A salicylic acid glycosyltransferase and uses thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a salicylic acid glycosyltransferase and use thereof. The present application finds a new gene (nucleotide sequence as shown in SEQ ID NO. 3) in highland barley, the protein (amino acid sequence as shown in SEQ ID NO. 4) expressed by the gene can convert salicylic acid into salicylic acid glucoside, and improve the level of highland barley resistance to powdery mildew. The present application also performs in-vitro expression on the gene fragment, obtains the salicylic acid glycosyltransferase, and successfully prepares salicylic acid glucoside by taking glucose as a glycosyl donor and salicylic acid as a receptor in an in-vitro reaction. The present application also transfers the gene into tobacco, so that the salicylic acid glycosyltransferase is also expressed in the tobacco plant, further produces salicylic acid glucoside, and improves the value. The new gene, the recombinant vector, the recombinant bacteria, the protein and the transgenic plant provided by the present application all have good application prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a salicylate glycosyltransferase and its uses. Background Technology

[0002] Salicylic acid (SA) is a class of plant phenolic hormones that play a central role in plant immune defense. It is widely involved in the establishment of systemically acquired resistance (SAR), activation of disease resistance signaling pathways, regulation of pathogenesis-related protein (PR) expression, and various defense responses to biotic stresses such as fungal, bacterial, and viral infections. Based on its form and activity, salicylic acid can be divided into free salicylic acid (with biological activity) and bound salicylic acid (such as salicylate glucoside and salicylate methyl ester). Free salicylic acid is considered a key signaling molecule for initiating plant immune responses and plays an indispensable regulatory role in plant defense against pathogens. However, the level of active salicylic acid in plants must be precisely regulated to balance the defense response with normal growth and development, avoiding excessive energy consumption and growth inhibition caused by sustained high levels of immune activation. Plants dynamically regulate salicylic acid through multiple metabolic pathways, with glycosylation modification being one of the main mechanisms for inactivation and storage. The reaction is catalyzed by a class of enzymes called salicylic acid glycosyltransferases (SAGTs), which transfer a sugar group (such as glucose) from an activated sugar donor (such as UDP-glucose) to the hydroxyl group of salicylic acid to form salicylic acid glycosides.

[0003] Salicylic acid glycosides (such as salicylic acid-2-O-glucoside) are generally considered a reversible storage form. Under pathogen infection or immune stimulation conditions, these glycosides can be hydrolyzed by specific glycosidases to release free salicylic acid, thereby rapidly increasing the level of active hormones and enhancing plant disease resistance. Therefore, salicylic acid glycosides are not only important products of salicylic acid metabolism, but also constitute a rapidly mobilized "defense signal reserve" in plants, playing a key role in dynamically regulating the intensity and timing of immune responses. Although glycosylation plays an important role in salicylic acid metabolism and disease resistance regulation, the molecular mechanisms of glycosyltransferases that catalyze salicylic acid-specific glycosylation and their functions in disease resistance pathways are still poorly understood.

[0004] As an important highland grain crop, highland barley is often threatened by various diseases during its growth, which seriously affects its yield and quality. If the metabolic flow of salicylic acid in highland barley can be regulated to promote its conversion into an active or mobilizable reserve form, it is expected to enhance the basic disease resistance and immune response efficiency of highland barley. This has important application value for improving the stress resistance and stable yield of highland barley. Summary of the Invention

[0005] The purpose of this invention is to provide a salicylate glycosyltransferase and its uses.

[0006] The present invention provides a gene fragment, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a recombinant vector comprising a gene fragment with a nucleotide sequence as shown in SEQ ID NO.3.

[0008] The present invention also provides a recombinant cell comprising the above-described recombinant vector.

[0009] Preferably, the recombinant cells are selected from bacterial or plant cells.

[0010] The present invention also provides a protein encoded by a gene fragment with the sequence shown in SEQ ID NO.3.

[0011] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO.4.

[0012] The present invention also provides the use of the aforementioned gene fragments, recombinant vectors, recombinant cells, or proteins in the preparation of salicylate glucoside.

[0013] The present invention also provides the use of the aforementioned gene fragments, recombinant vectors, or recombinant cells in the preparation of transgenic plants for the production of salicylate glucoside.

