Cytochrome p450 gene HvCYP94C1 and application thereof in improving resistance of highland barley to powdery mildew

Abstract

This invention provides a cytochrome P450 gene HvCYP94C1 and its application in improving the resistance of highland barley to powdery mildew, belonging to the field of agricultural biotechnology. The cytochrome P450 gene HvCYP94C1 of this invention can hydroxylate JA and JA-Ile to 12-OH-JA-Ile and 12-COOH-JA-Ile, respectively. Silencing this gene in powdery mildew-sensitive materials can significantly increase the jasmonic acid content, thereby improving the disease resistance of the materials, showing excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a cytochrome P450 gene HvCYP94C1 and its application in improving the resistance of highland barley to powdery mildew. Background Technology

[0002] Jasmonic acid-isoleucine (JA-Ile) is the core active hormone in the plant jasmonic acid signaling pathway. It releases transcriptional repression by binding to the COI1-JAZ co-receptor complex, thereby initiating the expression of downstream defense genes. However, the activity of JA-Ile is not static; it can be catalyzed by cytochrome P450 monooxygenase to successively generate 12-hydroxy-JA-Ile (12-OH-JA-Ile) and 12-carboxyl-JA-Ile (12-COOH-JA-Ile). Both products lose their affinity for COI1-JAZ and are considered "inactive" jasmonic acid. This hydroxylation / carboxylation pathway is a conserved major JA-Ile degradation pathway in higher plants, playing a decisive role in signal intensity and duration.

[0003] Powdery mildew of highland barley is a common disease caused by the powdery mildew fungus *Blumeria graminis*. It primarily damages the leaves, affecting photosynthesis and growth, ultimately leading to reduced yield and quality. In research on resistance to powdery mildew in highland barley, the plant hormone jasmonic acid (JA) and its derivatives play a crucial role in regulating the plant's immune response. Studies have shown that JA-Ile participates in regulating the plant's defense response to powdery mildew stress, and its degradation products, 12-hydroxy-JA-Ile (12-OH-JA-Ile) and 12-carboxyl-JA-Ile (12-COOH-JA-Ile), may indirectly affect highland barley's resistance to powdery mildew by regulating JA-Ile levels. When highland barley is infected by powdery mildew, the accumulation of JA-Ile induces the expression of a series of defense genes, thereby enhancing the barley's disease resistance. However, 12-OH-JA-Ile and 12-COOH-JA-Ile, as inactivated forms of JA-Ile, may weaken the signal intensity of JA-Ile, thereby affecting the barley's defense against powdery mildew.

[0004] To date, Arabidopsis CYP94B3 and CYP94C1 have been confirmed to be responsible for the initial hydroxylation and subsequent carboxylation of JA-Ile, respectively, but the function of homologous genes in gramineous crops still lacks systematic verification. Summary of the Invention

[0005] The purpose of this invention is to provide a cytochrome P450 gene HvCYP94C1 and its application in improving the resistance of barley to powdery mildew.

[0006] This invention provides a cytochrome P450 gene HvCYP94C1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a recombinant plasmid comprising the cytochrome P450 gene HvCYP94C1 described above.

[0008] Furthermore, the vector for the recombinant plasmid is the recombinant yeast expression vector pYeDP60.

[0009] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the above-described recombinant plasmid.

[0010] Furthermore, the recombinant bacteria is the engineered yeast strain WAT11.

[0011] The present invention also provides a yeast expression of exogenous protein HvCYP94C1, the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] Furthermore, the method for preparing the yeast-expressed exogenous protein HvCYP94C1 is as follows:

[0013] Using cDNA as a template, the full-length HvCYP94C1 gene was amplified using the listed primers. The PCR product was purified and cloned into the entry vector pDONR207; positive clones with correct sequencing were transformed into the target yeast expression vector pYeDP60 via Gateway recombination (Invitrogen). The recombinant plasmid was transformed into T1 phage-resistant chemocompetent cells. To maintain the catalytic activity of HvCYP94C1, it relies on NADPH-cytochrome P450 reductase (ATR). Therefore, the plasmid was extracted and transformed into the engineered yeast strain WAT11, which already carries the Arabidopsis ATR gene.

