Glycosyl transferase gene UGT90A2 and application thereof

Abstract

The invention discloses a glycosyl transferase gene UGT90A2 and application of the glycosyl transferase gene UGT90A2. The amino acid sequence of protein coded by the glycosyl transferase gene UGT90A2 is as shown in SEQ ID NO. 2. The fusarium wilt resistance of a UGT90A2 overexpressed transgenic hairy root plant obtained through a transgenic means is obviously improved, and an inoculation experiment shows that the UGT90A2 overexpressed transgenic hairy root plant is good in overall growth state, has no obvious fusarium wilt disease spot symptom and can be applied to plant disease-resistant molecular breeding and creation of high-resistance plant new germplasm.

Description

Technical Field

[0001] The present application relates to the technical field of plant genetic engineering, and in particular to a glycosyltransferase gene UGT90A2 and an application thereof. Background Art

[0002] Tung oil tree (Vernicia fordii) is an important industrial oil tree species native to my country. It grows fast, bears early, has high yield and good quality tung oil. Tung oil produced by tung oil tree is one of the best plant drying oils. It has excellent properties such as corrosion resistance and acid and alkali resistance. It is widely used in industrial fields such as printing, inking, integrated electronic version, etc. With the continuous advancement of science and technology, people's development and utilization of tung oil tree has been continuously deepened, the number of deep-processed products has been increasing, the degree of comprehensive utilization has been getting higher and higher, and the demand for tung oil in the market has gradually increased. According to statistics, the annual domestic demand for tung oil is at least 200,000 tons, but the current output of tung oil in my country is far from meeting the demand, and there is a large gap. Therefore, tung oil tree has great development potential. In addition, due to the unique natural conditions of my country's tung oil producing areas, my country's tung oil output and quality rank first in the world, and it has always dominated the international market and is an important foreign exchange earning product for the country. However, the tung oil planting industry is deeply harmed by wilt disease.

[0003] Tung oil wilt disease is commonly known as "tung plague" and its pathogen is Fusarium oxysporum Schl. Since it was first discovered in Liuzhou, Guangxi in 1939, tung oil wilt disease has spread to more than 90 counties and cities in 8 provinces across the country, causing devastating damage to the tung oil industry, huge economic losses, and seriously threatening nearly one million hectares of tung oil forests and a large number of tung oil seedlings across the country. The mortality rate of tung oil wilt disease is as high as 80% or more, which seriously affects and hinders the production and development of tung oil, and is also one of the main reasons for the annual decline in tung oil production in my country. At present, there is no method that can completely cure the outbreak and spread of tung oil wilt disease, which hinders the prevention and control of tung oil wilt disease and the advancement of tung oil wilt disease resistance breeding. Therefore, how to further cultivate high-resistant and high-quality cultivars is of great significance.

[0004] Plant molecular breeding provides a new approach for the directional breeding and genetic improvement of tung oil tree. Discovering and regulating key genes that improve disease resistance and conducting functional verification are the basis for plant molecular breeding. Plants have evolved the remarkable ability to synthesize a large number of secondary metabolites, which are an indispensable component of plant growth, development, reproductive strategies and defense mechanisms. Secondary metabolites have long been known to interact with pathogens, highlighting their key role in mediating plant defense responses. Secondary metabolites produced by plants, such as alkaloids, flavonoids and triterpenoids, can inhibit pathogen growth or regulate plant immune signals. In these key physiological processes, UDP-glycosyltransferases (UGTs) play a pivotal role.

[0005] Among UGTs, gene families (indicated by the first number after the UGT designation, e.g., UGT95) are defined by sharing ~45% or greater amino acid sequence identity. Subfamilies within a UGT family (indicated by letters following the family number, e.g., UGT95B) have ~60% or greater amino acid sequence identity. In the model plant Arabidopsis thaliana, 123 UGTs were identified, belonging to 21 gene families, divided into 14 phylogenetic groups (AN) and outgroups. The contribution of UGTs under biotic stresses, especially in plant-pathogen interactions, has been described through the production of secondary metabolites, such as flavonoids, isoflavones, etc. For example, studies have found that the HvUGT-10W1 gene isolated from a barley variety (10W1) resistant to Fusarium head blight (FHB) is closely related to FHB resistance; overexpression of AtUGT85A5 in tobacco can improve tolerance to salt stress; and constitutive overexpression of the AtUGT73C5 gene improves the tolerance of Forsythia suspensa to the deoxynivalenol mycotoxin produced by F.oxysporum. However, there have been no reports on the UGT90s subfamily in disease resistance, nor have there been any studies on using the UGT family in tung oil to improve resistance to Fusarium wilt. Summary of the invention

[0006] The present invention provides a glycosyltransferase gene UGT90A2, and the amino acid sequence of the protein encoded by the glycosyltransferase gene UGT90A2 is shown in SEQ ID NO.2.