[0014] The present invention also provides a method for preparing salicylate glucoside, which uses a protein with an amino acid sequence as shown in SEQ ID NO.4, with glucose as a glycosyl donor and salicylic acid as a glycosyl acceptor, to prepare salicylate glucoside.

[0015] This invention also provides a method for constructing a transgenic plant that produces salicylate glucoside, comprising the following steps: Take the gene fragment with the nucleotide sequence shown in SEQ ID NO.3, transform it into a plant, and obtain a plant that expresses the protein with the amino acid sequence shown in SEQ ID NO.4.

[0016] This invention discovers a novel gene in highland barley—the highland barley salicylate glycosyltransferase gene (nucleotide sequence shown in SEQ ID NO. 3). The protein expressed by this gene—salicylate glycosyltransferase (amino acid sequence shown in SEQ ID NO. 4)—can convert salicylic acid into salicylate glucoside, thereby improving the stress resistance of highland barley. This invention also obtained salicylate glycosyltransferase through in vitro expression of this gene fragment. In the in vitro reaction, glucose was successfully used as the glycosyl donor and salicylic acid as the acceptor to prepare salicylate glucoside. This invention further transfers this gene into tobacco, enabling the tobacco plant to also express salicylate glycosyltransferase, further producing salicylate glucoside and increasing its value. The novel gene provided by this invention, along with its recombinant vector, recombinant bacteria, protein, and transgenic plants, all have promising application prospects.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 Electrophoresis image of purified HAVUSG2388800 protein (protein size 78.4 kDa).

[0020] Figure 2 The in vitro functional identification of HOVUSG2388800 is as follows: A is the in vitro enzyme activity analysis of the target protein HOVUSG2388800; B is the identification of secondary fragments of the product SA-glucoside.

[0021] Figure 3 To identify the in vivo tobacco transfer function of HOVUSG2388800.

[0022] Figure 4 To verify the powdery mildew resistance of the silenced HAVUSG2388800 barley line, A represents the expression of HAVUSG2388800 in the wild-type line (WT) and the silenced line (hovusg2388800) (n=10); B represents the leaf lesion area (number of infected leaves / total number of leaves) of the wild-type line (WT) and the silenced line (hovusg2388800). 100% (n = 27); C represents the SA-glucoside content in the leaves of wild-type strain (WT) and silent strain (hovusg2388800).

[0023] Figure 5 The development of powdery mildew mycelium was observed in the silent strain (hovusg2388800, left) and the wild strain (WT, right) 7 days after powdery mildew infection.

[0024] Figure 6 The effects of powdery mildew infection on the expression of the HOVUSG2388800 gene and the accumulation of SA-glucoside in highland barley.

[0025] Figure 7 To investigate the effects of powdery mildew infection on the expression of the HOVUSG1249400 and HOVUSG2388900 genes in highland barley.

[0026] Figure 8 The in vitro functional identification of the homologous gene of HOVUSG2388800 was performed, where A is the in vitro enzyme activity analysis of the protein expressed by the HOVUSG1249400 gene; and B is the in vitro enzyme activity analysis of the protein expressed by the HOVUSG2388900 gene. Detailed Implementation

[0027] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0028] Example 1: Preparation of the target gene and expression of the target protein 1. Preparation of the target gene HOVUSG2388800 Two grams of fresh barley leaves were weighed, and barley RNA was extracted. cDNA was synthesized using Thermo Fisher's M-MLV Reverse Transcriptase. Primer pairs were designed for PCR amplification to obtain the target gene HOVUSG2388800. The PCR product was purified using a gel extraction kit (Gel Extraction Kit D2500-02, OMEGA).

[0029] The primer pair sequences are as follows: F: ttcaccatttacgaacgatagcatATGTGTGATCAACTGATCATCCAAACT(SEQ ID NO.1), R: cgggggtaccgtcgacggatccTCAGCCCACCCGGC (SEQ ID NO. 2).

