different functions, and their molecular mechanisms in plant immunity have not been explored. SUMMARY

[0006] The application provides a flavonoid glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO. 2, or a sequence complementary to the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO. 2. Further, the nucleotide sequence of the flavonoid glycosyltransferase gene is shown in SEQ ID NO. 1.

[0007] The application provides a flavonoid glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] The application provides a recombinant vector, an expression cassette or a recombinant bacterium containing the aforementioned flavonoid glycosyltransferase gene.

[0009] The application provides an application of the aforementioned flavonoid glycosyltransferase gene, flavonoid glycosyltransferase, recombinant vector, expression cassette or recombinant bacterium in the production of myricetin 3-O-glucoside.

[0010] The application provides an application of the aforementioned flavonoid glycosyltransferase gene, flavonoid glycosyltransferase, recombinant vector, expression cassette or recombinant bacterium in the regulation of the resistance of a plant to fusarium wilt, characterized in that the pathogenic bacterium of the fusarium wilt is fusarium oxysporum.

[0011] Preferably, the plant is a tung tree.

[0012] The application provides a method for improving the fusarium wilt resistance of a plant, comprising one or more of the following (1)-(3):

[0013] (1) introducing the aforementioned flavonoid glycosyltransferase gene into a recipient plant by a transgenic means and making it express, to obtain a transgenic plant with higher fusarium wilt resistance than the recipient plant;

[0014] (2) increasing the expression amount of the aforementioned flavonoid glycosyltransferase in a recipient plant by a transgenic means, to obtain a transgenic plant with higher fusarium wilt resistance than the recipient plant;

[0015] (3) introducing the aforementioned recombinant vector, expression cassette or recombinant bacterium into a recipient plant by a transgenic means and increasing the expression amount of the flavonoid glycosyltransferase in the recipient plant, to obtain a transgenic plant with higher fusarium wilt resistance than the recipient plant.

[0016] Further, the aforementioned flavonoid glycosyltransferase gene is introduced into a recipient plant by an agrobacterium transformation method.

[0017] The transformed agrobacterium competent cell is introduced into a plant body by rootstock injection.

[0018] The beneficial effects of the present application include: the present application combines the root system of oil camphor after Fusarium oxysporum f. sp. vasinfectum (Fof-1) infection Metabolites such as flavonoids (important differential metabolites include: myricetin 3-O-glucoside and kaempferol-glucoside, catechin, etc.) The synthesis of significantly enhanced characteristics, 11 UGTs were screened according to the transcriptome analysis and showed high expression after Fof-1 infection. Through the correlation analysis (Pearson's r) between the level of flavonoid metabolites and the transcription level of 11 UGTs, it is found that the increase of flavonoid glycoside accumulation after infection may be related to the activity of VfUGT87H9. In addition, the VfUGT87H9 gene shows strong root-specific expression and rapid transcription induction after Fof-1 infection. The in vitro enzymatic test of the VfUGT87H9 recombinant protein obtained by recombinant expression proves the ability of VfUGT87H9 to catalyze the glucosylation reaction of myricetin to produce myricetin 3-O-glucoside. The transgenic plants overexpressing VfUGT87H9 show stronger disease resistance compared with the control, and the accumulation level of myricetin-glucoside in the roots of VfUGT87H9 overexpressing plants is significantly increased after Fof-1 infection. The in vitro antibacterial test of Fusarium oxysporum f. sp. vasinfectum (Fof-1) of oil camphor further shows that the flavonoid glycoside transferase VfUGT87H9 catalyzes the glucosylation reaction of the substrate myricetin to produce myricetin 3-O-glucoside, which can effectively inhibit the hyphal growth of Fusarium oxysporum. Therefore, the present application first proves that VfUGT87H9 is a flavonoid glycoside transferase, which actively regulates the disease resistance of plants by maintaining the flavonoid glycoside balance in the roots of oil camphor, especially the resistance of oil camphor to Fusarium wilt caused by Fusarium oxysporum. The flavonoid glycoside transferase VfUGT87H9 gene provided by the present application provides a candidate gene for the biosynthesis of flavonoid glycosides (myricetin 3-O-glucoside), which can be applied in the creation of high-resistance plant new germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Key differential metabolites of oil camphor roots before and after inoculation with Fof-1; wherein A is the phenotypic change of oil camphor before and after infection with the Fusarium wilt pathogen Fof-1, B is the enrichment analysis of differential metabolites in the F0 vs F1 group, C is the differential metabolite bar chart of the top 20 differential metabolites (key flavonoid metabolites);

[0020] Figure 2 Key UGTs of oil camphor roots based on the oil camphor genome after infection with the pathogen Fof-1; wherein A is the transcription level of UGT genes at different periods after infection with the pathogen Fof-1 (the red box marks the genes highly expressed at F1 period screened in the present application), B is the correlation between the expression of 11 UGTs and the content of differential metabolites (key metabolites) at F1 period after infection with the pathogen Fof-1;