[0007] Furthermore, the glycosyltransferase gene UGT90A2 is derived from Aleurites fordii, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides a recombinant vector, an expression cassette or a recombinant bacterium containing the aforementioned glycosyltransferase gene UGT90A2.

[0009] The present invention also provides an application of the aforementioned glycosyltransferase gene UGT90A2 in breeding for improving plant resistance to wilt disease, wherein the pathogen of the wilt disease is Fusarium oxysporum.

[0010] The present invention also provides a method for improving the resistance of plants to wilt disease, wherein the glycosyltransferase gene UGT90A2 is introduced into a recipient plant by means of transgenic means and expressed, thereby obtaining a transgenic plant having a higher resistance to wilt disease than the recipient plant.

[0011] Specifically, the glycosyltransferase gene UGT90A2 is introduced into the recipient plant by Agrobacterium transformation. The transformed Agrobacterium competent cells are injected into the root and stem. The plant is preferably Tung oil tree.

[0012] The beneficial effects of the present invention include: the inventors found through preliminary studies that the F1 period (from the second day after inoculation to the stage without symptoms) is the critical period for tung oil plants to resist pathogens. By infecting tung oil plants with pathogenic bacteria Fof-1, KEGG enrichment analysis and heat map analysis were performed on genes with differential upregulated expression (F1 period), and it was found that the expression spectrum of UGT90A2 had a strong response to pathogens after Fof-1 infection, and further analysis found that UGT90A2 was the only hub gene among all genes whose expression was significantly increased during the critical disease resistance period (F1 period) of Fof-1 infection. Based on the above research, the present invention proposes a method for improving the resistance of tung oil plants to Fusarium wilt using the UGT glycosyltransferase gene (UGT90s subfamily) UGT90A2. The resistance to Fusarium wilt of transgenic hairy root tung oil plants with overexpression of UGT90A2 was significantly improved. Inoculation experiments showed that the overall growth state of transgenic hairy root tung oil plants with overexpression of UGT90A2 was good, and there were no obvious symptoms of wilt spots. Further studies have found that the UGT90A2 gene can enhance the resistance of tung oil tree to Fusarium oxysporum by catalyzing quercetin to improve the flavonoid metabolic pathway, and can be used in the molecular breeding of disease resistance in tung oil tree and the creation of new highly resistant tung oil tree germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the phenotypic symptoms of tung oil tree susceptible varieties at four stages (F0, F1, F2, and F3) after inoculation with the pathogen Fof-1;

[0014] Figure 2 After the pathogen Fof-1 infected the roots of tung oil trees, KEGG enrichment analysis and heat map analysis were performed on the differentially upregulated genes (F1 period);

[0015] Figure 3 This is an analysis of the specific expression of UGT90A2 in the root tissues of tung trees (susceptible varieties) at different stages (F0, F1, F2, F3) after inoculation of Fusarium oxysporum Fof-1;

[0016] Figure 4 This is the core co-expression gene network diagram of genes and their associated genes that respond to Fof-1 infection in tung oil tree roots;

[0017] Figure 5 It is the PCR amplification product of UGT90A2 in tung oil root in Example 2 of the present invention;

[0018] Figure 6 The figure is a transgenic hairy root tung oil plant overexpressing the UGT90A2 gene and a relative expression analysis figure; Figure A and C are the control group and OE-UGT90A2 group plants, respectively, Figure B and D are the enlarged root pictures of Figure A and C, respectively, and Figure E is the relative expression level of the UGT90A2 gene;

[0019] Figure 7 The wilting conditions of the transgenic hairy root plants in the control group and the OE-UGT90A2 group after inoculation with Fusarium oxysporum and the relative expression level of the UGT90A2 gene are analyzed; Figures A and B are the wilting conditions of the control group before and after inoculation, Figures C and D are the wilting conditions of the OE-UGT90A2 group before and after inoculation, and Figure E is the relative expression level of the UGT90A2 gene before and after inoculation with Fusarium oxysporum;