[0030] The nucleotide sequence (SEQ ID NO.3) of the target gene HOVUSG2388800 is as follows: ATGTGTGATCAACTGATCATCCAAACTTCTTCGTCCCATTCGCTGCAAAGAATAGAAGAACGCAACAGCAGCCGTCACCATACATATCGACCATTTATTTCATCATCAAACCAGTGTCCAAATGTCCACGCAACGCGCGATGCTCTGTGCAATTGCGGGAACAGTTCAATCCATGTCCATGGCTCCAGTTTCCAGCCAACTGATCACCCATCACTTGGAGTTGGAGCCGCCGTACTTGGCGACGAACTCGGCGATGTTCCGGTCGGAGCTGCCGCCCGCCCTGCTGGCCGCCCGTGCCTTCTCCGCCCACGCGGCGGCGTTCCTCCTGAACTCGCCGCTCCGCTCGCCGGCCATCACCGCCTCGATCCCGCCGGACACCTCCCCCCTCCGCGCGAGCCCGTCCTGGGCCGCGGGGCGCACGCGCACACCCGCCCGCCACACGGCCTCCACGTACTTGGCGTTCATCGGCTGGTCCGTCCACTGCGGCAGCGCCACCATGGGCACGCCGGCCACCAGCGCCTCCGACGTCGAGTTCCAGCCGCAGTGCGTCAGGAAGCACCCCACGGCCGGGTGCGCCAGCACCTCCAGCTGCGGGCACCACGACACCAGCATCCCGCCGCTCGCCGCGACGGCGTCGCCGTAGCCGGCGGGCAGCTTGTGCGACTCCGACTCGCGCACGGCCCAGAGGAACGGGCGGCCGGCGTCCAGGAGGCCGTGCGCCACCTCGCGCATCTCCGCCGGGTCCAGGTTGGAGAGGCTCCCGAAGGAGGCGAACACCACGGAGCTCGCCGGGTGGGCTGA。

[0031] The target gene HOVUSG2388800 of the present invention can be obtained by the above method or directly synthesized.

[0032] 2. Construction of the vector The gene HOVUSG2388800 was transferred into the vector pGEX-6P-1 to obtain the recombinant vector.

[0033] 3. Construction of recombinant strains The recombinant vector was transferred into Escherichia coli Transetta(DE3) strain to obtain a recombinant strain containing the target gene.

[0034] 4. Expression of the target protein (1) PCR detection of positive clones, plasmid extraction and sequencing.

[0035] (2) Transform Escherichia coli transeta(DE3) with the correctly sequenced plasmid vector by heat shock and resistant CN.

[0036] (3) At 9:00 AM, two normal-sized clones were randomly selected and placed in 5 mL of LB medium containing ampicillin (80 μg / mL) (LB medium formulation is shown in Table 1). The medium was shaken at 37°C until 4:00 PM. One clone was selected, and 4 mL of activated bacterial solution (concentration of 1×10⁶~10⁷ cfu / mL) was transferred to a 200 mL bottle of LB medium (LB medium formulation is shown in Table 1) at a ratio of 1:50. The culture was incubated on a large shaker at 37°C and 200 rpm. After 3-4 hours, 2 μL of 1M IPTG inducer was added to the 200 mL medium. The culture was induced overnight at 20°C and 160 rpm. The remaining 1 mL of bacterial solution was used for preservation.

[0037] Table 1 LB medium formulation (4) Collect the bacterial cells at 8:00 a.m. the next day, put them into a 500mL centrifuge bottle, and centrifuge at 4000rpm for 10min.

[0038] (5) Resuspend the bacterial cells in 50 mL of Lysis buffer, vortex to mix, transfer to a 50 mL centrifuge tube, add 50 μL of LPMSF and 10 μL of β-mercaptoethanol respectively, mix well and place on ice.

[0039] (6) The Escherichia coli cell disruption experiment was conducted using a high-pressure disruptor.

[0040] (7) After the sample is broken down, take 20 μL as the total protein sample. Then take 1 mL of the sample, centrifuge at 4℃ and 13000 rpm for 10 min, and take 20 μL of the supernatant as the supernatant sample. Add an equal volume of 2 Loading buffer, boiling for 5 minutes, and SDS-PAGE electrophoresis to detect protein expression. The remaining supernatant can be temporarily stored at -20°C. Uncentrifuged samples can be stored at -80°C.

[0041] (8) After SDS-PAGE electrophoresis, add Coomassie Brilliant Blue staining solution, microwave for 1 minute, then stain for half an hour, and add destaining solution to destain. Change the destaining solution every 1 hour until the protein bands are clear, then transfer to distilled water.