[0021] Figure 3 A, WGCNA analysis of 6 UGTs in V. fordii flavonoid biosynthesis pathway, B, relative expression pattern heatmap of 11 VfUGTs in different tissues of V. fordii based on FPKM values;

[0022] Figure 4 A, relative expression of VfUGT87H9 gene in V. fordii roots at different time after Fof-1 infection, B, expression of VfUGT87H9 gene in different tissues (root tip, main root, lateral root, stem, leaf bud, young leaf, mature leaf, flower, fruit, pericarp, kernel) of V. fordii;

[0023] Figure 5 A, SDS-PAGE detection B, Western detection, C, UPLC analysis of VfUGT87H9 reaction products with myricetin as substrate, D, UPLC-QqQ-MS / MS analysis of enzyme reaction products;

[0024] Figure 6 A, phenotype of V. fordii transgenic control plants and overexpression-VfUGT87H9 transgenic plants, B, disease symptoms of control plants and overexpression-VfUGT87H9 transgenic plants without inoculation and after Fof-1 infection (8 dpi), C, relative expression level of VfUGT87H9 in roots of overexpression-VfUGT87H9 and control groups without inoculation and after Fof-1 infection (8 dpi), D, content of myricetin and myricetin 3-O-glucoside (ng.g -1 FW) in roots of V. fordii transgenic control plants and overexpression-VfUGT87H9 transgenic plants;

[0025] Figure 7 A, colony growth status chart at different time, B, colony diameter change curve chart at different time, C, effect of control group crude extract on Fof-1 mycelial morphology, D, effect of overexpression-VfUGT87H9 group crude extract on Fof-1 mycelial morphology, E, effect of myricetin-Glu on Fof-1 mycelial morphology;

[0026] Figure 8 Growth of Fof-1 on growth medium containing different concentrations of myricetin 3-O-glucoside. DETAILED DESCRIPTION

[0027] The present invention will be further illustrated and described below in conjunction with the embodiments, but the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1: Enrichment of flavonoid metabolites in tung oil root after infection with pathogen Fof-1

[0031] Pathogen Fof-1 infection of tung oil plants and root sample collection

[0032] a. Under sterile conditions, the pathogen of tung oil wilt, strain Fof-1 (the pathogen was isolated and preserved by the research team from infected tung oil roots in Guangxi Zhuang Autonomous Region) was inoculated onto PDA medium. After activation in a 28°C incubator for 3-5 days, 5 mm bacterial blocks were picked and inoculated into SNA medium. Incubated at 28°C for 7 days, a small amount of sterile water was added to the plate, and the spores were brushed off with a sterilized soft brush. The excess medium was filtered through three layers of gauze to prepare 1 L of a 2×10 6 / mL of Fof-1 spore suspension.

[0033] b. Take seedlings of susceptible tung oil varieties that have grown to 3-4 leaves. Carefully brush away the soil around the roots of the tung oil plants and wash them. Rinse three times with sterile water. Then, use a pipette to draw 10 mL of the Fusarium oxysporum spore suspension prepared in step a and evenly apply it around the roots. Replant the inoculated plants in pots containing sterilized organic matter (ensure the soil is moist enough to allow moisture to escape when pinched) and incubate them in an artificial climate chamber. The photoperiod is 16 days of light and 8 days of darkness, with humidity maintained at approximately 90% (to promote spore growth, maintain soil moisture and water regularly). Light intensity is 5000 lx. Observe and record the growth, phenotypic changes, and symptom responses of the tung oil plants every 24 hours until wilt symptoms develop.

[0034] like Figure 1 As shown in A, during the infection of the pathogen Fof-1 on susceptible tung varieties, tung was infected with wilt disease and symptoms appeared 8 days after inoculation (8 dpi, where dpi: days post-infestation) ( Figure 1The two pictures on the left in center A are before pathogen inoculation, and the two pictures on the right are 8 days after pathogen inoculation).

[0035] Tung oil tree wilt is a soil-borne disease. The pathogen invades tung oil tree through the roots and spreads upward through the vascular bundle to infect the trunk, branches, leaf tips, leaf veins and fruits, causing partial or whole plant root rot, branch withering, leaf wilt and yellowing, and ultimately causing the plant to wither and die. Therefore, the root is selected as the key focus site in this application, which is conducive to the cultivation of high-resistance and high-quality cultivars for tung oil tree wilt disease. The collection of samples is carried out according to the symptoms of tung oil tree. It is intended to collect (with the symptoms that appear during infection as indicators) tung oil tree root materials that are not infected (F0, within 1 day after inoculation) and in the early stage of infection (F1, the second day after inoculation to the stage where no symptoms appear), the middle stage of infection (F2, the fifth to eighth day after inoculation, lesions appear on the stem, and the leaves wilt slightly), and the late stage of infection (F3, after the 9th day, obvious chlorosis and necrosis appear on the leaves) for experiment. Roots collected at each stage were gently washed with water, dried with filter paper, and quickly ground in liquid nitrogen to ensure thorough grinding. The samples were kept frozen and not allowed to melt during the grinding process. The ground samples were labeled and stored in a -80°C freezer in the laboratory. They were then processed using a LABCONCO freeze dryer (freeze dryer: freeze dryer with a stable -40°C temperature and a system vacuum of 0.120 mBar or less) for subsequent metabolomic and transcriptomic analysis.