[0020] Figure 8 The graphs show the growth inhibition of Fusarium oxysporum by crude extracts of transgenic hairy root plants overexpressing the UGT90A2 gene of Tung oil trees, wherein Figure A shows the growth of colonies at different stages, and Figure B shows the changes in colony diameters at different stages;

[0021] Fig. 9 The SDS-PAGE and Western blot results of UGT90A2 protein prepared and purified by prokaryotic expression, wherein Figure A is the SDS-PAGE electrophoresis result, and Figure B is the Western blot result, Lane M: Marker; Lane 1: eluted protein;

[0022] Fig.10 Liquid chromatogram of quercetin catalyzed by glycosyltransferase UGT90A2. DETAILED DESCRIPTION

[0023] The present invention is further illustrated and described below in conjunction with embodiments, but the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1: Preliminary screening of genes resistant to Fusarium wilt in tung oil trees

[0027] (1) Infection of tung oil tree with pathogen Fof-1 and collection of root samples

[0028] a. Inoculate the tung oil wilt pathogen Fof-1 onto PDA medium under sterile conditions. After activation in a 28°C incubator for 3-5 days, pick a 5 mm bacterial block and inoculate it into SNA medium. Incubate at 28°C for 7 days. Add a small amount of sterile water to the plate, brush off the spores with a sterilized soft brush, and filter out the excess medium with three layers of gauze. Prepare 1L of 2×106 A suspension of Fusarium oxysporum spores at a concentration of 100 / mL.

[0029] b. Take seedlings of susceptible tung oil varieties that have grown to 3 to 4 leaves, carefully brush off the soil around the roots of tung oil plants, wash them, rinse them with sterile water 3 times, use a pipette to absorb 10 mL of the spore suspension of Fusarium oxysporum prepared in step a, and apply it evenly around the roots of tung oil plants. Replant the inoculated plants in pots containing sterilized organic matter (ensure the soil moisture, so that water overflows when you pinch the soil with your hands), and cultivate them in an artificial climate box. The photoperiod is still 16 days of light and 8 days of darkness. At this time, the humidity is controlled at about 90%, and the light intensity is 5000 lx. (To maintain better growth of spores, pay attention to maintaining soil moisture, water regularly, and water the soil and trays). Observe and record the growth of tung oil plants, phenotypic changes, and symptom reactions every 24 hours) until the symptoms of wilt disease occur.

[0030] like Figure 1 As shown in the figure, during the infection of the pathogen Fof-1 in the susceptible tung oil variety, tung oil was infected with wilt disease and symptoms appeared 7 days after inoculation (7 dpi, where dpi: days post-infestation). The phenotypic changes show that the F1 period is the key period for tung oil to resist pathogens.

[0031] Fusarium wilt of tung oil is a soil-borne disease. The pathogen invades tung oil through the roots and spreads upward through the vascular bundle to infect the trunk, branches, leaf tips, 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 in this application, which is conducive to the cultivation of high-resistance and high-quality cultivars for tung oil wilt disease. The collection of samples is carried out according to the symptoms of tung oil, and it is intended to collect (with the symptoms that appear during infection as an indicator) uninfected (F0, within 1 day after inoculation) and early infection (F1, the second day after inoculation to the stage where no symptoms appear), mid-infection (F2, the fifth to eighth day after inoculation, lesions appear in the stem, and the leaves wilt slightly), late infection (F3, after the 9th day, the leaves show obvious chlorosis and necrosis) tung oil root materials for experiment. The roots collected in each period were gently washed with water, and the water was absorbed by filter paper. They were quickly ground in liquid nitrogen to ensure that they were fully ground. The samples were kept frozen and not melted during the grinding process. The ground samples were marked and stored in a -80℃ refrigerator in the laboratory, and then processed with a LABCONCO freeze dryer (freeze dryer: cold-sink stable -40℃, system vacuum reaches or is lower than 0.120mBar), and transcriptome detection was performed later.

[0032] (2) Expression profiles of different genes in tung oil plants in response to Fof-1 infection

[0033] To investigate the expression profiles of key disease resistance genes in the roots of Tung oil tree during Fof-1 infection (SRA database (accession number SRP116874)). Gene expression levels were normalized to the RPKM values ​​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 down-regulation and up-regulation of genes.