[0042] (9) Purification of GST-tagged fusion protein. Centrifuge all lysed but not centrifuged samples. Mix the supernatant with 1 mL of resin at 4°C for 3 h. After mixing, pass the mixture through the chromatography column twice for better results. First, rinse the resin (Glutathione Sepharose™ 4B, GE) with pre-cooled Lysis buffer, and simultaneously check the eluent with Bradford Assay until the blue color disappears, indicating that the contaminating protein has been washed away. Then, elute the target protein with 15 mmol / L reduced glutathione solution (0.09 g dissolved in 20 mL lysis buffer), adding 1 mL each time. Collect approximately 1 mL from the bottom of the chromatography column using 1.5 mL centrifuge tubes, labeling each tube as E1, E2, E3, E4, E5, and E6, until Bradford Assay indicates that the eluent contains no protein. Unused reduced glutathione solution was used to elute the resin completely, and then rinsed with Lysis buffer, ddH2O, and 20% ethanol, and stored in 20% ethanol.

[0043] (10) The collected target protein was detected by SDS-PAGE, yielding a band of 78.4 kDa. Figure 1 The molecular weight of the GST tag is 26 kDa, and the molecular weight of the remaining target protein is 52.4 kDa, which is the same as the molecular weight calculated from the amino acids. This indicates that the present invention has prepared a target protein with a GST tag.

[0044] The amino acid sequence (SEQ ID NO.4) of the target protein (HOVUSG2388800 protein) is as follows: MASHGEDAQEVVTGTDGGHVLLLPYPSQGHVHPMLQFAKRLAHHGVRPTLAVSRYILATCKPDAAAVGAVRLAAVSDGCDAGGFGQCNDVTAYLGLLEAAGSETLAELLRAEAAEGRPV RAVVYDAFLPWARGVAQRHGAAAVAFFTQPCAVNVVYGHVWCERVGVPVEAGSTVVGLPGLPALEPEGLPWFLKVGPGPYPGYFEMVMSQFKGLELADDVLVNSFYELEPEEAAYMASA WRAKTIGPTVPASYVGDDRMPSDTKYGFHLFELTAAPCVSWLSAHPASSVVFASFGSLSNLDPAEMREVAHGLLDAGRPFLWAVRESESHKLPAGYGDAVAASGGMLVSWCPQLEVLAH PAVGCFLTHCGWNSTSEALVAGVPMVALPQWTDQPMNAKYVEAVWRAGVRVRPAAQDGLARRGEVSGGIEAVMAGERSGEFRRNAAAWAEKARAASRAGGSSDRNIAEFVAKYGGSNSK.

[0045] Example 2 Construction of transgenic tobacco (1) Transient expression vector containing the target gene HOVUSG2388800 (transient expression vector pEAQ, from John Innes Centre) was transformed into Agrobacterium (EHA105); (2) Select positive Agrobacterium clones and place them in 500 μL of LB medium containing the corresponding antibiotic (ampicillin 80 μg / mL) (LB medium formula is shown in Table 1) and incubate for 20-24 hours; (3) Transfer 200 μL to 5 ml of LB medium containing the corresponding antibiotic (ampicillin 80 μg / mL) (LB medium formula is shown in Table 1), and shake at 28℃ and 220 rpm until OD = 2.0.

[0046] (4) Collect the bacterial cells by centrifugation at 10,000 rpm at room temperature for 2 min, resuspend the bacterial cells in the pre-prepared conversion buffer, and shake on a shaker for 3 h; the working solution composition and concentration of the buffer are as follows: 10 mM MES (pH 5.7), 10 mM MgCl2, 100 μM UDP-glucose.

[0047] (5) Take a prepared 1 mL syringe, remove the needle, select a syringe with a smooth outlet to draw in the bacterial solution, take a 1-month-old Nicotiana benthamiana, hold the leaf with your hand, and inject from the underside of the leaf to allow Agrobacterium to penetrate in.

[0048] (6) Tobacco injected with Agrobacterium was cultured in the dark for 24 hours, and then transferred to a tobacco incubator for light culture for 24-48 hours to obtain transgenic tobacco.

[0049] The following experimental examples illustrate the beneficial effects of the present invention.