[0036] Our previous research has shown that the F1 stage is a critical period for tung oilseed plants to resist pathogens. Therefore, our research focused on metabolites that were recurrent in V. fordii and highly expressed during the F1 stage. Subsequently, we conducted an enrichment analysis of the differentially expressed metabolites and identified key pathways for the biosynthesis of phenylpropiodones, flavonoids, and other plant secondary metabolites. In particular, differentially expressed genes (DEGs) in the "flavonoid biosynthesis" pathway were significantly enriched during the F1 stage.

[0037] pass Figure 1 The enrichment analysis of differential metabolites in the F0 vs F1 groups of B revealed that the “flavonoid biosynthesis” pathway was significantly enriched. Figure 1Hesperidin, Myricetin 3-O-glucoside, Kaempferol-3-glucoside, (-)-Epicatechin, cyanidin-3-O-β-D-glucoside, Quercetin-3-β-D-glucoside were significantly increased in F1 stage. The results showed that V. fordii might produce specific differential metabolites (flavonoids) to resist Fof-1 infection in F1 stage. These unique flavonoids in V. fordii were speculated to play an important role in resisting Fof-1 infection.

[0038] Example 2, Preliminary screening of resistance genes to fusarium wilt in V. fordii

[0039] (1) Expression profile of different genes in V. fordii in response to Fof-1 infection

[0040] To study the expression profile of key resistance genes in the roots of V. fordii during Fof-1 infection, we normalized the expression level to the RPKM value of four pathogen infection stages: uninfected stage (F0), early infection stage (F1), mid-infection stage (F2), and late infection stage (F3). The differential expression of genes was visualized using the R library (heatmap) to illustrate the downregulation and upregulation of genes. After the roots of V. fordii were infected by the pathogen Fof-1, we performed enrichment analysis on the differentially upregulated expression genes in F1 stage, and then performed clustering analysis on the identified genes, as shown in Figure 2 Fig. 11A. As can be seen from the figure, 11 VfUGTs were highly expressed after V. fordii responded to Fof-1 (Fig. 11A, lower right corner of the cluster (red display) is the 11 VfUGTs we identified as highly expressed during F1 (key resistance period) development). Figure 2 Fig. 11A, lower right corner of the cluster (red display) is the 11 VfUGTs we identified as highly expressed during F1 (key resistance period) development). Therefore, these 11 VfUGTs were initially considered as candidate genes showing a response pattern to Fof-1 resistance in V. fordii.

[0041] (2) Correlation analysis of the transcription level of 11 VfUGTs in V. fordii and key metabolites

[0042] This application also compared the transcript levels of VfUGTs with the expression levels of different metabolites (key metabolites) through correlation coefficient (Pearson r) analysis to identify candidate VfUGTs associated with the accumulation of essential flavonoids. A correlation coefficient exceeding 0.6 during F1 and a gene expression level expressed in FPKM greater than 10 were used as criteria for strong correlation between VfUGTs and key metabolites. The results are shown in Figure 2. Figure 2 As shown in B. As can be seen from the figure, at the F1 stage, gene VfUGT87H9 was associated with several key differential compounds (including myricetin-3-O-glucoside, kaempferol-3-O-glucoside, hesperidin, quercetin-3-β-D-glucoside, epicatechin and cyanidin-3-O-glucoside).

[0043] Vf03g02396 is highly correlated with myricetin-3-O-glucoside; Vf01g00761 is correlated with quercetin-3-β-D-glucoside; Vf03g02399 is correlated with phloretin; Vf03g01371 is correlated with myricetin-3-O-glucoside; and Vf03g02397 is correlated with quercetin-3-β-D-glucoside. Therefore, genes VfUGT87H9, Vf03g02396, Vf01g00761, Vf03g02399, Vf03g01371, and Vf03g02397 can be preliminarily identified as candidate genes encoding flavonol glycoside UGTs and are speculated to be associated with resistance metabolites to tung wilt disease.

[0044] (3) Network analysis of six VfUGTs in response to Fof-1 infection in tung oil root

[0045] To identify key tung resistance genes associated with other genes, we used the WGCNA (weighted gene co-expression network analysis) R package to analyze weighted gene co-expression modules, with a cutoff value greater than 0.60. Subsequently, we constructed a core co-expression gene network of key tung wilt resistance genes (VfUGT87H9, Vf03g02396, Vf01g00761, Vf03g02399, Vf03g01371, and Vf03g02397) and their associated genes using Cytoscape software.

[0046] The results are as follows Figure 3 Figure A. Analysis using weighted gene co-expression network analysis (WGCNA) showed that functionally related co-expressed gene modules associated with Fof-1 resistance were divided into eight modules. VfUGT87H9 was associated with 874 genes in the co-expression network and was the most important hub gene (TOP 1 hub gene) among these key genes.