[0034] like Figure 3 As shown in the figure, the expression of UGT90A2 was significantly upregulated after infection with the pathogen Fof-1, especially in the F1 period. Figure 2 The results of enrichment analysis and heat map analysis of differentially upregulated genes (F1 period) after the pathogen Fof-1 infected the roots of tung oil trees indicated that the UGT90A2 gene may play an important role in disease resistance after the pathogen Fof-1 infected tung oil trees, and clarified that UGT90A2 was associated with resistance to tung oil tree wilt.

[0035] (3) VfUGTs network analysis in response to Fof-1 infection in tung oil tree roots

[0036] To identify important tung oilseed disease resistance genes associated with other genes, we used the WGCNA (weighted gene co-expression network analysis) R package to analyze the weighted gene co-expression modules with a cutoff value greater than 0.60. Subsequently, the core co-expression gene network of the key genes for tung oilseed disease resistance and their associated genes was constructed using Cytoscape software.

[0037] The results are as follows Figure 4 As shown in the figure, the analysis using the weighted gene co-expression network analysis (WGCNA) method showed that the functionally related co-expressed gene modules associated with Fof-1 resistance were divided into six modules. UGT90A2 was associated with 2115 genes in the co-expression network. It is worth noting that UGT90A2 is the most important hub gene (TOP 1 Hub gene) among these key genes, which exerts its effect by associating with other genes.

[0038] Example 2: Gene cloning of UGT90A2 and overexpression in Tung oil plants

[0039] (1) RNA extraction

[0040] The root tissue of Tung oil tree was taken, ground into powder with liquid nitrogen, and RNA was extracted using the RN38 EASY spinplus plant RNA rapid extraction kit. The first-chain cDNA was synthesized according to the instructions of the Goldenstar RT6 cDNA Synthesis Kit. According to the nucleotide sequence of UGT90A2 obtained by sequencing the Tung oil tree genome, primers primer F (SEQ ID NO.3) and primer R (SEQ ID NO.4) were designed, and the cDNA sequence of UGT90A2 was amplified using MCLAB high-fidelity enzyme.

[0041] PCR reaction system:

[0042]

[0043] Reaction procedure: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min.

[0044] Purification and recovery of target DNA band

[0045] PCR products were separated by electrophoresis on 1% agarose gel. The gel block containing the target fragment was cut out, weighed, and purified and recovered using a DNA gel recovery kit (the amplified target fragment was 1467 bp, and the sequence was shown in SEQ ID No: 1 nucleotide) and the concentration of the recovered PCR product was detected ( Figure 5 ).

[0046] (2) The target fragment recovery product was connected with the overexpression vector pCAMBIA1300S (GFP) to construct the overexpression vector UGT90A2-pCAMBIA1300S

[0047] Reaction system:

[0048]

[0049] Add the sample at room temperature, mix gently, centrifuge briefly, and let stand at 25°C for 10 min.

[0050] Take 5 μL of the ligation product and add it to 50 μL of E. coli DH5α competent cells in an ice-water mixture. Flick gently to mix and place in an ice bath for 30 minutes. Heat shock at 42°C for 60 seconds. Let stand on ice for 2-3 minutes. Add 700 μL of LB liquid medium without antibiotics. Shake and culture at 200 rpm and 37°C for 1 hour. Centrifuge at 3,000 rpm for 1 minute. Discard the supernatant, suspend the cells and apply an appropriate amount of the bacterial solution on a plate containing 50 mg·L -1 Kan's LB solid medium was incubated at 37°C overnight until a single colony grew.