[0050] Experimental Example 1: Enzyme Activity Detection of Target Protein The target protein prepared according to the method in Example 1 was used for enzyme activity assay. Salicylic acid (glycosyl acceptor) was added to 100 μL Tris-HCl buffer (100 mM, pH 7.4) to a final concentration of 200 μM, and UDP-glucose (glycosyl donor) was added to a final concentration of 100 μM. 500 ng of the target protein was then added for in vitro acyltransferase assay. After incubation for 10 min, 300 μL of ice-cold methanol was added to stop the reaction. The reaction mixture was filtered through a 0.2 μm filter (micropores) and analyzed by LC-MS.

[0051] The results showed that the substance produced by the reaction was salicylate glucoside. Figure 2 This indicates that the target protein of the present invention has the ability to catalyze the glycosylation of salicylic acid to salicylate glucoside.

[0052] Experimental Example 2: Production of Salicylate Glucoside from Genetically Modified Tobacco 1. Experimental Methods (1) Construction of genetically modified tobacco Transgenic tobacco was constructed according to the method in Example 2 (three batches, namely OX-1, OX-2 and OX-3).

[0053] Following the method of Example 2, the injected bacterial solution was replaced with an equal volume of conversion buffer as a control tobacco (CK).

[0054] (2) Product collection and purification Leaf fragments from the Agrobacterium-infiltrated region were cut and placed in pre-weighed EP tubes containing steel balls. These tubes were labeled and quickly placed in liquid nitrogen for lyophilization. After lyophilization, the samples were ground using a grinder (MM 400, Retsch) at 30Hz for 60 seconds. The ground sample powder was then transferred to 2ml EP tubes. The weight of each EP tube was weighed using an electronic balance and recorded. An appropriate amount of the ground sample (range 30-60mg) was added to each EP tube, weighed, and recorded. The net weight of all samples in the EP tubes was calculated. Given the net weight of each sample, the formula V = net weight of sample (mg) was applied. Add 12 μL / mg of 70% MeOH solution to ice at 4°C. Mix well, vortex for 15 seconds, repeating this process every half hour for a total of 4 vortexes. Extract at 4°C for at least 12 hours. Then centrifuge. Pre-cool the centrifuge to 4°C, set the time to 10 minutes and the speed to 12000 rpm. Vortex the sample and then centrifuge, ensuring symmetrical balance. After centrifugation, aspirate the supernatant. Filter the supernatant through a microporous membrane (0.22 μm pore size) and transfer it to a sample vial for LC-MS analysis.

[0055] (3) Detection of target product Place the vials containing the sample extract into the sample tray of the autosampler, and record the position of the injection port corresponding to each vial number. Simultaneously, open Analyst Software, double-click Hardware Configuration, select LCMS-V (with switching valve mode), click Activate Profile, select Acquire Mode, click Acquire, and then click the Equilibrate button above the image. The time is generally set to 3 minutes. This operation is to preheat the instrument, ensuring that the high-pressure pump, column, column oven, and ion source temperatures reach the conditions set in the method. Once all instrument components are in the Ready state, the Start Sample button in the function area will become clickable, indicating that the instrument and analytical conditions are normal. Then click Start Sample to begin the sample run. Before the first run, submit four blank samples.

[0056] 2. Results Experimental results showed that, compared with non-transgenic tobacco (CK), the content of salicylate glucoside in the transgenic tobacco of this invention was significantly increased, and the peak value of salicylate glucoside reached 6.2 × 10⁻⁶. 5 ( Figure 3 The results indicate that the target protein HOVUSG2388800 has high activity. These experimental results demonstrate that the present invention transfers the gene HOVUSG2388800 into tobacco, enabling the tobacco plant to express salicylate glycosyltransferase and inducing the accumulation of salicylate glucoside in tobacco.

[0057] Experimental Example 3: The silencing of the HOVUSG2388800 barley strain significantly increased its resistance to powdery mildew. 1. Construction of Silent Barley Lines (1) Carrier construction Using cDNA from the powdery mildew-susceptible barley variety ZQ13 as a template, the HOVUSG2388800 gene fragment was amplified using the primers listed below. After enzyme digestion, the fragment was inserted into the barley stripe mosaic virus γ genome (BSMV-γ) to construct the recombinant viral vector BSMV:HOVUSG2388800. Simultaneously, BSMV:TaPDS containing the tomato carotenoid cleavage dioxygenase gene (TaPDS) was prepared as a gene silencing efficiency and a positive control for viral infection. The primer sequences are as follows: F: TTTCTAAGGAAGGGCCAGGGACATCGTCG (SEQ ID NO.5); R: TTAACCACCACCACCGGGCGTGGTAGGGTGACCC (SEQ ID NO. 6).