[0047] (4) Key VfUGTs gene expression pattern in different tissues

[0048] To determine the UGTs responsible for catalyzing flavonoid glycosylation in the roots of V. fordii, the expression patterns of key VfUGTs were also analyzed, and the results showed that these UGTs were widely expressed in different tissues of V. fordii, such as buds, main roots, lateral roots, stems, leaves, flowers, fruits and kernels (Fig. 2B). Among the different developmental stages of the V. fordii transcriptome in different tissues, VfUGT87H9 had the highest expression level at the F1 stage, and the highest expression level in the lateral roots at the F1 stage (Fig. 2C). Figure 3 This high expression in lateral roots is crucial, and it is speculated that VfUGT87H9 may be related to the resistance of Fof-1, and is related to the biosynthesis of root flavonol glycosides. Figure 4

[0049] Example 3, Catalytic substrate study of glycosyltransferase VfUGT87H9

[0050] (1) Construction of expression vector pET32a of glycosyltransferase VfUGT87H9 gene and transformation of prokaryotic cells, including the following steps:

[0051] The cloned target fragment (the nucleotide sequence of VfUGT87H9 obtained according to the genome sequencing of V. fordii, the sequence of which is shown as SEQ ID NO. 1) was transformed into the linearized pET32a vector by homologous recombination. The reaction conditions for ligation were as follows: after mixing the reaction system, it was incubated at 37°C for 30 min, and then at 20°C for 1 h. 5 μL of the reaction solution was added to 50 μL of E. coli DH5α competent cells, mixed, and incubated on ice for 30 min. Then, it was gently taken out, heated at 42°C for 60 s, immediately placed in ice water for 2 min, and then 500 μL of LB medium was added and incubated at 37°C for 1 h. 100 μL of the bacterial solution was spread on an LB plate containing Amp, and incubated overnight. The positive colonies selected by antibiotic (Amp) were used to extract plasmids. The constructed expression vector VfUGT87H9-pET32a was transformed into E. coli BL21 strain.

[0052] (2) Protein purification and protein detection of glycosyltransferase VfUGT87H9-pET32a recombinant protein, including the following steps:

[0053] ​The prepared E. coli (BL21) single colony containing recombinant plasmid VfUGT87H9-pET32a was inoculated into 3 mL of LB liquid medium (Amp resistant), and cultured at 37°C overnight and then stored at -20°C. 100 μL of the strain stored at -20°C was inoculated into 100 mL of LB liquid medium (Amp resistant), and cultured overnight. 100 mL of the bacterial liquid was inoculated into 2000 mL of LB liquid medium, and cultured at 37°C to an OD600 of about 0.6, and then the culture temperature was reduced to 30°C. IPTG inducer was added to a final concentration of 0.5 mM, and the culture was continued at 30°C for 3 h. The bacterial body was collected by centrifugation at 8000 rpm for 3 min, resuspended in 50 mL of pre-cooled NTA-0 buffer, and then ice-bathed for 30 min. The bacterial body was broken by ultrasonic wave, and the parameters were set as follows: power 200 W, working for 3 s, pausing for 4 s, and 99 cycles. The supernatant and the precipitate were collected by centrifugation at 16000 rpm at 4°C for 50 min. A small amount of the supernatant and the precipitate were subjected to SDS-PAGE detection, and the remaining supernatant and the precipitate were stored at 4°C.

[0054] The supernatant protein solution was filtered with a 0.22 μm filter for standby use. A Ni-NTA column was prepared. The supernatant protein solution was loaded at a flow rate of 1 mL / min. The column was washed with NTA-0 buffer (pH = 8.0) until the effluent did not contain protein (G250 detection solution did not change color). Elution was performed with 20 mM, 60 mM, 200 mM and 500 mM imidazole, respectively, and the eluate was collected until the G250 detection solution did not change color. The column material was washed with 3 times the column volume of deionized water, and the column was sealed with 20% ethanol. The collected eluate was subjected to SDS-PAGE electrophoresis detection and Western blot detection.

[0055] The results of SDS-PAGE and Western blot detection are shown in Figs. 1A and 1B. Figure 5 As shown in Fig. 1A, the size of the recombinant protein is about 71 kDa (including VfUGT87H9 protein 50 kDa + and vector GST tag 21 kDa), and the size of the recombinant protein is correct, indicating that the expression of the recombinant protein of glycosyltransferase VfUGT87H9 is successful. Figure 5

[0056] (3) Biochemical function of glycosyltransferase VfUGT87H9

[0057] ​As described in Example 2, step (2), there was a clear correlation between the expression level of VfUGT7H9 and the biosynthesis of various flavonol glycosides. These glycosides included cyanidin-3-O-glucoside, kaempferol-3-O-glucoside, hesperidin, quercetin-3-β-D-glucoside, epicatechin, and myricetin-3-O-glucoside. Transcription of glycosyltransferase genes is generally induced by their substrates. To understand the way VfUGT87H9 influences these important differentiating compounds, the present application evaluated its potential glycosylation activity on these key differentiating compounds.