[0051] PCR detection of positive clones

[0052] Use a sterile pipette to pick up a single colony and transfer it to 1 mL of LB liquid culture medium containing 50 μg / mL antibiotics. Cultivate at 200 rpm and 37°C for 10 hours until the bacterial solution becomes turbid. Use M13 universal primers to perform PCR identification on the bacterial solution of positive clones. The reaction system is the same as step 2 (1). Reaction procedure: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 25 cycles; finally, extension at 72°C for 5 minutes. After detecting the PCR product by 1% agarose gel electrophoresis, select the bacterial solution with the same band size as the target fragment for sequencing. Use DNAMAN7 software to align the sequenced sequences with the UGT90A2 sequence of the tung oil plant transcriptome. The alignment results are completely consistent. The nucleotide sequence of the glycosyltransferase gene UGT90A2 is shown in SEQ ID NO.1, encoding 488 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0053] UGT90A2-pCAMBIA1300S plasmid extraction: Pick a single colony of positive clones and inoculate it into 50mL LB liquid medium (50μg / mL Kan), and culture it at 37℃ and 200rpm overnight. Take 2-4mL of bacterial solution and use Axygen Plasmid Mini DNA Extraction Kit to extract plasmid for subsequent experiments. Centrifuge the remaining bacterial solution at 12000rpm for 1min, resuspend the precipitate with LB liquid medium containing 20% ​​glycerol, and store it in aliquots at -80℃.

[0054] (3) Overexpression of UGT90A2 in Tung oil plants

[0055] Screen the positive clones of the overexpression vector UGT90A2-pCAMBIA1300S and transform them into Agrobacterium K599 competent cells. Pick the positive Agrobacterium colonies and culture them until the OD600 is 0.8-1.0. Then centrifuge at 2250g for 1min to collect the cells. –1 , MgCl210 mM, MES10 mM), the OD600 was adjusted to 0.8, and the resuspensions collected twice were mixed in a 1:1 volume ratio and allowed to stand at room temperature for 2 h (incubation) for use.

[0056] Tung oil wilt is a soil-borne disease that spreads through the roots of tung oil plants. The tung oil hairy root transformation technology can allow disease resistance genes to be overexpressed in the roots, which is conducive to the detection of disease resistance. Specifically, tung oil seedlings that have grown for about one month and are fully extended are selected, and the mixed bacterial solution is injected from the rhizomes of tung oil plants using a 1mL syringe (without the needle). The empty pCAMBIA1300S-Vector (GFP) bacterial solution is used as a control (Control), and the bacterial solution (OE-UGT90A2) containing the target vector UGT90A2-pCAMBIA1300S (GFP) to be detected is injected into the rhizomes of tung oil seedlings with the same growth conditions to ensure the same growth background. 20-30 tung oil seedlings are injected for each treatment, and more than 4 biological replicates are performed. Samples are taken after 30-40 days, and transgenic hairy root positive plants are identified and screened. RNA was extracted from the hairy roots grown in the control group and OE-UGT90A2 group, and the expression levels of the control and OE-UGT90A2D were detected by qRT-PCR.

[0057] The results are as follows Figure 6 As shown, from Figure 6 As can be seen in Figures AD, strong green fluorescence can be observed in the roots of all transgenic hairy root-positive plants, proving that both the control and OE-UGT90A2 were transferred into tung trees and that both the control and OE-UGT90A2 induced hairy roots. The transgenic positive hairy roots of the control group and OE-UGT90A2 were screened, and the results showed that there was no obvious visual difference between the two. Figure 6 As can be seen from Figure E, after the UGT90A2 gene was introduced into tung oil tree through transgenic hairy root technology, the gene was overexpressed in the roots of tung oil tree (** in the figure represents 0.001<=p<0.01).

[0058] Example 3: Resistance detection of transgenic hairy root plants after inoculation with Fusarium oxysporum

[0059] (1) Inoculation of transgenic hairy root plants with Fusarium oxysporum

[0060] Dilute the spores of Fusarium wilt pathogen Fof-1 to 1×10 per ml with sterile distilled water. 4 Concentration of spores. The Tung oil plants of the control group and the transgenic hairy root-positive Tung oil plants of the OE-UGT90A2 group obtained in Example 2 were removed from the soil together with their intact root systems, washed with sterile water, and then immersed in the Fof-1 spore solution for 30 minutes.

[0061] (2) Place the plants back into the pots and soil used for planting and culture them in an artificial incubation climate box at 26°C, with a relative humidity of more than 85% and a light-dark cycle of 16 hours and 8 hours, respectively.

[0062] (3) Observe and record the growth, phenotypic changes, and symptom responses of tung trees. Samples were taken from the tung trees in the control group and the transgenic hairy roots in the OE-UGT90A2 group before and 7 days after inoculation (the materials here are not the same as those in Implementation Case 3), and RNA was subsequently extracted (control and OE-UGT90A2). The expression levels of the control and OE-UGT90A2D before and after inoculation with Fusarium oxysporum were detected by qRT-PCR.