[0058] (2) Viral in vitro transcription After linearizing the recombinant plasmid, an in vitro transcription reaction was performed to obtain viral positive strand RNA; equal amounts of BSMV-α, BSMV-β and BSMV:HOVUSG2388800 RNA were mixed for later use.

[0059] (3) Seedling preparation Six uniformly grown three-leaf stage barley seedlings (ZQ13) were selected and inoculated after 12 hours of dark acclimatization.

[0060] (4) Virus inoculation The friction inoculation method was used: emery was evenly sprayed on the surface of 2-3 newly unfolded leaves, and then 5 μL of viral RNA mixture was taken with a disposable pipette tip and gently wiped along the main vein of the leaf 2-3 times in one direction; the control group was treated with BSMV-γ empty vector RNA in the same way.

[0061] (5) Cultivation and microscopic observation of mycelia After inoculation, the plants were kept in darkness and kept moist for 24 hours (temperature 22℃, relative humidity 85%), and then transferred to a light incubator (16 h light / 8 h darkness, 22℃). The bleaching phenotype of the TaPDS control leaves was observed 7 days after inoculation (when...). When the TaPDS gene is silenced, carotenoid synthesis is inhibited, and chlorophyll loses its protection. Under strong light, leaves undergo photobleaching (manifesting as white patches) to verify virus infection and silencing efficiency. Subsequent experiments detected the relative expression levels of HOVUSG2388800 between the silenced strain and the wild-type (WT) 7 days after powdery mildew infection to determine the successfully constructed silenced strain. A silenced strain with a significantly downregulated relative expression level of HOVUSG2388800 compared to the wild-type WT (p<0.01) was considered a successfully constructed silenced strain.

[0062] 2. Material Collection (1) Inoculation with powdery mildew Powdery mildew was inoculated onto silent barley strains and wild-type control strains: The powdery mildew pathogen was propagated in advance using susceptible barley varieties to obtain diseased plants covered with fresh spores as the inoculum source. Seedlings (3-leaf stage) to be inoculated were placed in an inoculation box, and diseased plants were gently shaken above the seedlings to distribute conidia evenly onto the leaves. After inoculation, the plants were sprayed with water to maintain humidity and cultured at 15-20℃ with a relative humidity of 70%-90%.

[0063] (2) Gene expression detection Seven days after powdery mildew inoculation, newly developed leaves were collected and immediately placed in pre-weighed EP tubes containing steel beads, labeled, and rapidly frozen in liquid nitrogen for lyophilization. 30-60 mg of powder was accurately weighed into a new EP tube, and after calculating the net weight, 12 μL / mg of pre-cooled 90% methanol (v / w) at 4 °C was added. The tube was vortexed for 15 s at 4 °C on an ice bath, repeated every 30 min for a total of 4 times, followed by static extraction at 4 °C for ≥12 h. After extraction, the tube was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was filtered through a 0.22 μm microporous membrane. The filtrate was collected in a sample vial. The sample was divided into two parts: one part was used for total RNA extraction, and the relative expression level of the target gene HAVUSG2388800 was detected by real-time quantitative PCR (qRT-PCR). -ΔΔCt Gene expression levels in silent lines were calculated using a method with three technical replicates for each sample (leaf samples from three biological replicates of each line were taken, RNA was extracted from each replicate, and quantitative fluorescence analysis was performed). Results are expressed as mean ± standard deviation. Another portion of the samples was used for LC-MS / MS analysis to detect changes in the content of target metabolites.

[0064] (3) Observation of powdery mildew phenotype mycelia Seven days after inoculation, the changes in powdery mildew mycelium on the leaves of the normal control and silent strains were observed and photographed under a microscope.