[0058] The enzymatic reaction system was 0.5 M Tris-HCl (pH 8.0), 25 mM; MgSO4, 50 mM; KCl, 200 mM; DTT, 5 mM; UDP-glucose, 0.1 mM; and 10 mM of each substrate was added. 20 ng of purified VfUGT87H9 protein was added and incubated at 30°C for 3 h. The HPLC analysis conditions were as follows: instrument, Shimadzu LC-20AT; diode array detector, SPD-M20A; system controller, CBM-20A; degasser, DGU-20A3; and autosampler, SIL-20A.

[0059] Detection method: column, Agela Venusil C18 Plus 50*2.1 mm, 5 μm; flow rate, 0.4 mL / min; aqueous phase, 0.1% formic acid; organic phase, methanol; needle washing liquid, methanol; column oven temperature, 35°C; autosampler temperature, 8.0°C; injection needle height, 2.00 mm; autosampler needle washing settings, After Draw; autosampler needle washing volume, 200.0 μL; autosampler needle soaking time, 3.00 ms; autosampler injection volume, 5.00 μL; qualitative and quantitative analysis, each component was searched and matched using the NIST08 standard spectral library, fragment comparison, and qualitative analysis was performed in combination with related literature reports and the relative retention time of each component; quantitative analysis, the relative content of each peak area was calculated according to the peak area normalization method.

[0060] The results are shown in Table 1. Recombinant VfUGT87H9 only had glycosylation activity on myricetin, and no activity was observed on the other substrates.

[0061] Table 1. Enzymatic analysis of VfUGT87H9 protein on different substrates

[0062]

[0063] Note: '+' indicates that enzyme activity was detected; '-': indicates that no enzyme activity was detected

[0064] After the reaction was completed, the obtained product was carefully detected by liquid chromatography-mass spectrometry (LC-MS). The glycosylation product was identified by carefully comparing the retention time and mass spectrometry (MS / MS) data of the glycosylation product with the retention time and mass spectrometry (MS / MS) data of the identified real compound. In the case where the commercial standard of the expected product cannot be obtained, its identity is determined according to the detection results of similar substrates and the corresponding mass spectrum (D). Figure 5 D). Specifically, qualitative and quantitative analysis was performed: each component was searched and matched with NIST08 standard spectral library, fragment comparison, and qualitative analysis was performed in combination with related literature reports, relative retention time of each component, etc. The relative content of each peak area was calculated by peak area normalization method. The peak time and ion characteristic peak can be matched with NIST08 standard spectral library, so the enzyme catalysis product can be qualitatively analyzed; due to the limited enzyme content, the experiment cannot be quantified, only the enzyme catalytic activity of the enzyme can be proved.

[0065] The results are shown in Table 1. Figure 5 As shown in Table 1, VfUGT87H9 protein can catalyze myricetin to be converted into the corresponding glucoside product. When myricetin is used as the substrate, VfUGT87H9 enzyme effectively catalyzes myricetin to be converted into myricetin-3-O-glucoside.

[0066] Example 4, Gene cloning and overexpression of VfUGT87H9 in Jatropha curcas

[0067] (1) RNA extraction

[0068] The root tissue of Jatropha curcas was taken, ground into powder with liquid nitrogen, and then RNA was extracted using RN38 EASY spin plus plant RNA rapid extraction kit. The synthesis of the first strand of cDNA was performed according to the instructions of Goldenstar RT6 cDNA Synthesis Kit reverse transcription kit. According to the nucleotide sequence (SEQ ID NO. 1) of VfUGT87H9 obtained by Jatropha curcas genome sequencing, primers primer F (SEQ ID NO. 3) and primer R (SEQ ID NO. 4) were designed, and the cDNA sequence of VfUGT87H9 was amplified using MCLAB high-fidelity enzyme.

[0069] PCR reaction system:

[0070]

[0071] Reaction procedure: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 60°C annealing for 15 s, 72°C extension for 30 s, a total of 35 cycles; 72°C extension for 5 min.

[0072] Purification and recovery of DNA target band

[0073] 1% agarose gel electrophoresis was performed to separate the PCR products. The gel block containing the target fragment was weighed and then purified and recovered using a DNA gel recovery kit (the amplified target fragment was 1368 bp, and the sequence is shown in SEQ ID No: 1 nucleotides). The concentration of the recovered PCR product was detected. Figure 5

[0074] (2) The target fragment recovery product was ligated with the overexpression vector pCAMBIA1300S (GFP) to construct the overexpression vector VfUGT87H9-pCAMBIA1300S.

[0075] Reaction system:

[0076]

[0077] The sample was added at room temperature, gently mixed, centrifuged briefly, and incubated at 25°C for 10 min.

[0078] 5 μL of the ligation product was added to 50 μL of E. coli DH5α competent cells in an ice water mixture, mixed gently, and then incubated in an ice bath for 30 min; 42°C heat shock for 60 s; incubate on ice for 2-3 min; add 700 μL of LB liquid medium without antibiotics; 200 rpm, 37°C shaking culture for 1 h; centrifuge at 3,000 rpm for 1 min; discard the supernatant, suspend the bacterial cells, and then take an appropriate amount of bacterial liquid and spread on LB solid medium containing 50 mg·L -1 Kan, and incubate at 37°C overnight until single colonies grow.