[0063] like Figure 7 As shown in the figure, on the 7th day after inoculation with Fusarium oxysporum, the plants in the control group wilted first and showed lesions and wilt symptoms, while the transgenic hairy root plants in the OE-UGT90A2 group had a good overall growth state (no obvious wilt lesions), indicating that compared with the control plants, the disease resistance of the UGT90A2 transgenic hairy root plants was significantly enhanced. Figure 7 As can be seen from Figure E, the expression level of UGT90A2 gene increased significantly after inoculation with Fusarium oxysporum, and the increase in expression in the OE-UGT90A2 group was more obvious than that in the control group (** in the figure represents 0.001<=p<0.01).

[0064] Example 4: Inhibitory effect of crude extracts from transgenic hairy root plants of Tung oil tree overexpressing UGT90A2 gene on the growth of Fusarium oxysporum

[0065] The tung oil plants of the control group and the transgenic hairy root-positive tung oil seedlings of the OE-UGT90A2 group obtained in Example 2 were carefully uprooted and thoroughly rinsed with sterile water. After drying in an oven at 65°C for 72 hours, the plants were crushed, 5 g of them were added to 50 ml of 95% ethanol and ultrasonically treated for 2 hours, then filtered, and then ultrasonically treated with 25 ml of anhydrous ethanol for 1 hour. The extract was then evaporated and concentrated using a rotary evaporator. It was then diluted with water to 6 ml and extracted with ethyl acetate to form a concentrated paste, which was then dissolved in ethyl acetate.

[0066] Equal amounts (400ul each) of crude extracts from the control group (Control) tung oil plants and the OE-UGT90A2 group of transgenic hairy root-positive tung oil seedlings were evenly applied on PDA culture medium. An equal amount of extractant ethyl acetate was applied as a blank control (Ethyl acetate). Each treatment was repeated 4 times. Then, a 3 mm diameter Fusarium oxysporum colony was inoculated on the culture medium of each treatment, and the culture was cultured at 28°C. Subsequently, the growth of the Fusarium oxysporum colony was monitored, and its diameter was measured using the cross method.

[0067] The results are as follows Figure 8As shown: Compared with the crude extracts of the blank control group and the control group Tung oil plants, the crude extracts of the transgenic hairy root positive Tung oil seedlings in the OE-UGT90A2 group had a significant inhibitory effect on the growth of Fusarium oxysporum.

[0068] Example 5. Study on the catalytic substrate of glycosyltransferase UGT90A2

[0069] (1) Constructing the expression vector pET32a of the glycosyltransferase UGT90A2 gene and transforming prokaryotic cells comprises the following steps:

[0070] The cloned target fragment was transformed into the pET32a linearized vector by homologous recombination. The ligation reaction conditions were as follows: the reaction system was mixed and incubated at 37°C for 30 minutes, then at 20°C for 1 hour, 5 μL of the reaction solution was added to 50 μL of E. coli DH5α competent cells, mixed and placed in an ice bath for 30 minutes, gently removed, heat-shocked at 42°C for 60 seconds, immediately placed in an ice bath for 2 minutes, and 500 μL of LB medium was added to culture at 37°C for 1 hour; 100 μL of the bacterial solution was spread on an LB plate containing Amp resistance and cultured overnight. The positive colonies obtained by antibiotic (Amp) screening were picked and the plasmid was extracted. The constructed expression vector UGT90A2-pET32a was transferred into the E. coli BL21 strain.

[0071] (2) Protein purification and protein detection of glycosyltransferase UGT90A2-pET32a recombinant protein include the following steps:

[0072] A single colony of Escherichia coli (BL21) containing the recombinant plasmid UGT90A2-pET32a was selected and placed in 3 mL LB liquid culture medium (Amp resistance), 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 LB liquid culture medium (Amp resistance) and cultured overnight with shaking; 100 mL of the bacterial solution was inoculated into 2000 mL LB liquid culture medium, cultured at 37°C until OD600 was about 0.6, and the culture temperature was lowered to 30°C; IPTG inducer was added to a final concentration of 0.5 mM, and the culture was continued at 30°C with shaking for 3 h; the bacteria were collected by centrifugation at 8000 rpm for 3 min, resuspended in 50 mL pre-cooled NTA-0 buffer, and placed in an ice bath for 30 min; the bacteria were broken by ultrasound, and the parameters were set to power 200 W, work 3 s, pause 4 s, and 99 cycles; 16000 rpm Centrifuge at 4°C for 50 min, collect the supernatant and precipitate; take a small amount of the supernatant and precipitate for SDS-PAGE detection, and store the remaining supernatant and precipitate at 4°C for later use.