[0065] 3. Detection of target product Place the vials containing the sample extract into the sample tray of the autosampler and record the position of the injection port corresponding to each vial number. A Waters Acquity UPLC system in series with a Waters Quattro Premier XE triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source, was used. Column: Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) and its guard column. Mobile phase: Phase A: 0.1% formic acid in water; Phase B: 0.1% formic acid in methanol. Gradient program: 0–2 min 95% A; 2–12 min linear ramp to 100% B; 12–14 min 100% B isocratic; 14–17 min return to 95% A equilibrium. Column temperature: 35 °C; flow rate: 0.35 mL / min. -1 Injection volume: 3 µL. Ion source parameters: Nitrogen gas was also used as the nebulizer (50 Lh). -1 ) and dry gas (900 L h) -1 Capillary voltage 3.2 kV; cone voltage 25 V; interface temperature 400 ℃; source temperature 135 ℃; positive / negative ion mode can be switched as needed. Multiple reaction monitoring (MRM, collision energy 20 V): • Negative ion mode: 12-OH-JA-Ile 338.2 → 130.1; SA-glucoside 352.2 → 130.1, • Positive ion mode: JA-Ile 322.4 → 129.7. Data acquisition and processing are performed using MassLynx software.

[0066] 4. Results This invention found that the expression of HOVUSG2388800 was significantly suppressed in resistant materials after infection with powdery mildew, while no significant change was observed in susceptible materials. To clarify the functional differences of HOVUSG2388800 in the SA inactivation cascade of highland barley, this invention compared the expression of HOVUSG2388800 and the metabolic profile of SA-related substances in the silent strain (hovusg2388800) and wild-type (WT) 7 days after powdery mildew infection. The results showed that 7 days after powdery mildew infection, the expression of HOVUSG2388800 in the leaves of wild-type (WT) and silent strain (hovusg2388800) was detected. It was found that the transcriptional level of the two silent strains (sample group numbers hovusg2388800-2 and hovusg2388800-5, respectively) was significantly downregulated compared with wild-type WT (p<0.01). Figure 4A) indicates that the two silent lines, numbered hovusg2388800-2 and hovusg2388800-5, were successfully constructed silent lines. Corresponding to the reduced expression, the proportion of leaf lesion area in the silent line (hovusg2388800) was only 40.1% of the total leaf area in the wild-type line (WT). Figure 4 B). Metabolic analysis showed that decreased expression of HOVUSG2388800 led to a sharp drop of 55.1% in SA-glucoside content (p<0.01). Figure 4 C). Therefore, silencing HOVUSG2388800 significantly blocked the final inactivation of SA, leading to the continuous activation of defense signals, which manifested as a reduction in lesion area and a significant inhibition of powdery mildew mycelial development in HOVUSG2388800-silenced lines, resulting in enhanced disease resistance. Figure 5 ).

[0067] The following comparative experiment selected two genes with the same annotation as HOVUSG2388800 and the highest homology for comparison: HOVUSG2388900 (E value: 5.6e). -166 ) and HOVUSG1249400 (E value: 2.6e -164 The sequences of the two genes are as follows: HOVUSG2388900 (SEQ ID NO.7):

[0068] HOVUSG1249400(SEQ ID NO.8):

[0069] Preparation of two homologous genes: The primer pairs for preparing the homologous gene HOVUSG2388900 are as follows, and the method for preparing the homologous gene is the same as in Example 1: F1: ttcaccatttacgaacgatagcatATGGTGCACGCCGAC (SEQ ID NO.9), R1: cgggggtaccgtcgacggatccTCATTTTGCCACGCGATATTTG (SEQ ID NO. 10).

[0070] The primer pairs for preparing the homologous gene HOVUSG1249400 are as follows, and the method for preparing the homologous gene is the same as in Example 1: F2: ttcaccatttacgaacgatagcatATGTCGGAAACGGAGACAACTTC (SEQ ID NO. 11), R2: cgggggtaccgtcgacggatccTCAACTTGTAGTTGGAGAATACTTGGC (SEQ ID NO. 12).