[0079] PCR detection of positive clone bacteria liquid

[0080] A sterile gun tip was used to pick a single colony and transfer it to 1 mL of LB liquid medium containing 50 μg / mL of antibiotic, and incubate at 200 rpm and 37°C for 10 h until the bacterial liquid was turbid. The bacterial liquid of the positive clone was subjected to PCR identification using M13 universal primers. The reaction program was as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 45 s, for a total of 25 cycles; finally, 72°C extension for 5 min. After detecting the PCR product by 1% agarose gel electrophoresis, the bacterial liquid with a band size consistent with the target fragment was selected for sequencing. The sequences obtained by sequencing were compared with the VfUGT87H9 sequence of the oil camellia transcriptome using DNA MAN7 software. The comparison results were completely consistent. The nucleotide sequence of the glycosyltransferase gene VfUGT87H9 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0081] ​Plasmid extraction for VfUGT87H9-pCAMBIA1300S: Inoculate a single positive colony into 50 mL of LB medium (50 μg / mL Kan) and incubate overnight at 37°C with shaking at 200 rpm. Extract the plasmid using the Axygen Plasmid Mini DNA Extraction Kit (2-4 mL) for subsequent experiments. Centrifuge the remaining culture at 12,000 rpm for 1 minute, resuspend the pellet in LB medium containing 20% ​​glycerol, and store in aliquots at -80°C.

[0082] (3) Overexpression of VfUGT87H9 in Tung oil

[0083] Positive clones expressing the VfUGT87H9-pCAMBIA1300S vector were screened and transformed into Agrobacterium K599 competent cells. A single positive Agrobacterium colony was picked and cultured to an OD600 of 0.8-1.0, and then centrifuged at 2250 g for 1 min to collect the cells. –1 , MgCl2 10mM, MES 10mM) to resuspend the bacterial solution, adjust the OD600 to 0.6, and then mix the two collected resuspensions in a 1:1 volume ratio and let it stand at room temperature for 2 h (incubation) for use.

[0084] Tung wilt is a soil-borne disease that spreads through the roots of tung trees. Using the tung hairy root transformation technique, disease resistance genes can be overexpressed in the roots, facilitating disease resistance testing. Specifically, fully expanded tung seedlings, approximately one month old, were selected and the mixed bacterial solution was injected into the rhizomes of the tung trees using a 1 mL syringe (with the needle removed). A bacterial solution containing an empty pCAMBIA1300S-Vector (GFP) vector was used as a control. The bacterial solution containing the target vector VfUGT87H9-pCAMBIA1300S (GFP) (OE-VfUGT87H9) was injected simultaneously into the rhizomes of tung seedlings of similar growth conditions to ensure a consistent growth background. Twenty to 30 tung seedlings were injected per treatment, with at least four biological replicates. Samples were collected 30-40 days later, and transgenic hairy root-positive plants were identified and screened. RNA was extracted from the hairy roots grown in the control group and the VfUGT87H9 overexpression group, and the expression levels of the control and the VfUGT87H9 overexpression group were detected by qRT-PCR.

[0085] The results are as follows Figure 6 As shown. Figure 6As can be seen in Figure A, strong green fluorescence can be observed in the roots of all transgenic hairy root-positive plants, proving that both the control and overexpressed-VfUGT87H9 were introduced into tung trees and that both the control and overexpressed-VfUGT87H9 induced hairy roots. Screening of transgenic positive hairy roots of the control group and overexpressed-VfUGT87H9 showed no obvious visual difference between the two. Figure 6 As can be seen from Figure C, after the VfUGT87H9 gene was introduced into tung oil tree through transgenic hairy root technology, the gene was overexpressed in the roots of tung oil tree.

[0086] Example 5: Resistance detection of transgenic hairy root plants after inoculation with Fusarium oxysporum

[0087] (1) Inoculation of transgenic hairy root plants with Fusarium oxysporum

[0088] Dilute the spores of Fusarium wilt pathogen Fof-1 to 1×10 per ml with sterile distilled water. 4 Spore concentration: The Tung oil plants of the control group and the Tung oil plants of the OE-VfUGT87H9 group obtained in Example 4 were removed from the soil with their intact root systems, rinsed with sterile water, and then immersed in a Fof-1 spore solution for 30 minutes.

[0089] (2) Place the plant back in the pot and soil used for planting, and culture it in an artificial incubation climate box at 26°C, with a relative humidity exceeding 85% and a light-dark cycle of 16 hours and 8 hours, respectively.

[0090] (3) Observe and record the growth, phenotypic changes, and symptom responses of tung trees. Samples were taken from the control tung trees and the transgenic hairy roots of the VfUGT87H9 overexpression group before and 7 days after inoculation (the materials here were different from those in Example 4). RNA was subsequently extracted (control and overexpression-VfUGT87H9). qRT-PCR was used to detect the expression levels of the control and overexpression-VfUGT87H9 genes before and after inoculation with Fusarium oxysporum.