[0014] Pick single colonies, incubate overnight at 29°C and 280 rpm in 10 mL of SGI medium until OD. 600 ≈1.2. Then, 1 mL of SGI culture was transferred to 10 mL of SLI medium and incubated overnight at 28 °C. The next day, the bacterial culture was diluted to OD using fresh SLI medium. 600 =0.4.

[0015] The present invention also provides a method for preparing the above-mentioned yeast expression exogenous protein HvCYP94C1, the method comprising the following steps:

[0016] (1) Construct the above-mentioned recombinant plasmid;

[0017] (2) The recombinant plasmid was transferred into the host cell, induced to express, purified, and the yeast-expressed exogenous protein HvCYP94C1 was obtained.

[0018] The present invention also provides the use of the above-mentioned cytochrome P450 gene HvCYP94C1, the above-mentioned recombinant plasmid, the above-mentioned recombinant bacteria, and the above-mentioned yeast expressing the exogenous protein HvCYP94C1 in the preparation of plant varieties resistant to powdery mildew.

[0019] Furthermore, the plant is one that reduces the content of 12-OH-JA-Ile and 12-COOH-JA-Ile and increases the content of JA-Ile.

[0020] Furthermore, the plant is tobacco or barley.

[0021] The present invention also provides a method for constructing powdery mildew-resistant plants, comprising the following steps:

[0022] (a) Constructing a recombinant viral vector containing the cytochrome P450 gene HvCYP94C1 as described in claim 1;

[0023] (b) The recombinant viral vector was transcribed in vitro to obtain viral RNA;

[0024] (c) Inoculate the viral RNA into the leaves of plant seedlings;

[0025] (d) Cultivate plants with reduced HvCYP94C1 gene expression, which are then considered powdery mildew resistant plants.

[0026] Furthermore, the method for constructing the recombinant viral vector includes the following steps:

[0027] Using cDNA from ZQ13, a barley variety susceptible to powdery mildew, as a template, the HvCYP94C1 gene fragment was amplified using primer sequences (F: TTTTCTAAGGAAGGGCCAGGCCCTGGCGCT; R: TTAACCACCACCACCGGGCGGAGAAGGGCTTG). After enzyme digestion, the fragment was inserted into the barley stripe mosaic virus γ genome (BSMV-γ) to construct the recombinant viral vector BSMV:HvCYP94C1.

[0028] Furthermore, the in vitro transcription includes the following steps:

[0029] After linearizing the recombinant plasmid, in vitro transcription was performed according to the manufacturer's instructions to obtain viral positive strand RNA; equal amounts of BSMV-α, BSMV-β and BSMV:HvCYP94C1 RNA were mixed for later use.

[0030] Furthermore, the method further includes the following steps:

[0031] Seedling preparation: Select plants with uniform growth and inoculate them after 12 hours of dark acclimatization.

[0032] Virus inoculation: The friction inoculation method is used: Spray carborundum evenly on the surface of 2-3 newly unfolded leaves, and then use a disposable pipette tip to pick up 5 μL of virus RNA mixture and gently wipe along the main vein of the leaf 2-3 times in one direction.

[0033] The present invention has achieved the following beneficial effects:

[0034] This invention discovers a novel gene in highland barley: the cytochrome P450 gene (HOVUSG4593100, HvCYP94C1). The expression level of this gene significantly decreased after infection with powdery mildew-resistant highland barley materials, while its expression level remained unchanged in susceptible materials. The protein expressed by this gene—highland barley cytochrome P450 hydroxylase—can convert JA-Ile into 12-OH-JA-Ile and 12-COOH-JA-Ile. This invention also silenced the expression of HvCYP94C1 in the susceptible highland barley material ZQ13. The silenced lines showed a significant decrease in the levels of 12-OH-JA-Ile and 12-COOH-JA-Ile, a significant increase in the content of JA-Ile, and a significant enhancement in disease resistance. The novel gene, its recombinant vector, recombinant bacteria, and silenced lines provided by this invention all have promising application prospects.