[0071] Comparative Example 1: Expression of the homologous gene HOVUSG2388800 in barley infected with powdery mildew. 1. Experimental Methods Total DNA was extracted from barley leaves at 2, 6, 36, 72, and 168 h after powdery mildew infection. The extracted DNA was used as a template for real-time quantitative PCR analysis using the TB Green chimeric fluorescence assay. The reaction system was 10 μL, containing 5 μL TB Green Premix Ex Taq (2×), 1 μL each of forward and reverse primers (primer concentration 2 μmol / L), and 3 μL sterile distilled water. The amplification reaction was performed on a Bio-Rad CFX96 quantitative PCR instrument. The program was set as follows: 95℃ pre-denaturation for 3 min; followed by 40 cycles of 95℃ denaturation for 15 s and 58℃ annealing extension for 30 s; after the reaction, a melting curve analysis program was added: 65℃ to 95℃ in increments of 5 s to verify the specificity of the amplified products. The barley housekeeping gene (GAPDH) was used as an internal control, and 2... -ΔΔCt The relative expression levels of the target genes HOVUSG2388900 and HOVUSG1249400 were calculated using a method. All samples were tested in triplicate.

[0072] 2. Results The HOVUSG2388800 gene was significantly induced to express under powdery mildew infection. Figure 6 ), while the expression of the two homologous genes did not show significant changes under infection conditions ( Figure 7 This indicates that HOVUSG2388800 is a specific response gene for resistance to powdery mildew in barley and may play an important role in barley's defense against powdery mildew stress.

[0073] Enzymatic activity of the homologous gene expressed by Comparative Example 2, HOVUSG2388800 To detect whether the expressed proteins of the two homologous genes of HOVUSG2388800 have the activity of catalyzing salicylic acid (SA) glycosylation, this invention carried out enzyme activity assay.

[0074] 1. Experimental Methods Two homologous gene expression proteins were prepared according to the method in Example 1, and their enzyme activity was detected according to the method in Experimental Example 1.

[0075] 2. Results In the reaction systems of HOVUSG1249400 and HOVUSG2388900 expressed proteins, only the substrate SA was detected, and no SA-glucoside product was observed. Figure 8 This result indicates that although both genes are annotated as SA glycosyltransferases and are homologs of HOVUSG2388800, their expressed proteins do not have the function of glycosylation modification of SA.

[0076] In summary, the protein expressed by the HOVUSG2388800 gene (nucleotide sequence shown in SEQ ID NO.3) of this invention (amino acid sequence shown in SEQ ID NO.4) can convert salicylic acid into salicylate glucoside, thereby improving the powdery mildew resistance level of highland barley. HOVUSG2388800 is a specific response gene for highland barley powdery mildew resistance. Using this gene fragment for in vitro expression, salicylate glycosyltransferase was obtained. In the in vitro reaction, glucose was successfully used as the glycosyl donor and salicylic acid as the acceptor to prepare salicylate glucoside. This invention also transfers this gene into tobacco, enabling the tobacco plant to express salicylate glycosyltransferase, further producing salicylate glucoside and increasing its value. The novel gene, recombinant vector, recombinant bacteria, protein, and transgenic plants provided by this invention all have good application prospects.

Claims

1. A gene fragment, characterized in that, The nucleotide sequence of the gene fragment is shown in SEQ ID NO.

3.

2. A recombinant vector, characterized in that, The recombinant vector contains a gene fragment with a nucleotide sequence as shown in SEQ ID NO.

3.

3. A recombinant cell, characterized in that, The recombinant cells comprise the recombinant vector of claim 2.

4. The recombinant cell according to claim 3, characterized in that, The recombinant cells are selected from bacterial or plant cells.

5. A protein, characterized in that, The protein is encoded by a gene fragment with the sequence shown in SEQ ID NO.

3.

6. The protein according to claim 5, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

4.

7. Use of the gene fragment of claim 1, the recombinant vector of claim 2, the recombinant cell of claim 3 or 4, or the protein of claim 5 or 6 in the preparation of salicylate glucoside.

8. Use of the gene fragment of claim 1, the recombinant vector of claim 2, or the recombinant cell of claim 3 or 4 in the preparation of transgenic plants for the production of salicylate glucoside.

9. A method for preparing salicylate glucoside, characterized in that, It uses a protein with an amino acid sequence as shown in SEQ ID NO.4, with glucose as the glycosyl donor and salicylic acid as the glycosyl acceptor, to prepare salicylate glucoside.

10. A method for constructing a transgenic plant that produces salicylate glucoside, characterized in that, Includes the following steps: Take the gene fragment with the nucleotide sequence shown in SEQ ID NO.3, transform it into a plant, and obtain a plant that expresses the protein with the amino acid sequence shown in SEQ ID NO.4.

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