[0091] like Figure 6 As shown in Figure B, 8 days after inoculation with Fusarium oxysporum, the control group (Control+Fof-01, Control+Fof-02, Control+Fof-03) plants wilted first and showed symptoms of lesions and wilt. The plants in the overexpression-VfUGT87H9 group (OE+Fof-01, OE+Fof-02, OE+Fof-03) were in good overall growth condition (no obvious symptoms of wilt and lesions), indicating that compared with the control plants, the disease resistance of VfUGT87H9 transgenic hairy root plants was significantly enhanced. Figure 6As can be seen from the middle C figure, the expression of VfUGT87H9 gene increased significantly after inoculation with Fusarium oxysporum, and the increase in the overexpression-VfUGT87H9 group was more obvious than that in the control group. In addition, from the middle D figure, it can be seen that compared with the control group, the content of myricetin-3-O-glucoside in the overexpression-VfUGT87H9 group increased significantly, indicating that VfUGT87H9 generated myricetin-3-O-glucoside in Vernicia fordii to resist the invasion of Fusarium oxysporum. Figure 6 As can be seen from the middle C figure, the expression of VfUGT87H9 gene increased significantly after inoculation with Fusarium oxysporum, and the increase in the overexpression-VfUGT87H9 group was more obvious than that in the control group. In addition, from the middle D figure, it can be seen that compared with the control group, the content of myricetin-3-O-glucoside in the overexpression-VfUGT87H9 group increased significantly, indicating that VfUGT87H9 generated myricetin-3-O-glucoside in Vernicia fordii to resist the invasion of Fusarium oxysporum.

[0092] Example 6, Inhibition of the growth of Fusarium oxysporum by the crude extract of the transgenic hairy root plants of Vernicia fordii overexpressing VfUGT87H9 gene

[0093] The control group Vernicia fordii plant and the overexpression-VfUGT87H9 group Vernicia fordii seedling plant obtained in Example 4 were carefully uprooted and washed thoroughly with sterile water. After drying in an oven at 65°C for 72 hours, the plants were crushed, 5g was added to 50ml of 95% ethanol and ultrasonically treated for 2 hours, then filtered, and 25ml of anhydrous ethanol was added and ultrasonically treated for 1 hour. Then the extract was concentrated by evaporation using a rotary evaporator. Subsequently, it was diluted with water to 6ml, extracted with ethyl acetate, and formed a concentrated paste, which was then dissolved in ethyl acetate.

[0094] Meanwhile, myricetin-3-O-glucoside (Myricetin-Glu) was extracted from the roots of the control group Vernicia fordii plant and the overexpression-VfUGT87H9 group Vernicia fordii seedling plant. The specific method is as follows: After the roots were ground in liquid nitrogen, about 0.3g of sample was weighed and added to 3.0ml of methanol solution. Mix well by vortexing for 30s and stand overnight. Centrifuge at 4°C, 6000r for 5min, take the supernatant; the precipitate is ultrasonically extracted once with 3.0ml of methanol, centrifuged to take the supernatant, and the extract is combined. Then blow dry with nitrogen, redissolve in 1.0ml of methanol, pass through a 0.22μm organic phase filter membrane, and perform on-machine detection. The chromatographic conditions are as follows: chromatographic column: Agilent Poroshell 120SB-C18; mobile phase: A phase: 0.1% formic acid water; B phase: acetonitrile; flow rate: 0.40ml / min; column temperature: 30°C; injection volume: 5μL; gradient elution. The mass spectrometry parameters we used are as follows: ionization mode: ESI-gas curtain gas: 35psi; spray voltage: -4500v; nebulizing gas pressure: 60psi; auxiliary gas pressure: 60psi; atomization temperature: 500°C.

[0095] Equal amounts (each 400 ul) of crude extracts of control Jatropha curcas plants and overexpression-VfUGT87H9 group Jatropha curcas plants (OE-VfUGT87H9) and myricetin-3-O-glucoside (Myricetin-Glu) were uniformly smeared on PDA medium. Each treatment was repeated 4 times. Then, 3 mm diameter Fusarium oxysporum colonies were inoculated on the medium of each treatment, and the culture was incubated at 28°C. Subsequently, the growth of Fusarium oxysporum colonies was monitored, and their diameters were measured using the cross method.