[0035] The gene fragments and recombinant vectors of this invention can be used to reduce the content of 12-OH-JA-Ile and 12-COOH-JA-Ile in highland barley, thereby achieving targeted improvement of highland barley.

[0036] The method for constructing silent barley lines in this invention provides an important reference for the targeted improvement of barley.

[0037] 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.

[0038] 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 embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0039] Figure 1 Analysis of HvCYP94C1 gene expression in resistant (GND7) and susceptible (ZQ13) barley materials after powdery mildew infection.

[0040] Figure 2 To assess the catalytic activity of recombinant HvCYP94C1.

[0041] Figure 3 To construct a silenced strain of HvCYP94c1 and to investigate how HvCYP94C1 weakens the jasmonic acid-mediated defense response against powdery mildew through JA-Ile inactivation. (A) RT-qPCR results showing the expression of HvCYP94C1 in WT and Hvcyp94c1 (n=10). Sampling time: 7 days after powdery mildew infection. (B) Lesion area of ​​WT and Hvcyp94c1 leaves (number of infected leaves / total number of leaves*100%) (n = 27). (CE) Content of JA-Ile (C), 12-OH-JA-Ile (D), and 12-COOH-JA-Ile (E) in WT and Hvcyp94c1 leaves collected 7 days after powdery mildew infection (n = 10).

[0042] Figure 4 Phenotypic comparison of the silent strain (Hvcyp94c1, left) and the wild strain (WT, right) 7 days after powdery mildew infection. Detailed Implementation

[0043] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0044] Example 1: Isolation and prokaryotic expression of the HOVUSG4593100 gene

[0045] This embodiment mainly describes the methods for obtaining the HOVUSG4593100 gene, constructing the vector, and expressing it in prokaryotes.

[0046] (1) Construction of gene fragments and vectors

[0047] Weigh 2 grams of fresh barley leaves, extract barley RNA, synthesize cDNA using Thermo Fisher's M-MLV Reverse Transcriptase, and design primers as follows:

[0048] F: aaaaagcaggcttaATGGGCGTCGAGGCTG;

[0049] R: agaaagctgggtaTCAGGTTGGTGGGCAGC.

[0050] The nucleotide sequence (SEQ ID NO.1) of the amplified target fragment HOVUSG4593100 gene is as follows:

[0051]

[0052] Using cDNA as a template, the full-length HvCYP94C1 gene was amplified using the listed primers. The PCR product was purified and cloned into the entry vector pDONR207; positive clones with correct sequencing were transformed into the target yeast expression vector pYeDP60 via Gateway recombination (Invitrogen). The recombinant plasmid was transformed into T1 phage-resistant chemocompetent cells. To maintain the catalytic activity of HvCYP94C1, it relies on NADPH-cytochrome P450 reductase (ATR). Therefore, the plasmid was extracted and transformed into the engineered yeast strain WAT11, which already carries the Arabidopsis ATR gene.

[0053] Pick single colonies, incubate overnight at 29°C and 280 rpm in 10 mL of SGI medium until OD reaches zero. 600 ≈1.2. Then, 1 mL of SGI culture was transferred to 10 mL of SLI medium and incubated overnight at 28°C. The next day, the bacterial culture was diluted to OD using fresh SLI medium. 600 =0.4. Take 5 mL of diluted bacterial culture, add JA-Ile substrate, and react at 28 ℃ for 16 h.

[0054] After the reaction, the bacterial culture was ultrasonically disrupted under ice bath conditions (20 cycles, 5 s sonication / 5 s interval). The pH was adjusted to 4.0 with concentrated HCl, and the product was then extracted with ethyl acetate; the acidic ethyl acetate phase was collected and dried under nitrogen. The residue was redissolved in methanol and analyzed by LC-MS / MS. Data acquisition and processing were performed using MassLynx software. The ion pairs monitored in negative ion mode were: JA m / z 209→59, 12-OH-JA m / z 227→163, 12-COOH-JA m / z 211→167, JA-Ile m / z 322→130, 12-OH-JA-Ile m / z 338→130, 12-COOH-JA-Ile m / z 352→130, and confirmed by comparison with the corresponding standards.