[0073] The supernatant protein solution was filtered with a 0.22 μm filter for later 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 contained no protein (the G250 detection solution did not change color); eluted with 20 mM, 60 mM, 200 mM and 500 mM imidazole respectively, and the eluate was collected in sections until the G250 detection solution did not change color; the column material was washed with 3 times column volume of deionized water, and the column was sealed with 20% ethanol; the collected eluate was subjected to SDS-PAGE electrophoresis and Western blot detection, and the results were as follows: Fig. 9 As shown, the induced expression of recombinant protein of glycosyltransferase UGT90A2 was successful, and the recombinant protein size was correct, which enabled us to conduct further tests on the biochemical function of UGT90A2.

[0074] (3) Biochemical function of glycosyltransferase UGT90A2

[0075] Quercetin was used as substrate, and the enzymatic reaction system was: 0.5M Tris-HCl (PH8.0), 25mM; MgSO4, 50mM; KCl, 200mM; DTT, 5mM; UDP-glucose, 0.1mM; quercetin, 10mM. 20ng of purified protein was added and placed at 30℃ for 3h. HPLC analysis conditions: The instrument was Shimadzu LC-20AT, diode array detector SPD-M20A, system controller CBM-20A, degasser DGU-20A3, and autosampler SIL-20A.

[0076] Detection method: Chromatographic column: Agela Venusil C18 Plus 50*2.1mm, 5μm. Flow rate: 0.4mL / min; Aqueous phase: 0.1% formic acid water; Organic phase: Methanol; Needle washing solution: Methanol. Column oven temperature: 35℃, Autosampler temperature: 8.0℃. Injection needle height: 2.00mm, Autosampler cleaning setting: AfterDraw, Autosampler needle washing volume: 200.0μL. Autosampler needle immersion time for cleaning: 3.00ms, Autosampler injection volume: 5.00μL. Qualitative and quantitative: Each component was searched and matched with the NIST08 standard spectral library, fragments were compared, and qualitative analysis was performed in combination with relevant literature reports and the relative retention time of each component. Quantitative analysis was performed by calculating the relative content of each peak area by peak area normalization method.

[0077] result( Fig.10) showed that glycosyltransferase UGT90A2 can catalyze the synthesis of quercetin from quercetin, and the peak time and ion characteristic peaks can match the NIST08 standard spectral library retrieval, so the enzyme-catalyzed product can be qualitatively identified; due to the limited enzyme content, the experiment cannot be quantitatively performed, and it can only be proved that the enzyme has enzyme catalytic activity.

Claims

1. A glycosyltransferase gene UGT90A2, characterized in that: The amino acid sequence of the protein encoded by the glycosyltransferase gene UGT90A2 is shown in SEQ ID NO.

2.

2. The glycosyltransferase gene UGT90A2 according to claim 1, characterized in that The nucleotide sequence of the glycosyltransferase gene UGT90A2 is shown in SEQ ID NO.

1.

3. The glycosyltransferase gene UGT90A2 according to claim 1, characterized in that The glycosyltransferase gene UGT90A2 is derived from Tung oil tree.

4. A recombinant vector, expression cassette or recombinant bacterium containing the glycosyltransferase gene UGT90A2 according to any one of claims 1 to 3.

5. Use of the glycosyltransferase gene UGT90A2 according to any one of claims 1 to 3 in breeding for improving plant resistance to Fusarium wilt, wherein the pathogen of the Fusarium wilt is Fusarium oxysporum.

6. A method for improving plant resistance to wilt disease, characterized in that: The glycosyltransferase gene UGT90A2 is introduced into a recipient plant by means of transgenic means and expressed, thereby obtaining a transgenic plant having a higher resistance to wilt disease than the recipient plant.

7. The method according to claim 6, characterized in that The glycosyltransferase gene UGT90A2 is introduced into the recipient plant by using the Agrobacterium transformation method.

8. The method according to claim 7, characterized in that The transformed Agrobacterium competent cells are injected into the roots and shoots.

9. The method according to claim 6, characterized in that The plant is Tung oil tree.

Images