[0096] The results are shown in A-B of Figure 7 The results are shown in A-B of

[0097] Fof-1 conidial suspensions with a total volume of 100 μL containing 10 6 spores per milliliter were prepared and incubated overnight. After the initial incubation period, the suspension was mixed with 100 milliliters of potato dextrose broth (PDB) liquid medium. The resulting mixture was then incubated at a constant temperature of 28°C for 24 hours to promote growth and development. Equal amounts (each 100 ul) of crude extracts of control Jatropha curcas plants, crude extracts of overexpression-VfUGT87H9 group Jatropha curcas plants (OE-VfUGT87H9), and myricetin-3-O-glucoside were added to the PDB liquid medium, temperature: 28°C, time: 24 hours, rotation speed: 80 rpm. To analyze the hyphal morphology using a scanning electron microscope (SEM), vacuum sections of the hyphal samples were first fixed in a 2.5% glutaraldehyde solution and incubated at a controlled temperature of 4°C overnight. After fixation, the Fof-1 hyphal samples were thoroughly rinsed three times with phosphate-buffered saline (PBS) to remove excess glutaraldehyde that could interfere with subsequent imaging. To further prepare the SEM samples, the samples were immersed in increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and finally 100%) through a series of consecutive ethanol gradients. Each concentration was maintained for 15 minutes, which helped effectively remove water from the samples. This dehydration step was crucial to prevent structural collapse and ensure optimal preservation of surface morphology during imaging. Subsequently, the Fof-1 hyphal samples were freeze-dried using a carbon dioxide critical point dryer. Finally, to enhance electrical conductivity and improve the imaging quality of the scanning electron microscope, a thin layer of gold palladium was sputter-coated onto the samples using a gold sputter coater. The Fof-1 hyphal sample organization was observed and electron micrographs were obtained using a scanning electron microscope (Phenom-world, Eindhoven, Netherlands).

[0098] The results are shown in A-B of Figure 7As shown in CE, the hyphae in the crude extract of the control group appeared intact and uniform, with no obvious damage. In contrast, the hyphae in the crude extract and myricetin-3-O-glucoside of the overexpression-VfUGT87H group showed obvious twisting and deformation, and some hyphae even broke. In addition, the fiber modifications attached to the outer cell wall were also significantly reduced. This shows that the flavonol glycosides in the VfUGT87H9 transgenic hairy roots have a direct inhibitory effect on Fof-1. More importantly, VfUGT87H9 plays a key role in the biosynthesis of flavonol glycosides (myricetin-3-O-glucoside) in tung oil tree roots, which helps plants resist the invasion of Fof-1.

[0099] Example 7: Antibacterial (Fof-1) Effect of Myricetin-3-O-Glucoside at Different Concentrations

[0100] To investigate the role of the flavonoid glycoside metabolite myricetin-3-O-glucoside in Fof-1 resistance, an in vitro inhibition assay was performed using DMSO (control) and myricetin-3-O-glucoside. Specifically, myricetin-3-O-glucoside at the specified concentration was added to a polystyrene-dioxide (PDA) plate. A 5-mm-diameter fungal colony was placed in the center of the PDA plate containing the desired concentration of myricetin-3-O-glucoside. The corresponding solvent, dimethyl sulfoxide (DMSO), was used as a control. Images were captured after 1-8 days of incubation at 28°C, and the diameter of the inhibition zone was measured daily to calculate the inhibition rate.

[0101] The results are as follows Figure 8 As shown in Figure 3, myricetin-3-O-glucoside had the strongest inhibitory effect on the mycelial growth of Fof-1 at a concentration of 19 μg / ml. It is noteworthy that myricetin-3-O-glucoside had a significant inhibitory activity on the growth of Fof-1.

Claims

1. A flavonoid glycosyltransferase gene, characterized in that: The sequence thereof is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

2. A flavonoid glycosyltransferase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, expression cassette or recombinant bacterium containing the flavonoid glycosyltransferase gene according to claim 1.

4. Use of the flavonoid glycosyltransferase gene according to claim 1, the flavonoid glycosyltransferase according to claim 2, or the recombinant vector, expression cassette or recombinant bacterium according to claim 3 in the production of myricetin 3-O-glucoside.

5. Use of the flavonoid glycosyltransferase gene according to claim 1, the flavonoid glycosyltransferase according to claim 2, or the recombinant vector, expression cassette or recombinant bacterium according to claim 3 in regulating plant resistance to Fusarium wilt disease, characterized in that: The pathogen of the wilt disease is Fusarium oxysporum.

6. The use according to claim 5, characterized in that The plant is tung oil tree.

7. A method for improving plant resistance to wilt disease, characterized in that: Include one or more of the following (1)-(3): (1) introducing the flavonoid glycosyltransferase gene of claim 1 into a recipient plant by genetic means and expressing it, thereby obtaining a transgenic plant having a higher resistance to wilt disease than the recipient plant; (2) increasing the expression level of the flavonoid glycosyltransferase according to claim 2 in a recipient plant by genetic means, thereby obtaining a transgenic plant having a higher resistance to wilt disease than the recipient plant; (3) The recombinant vector, expression cassette or recombinant bacteria described in claim 3 are introduced into a recipient plant by genetic means to obtain a transgenic plant having a higher resistance to wilt disease than the recipient plant.

8. The method according to claim 7, characterized in that The flavonoid glycosyltransferase gene is introduced into a recipient plant by using the Agrobacterium transformation method.

9. The method according to claim 8, characterized in that The transformed Agrobacterium competent cells are introduced into the plant body through root injection.

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