[0055] The amino acid sequence of the target protein (SEQ ID NO.2) is as follows:

[0056] MGVEAALHGGAGAAAEALALSLRPHVAGAFFALAACAVALAALLAVARTRPPWWCDCAVCEAYLTASWAGEFDNLCDWYAHLLRRSPSQTVHVHVLRNVLTANPVTVDHMLRARFDNYPKGKPFSAILAD LLGRGIFNVDGDAWLFQRKLAAAELASPALRAFAAGVVASELRGRLLPLLHSACSSSKVLDLQDVFRRFAFDCICKISGFGLDPGCLELSMPVSAFVDAFDTASMLSARRATAPMQIVWRLKRFFNVGDER KLRESVRLVDGFAAEVTRQRHKLGGAASGSDLLSRFMGSISDEKYLRDIVVSFMLAGRDTVASALTAFFLLLSDHPEVAAAIRDEVSRVTGDDDDRPRPSFSKLKDMHYVHAAMYESMRLFPPVQFDSKF AAGDDKLPDGTVVAKGTRVTYHAYAMGRMESVWGADCSEFRPERWLRDGQFVPVSPYRYPVFQAGVRVCVGKDLALEMKAVIVAVVRSFDIEAIDRSSRRPKFAPGLTATFAGGLPVRVRRRARGCPPT.

[0057] Example 2: Construction of Silent Barley Lines

[0058] F:TTTCTAAGGAAGGGCCAGGCCCTGGCGCT;

[0059] R: TTAACCACCACCACCGGGCGGAGAAGGGCTTG.

[0060] 1) Carrier construction

[0061] Using cDNA from ZQ13, a barley variety susceptible to powdery mildew, as a template, the HvCYP94C1 gene fragment was amplified using the primers described above. After enzyme digestion, the fragment was inserted into the barley stripe mosaic virus γ genome (BSMV-γ) to construct the recombinant viral vector BSMV:HvCYP94C1. Simultaneously, BSMV:TaPDS containing the tomato carotenoid cleavage dioxygenase gene (TaPDS) was prepared as a positive control for gene silencing efficiency and viral infection.

[0062] 2) Viral in vitro transcription

[0063] After linearizing the recombinant plasmid, in vitro transcription was performed according to the manufacturer's instructions to obtain viral positive strand RNA; equal amounts of BSMV-α, BSMV-β and BSMV:HvCYP94C1 (or BSMV:TaPDS, BSMV-γ) RNA were mixed for later use.

[0064] 3) Seedling preparation

[0065] Six uniformly grown three-leaf stage barley seedlings (ZQ13) were selected and inoculated after 12 hours of dark acclimatization.

[0066] 4) Virus vaccination

[0067] 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.

[0068] 5) Cultivation and Phenotypic Observation

[0069] After inoculation, the cells 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 to verify the virus infection and silencing efficiency.

[0070] 6) Gene expression detection

[0071] Newly developed leaves were collected 10-12 days after inoculation and flash-frozen in liquid nitrogen. Half of the materials were subjected to real-time quantitative PCR to detect the relative expression level of HvCYP94C1, with 2... -ΔΔCt The silencing efficiency was calculated using a method repeated three times, and the results are presented as mean ± standard deviation. Another part of the analysis involved LC / MS / MS detection of changes in the target product content.

[0072] 7) Observation of anti-powdery mildew phenotype

[0073] Ten to twelve days after inoculation, both the normal control and the silent strain were infected with powdery mildew. Phenotypic changes were observed and photographed seven days later.

[0074] The following experimental examples illustrate the beneficial effects of the present invention:

[0075] Experimental Example 1: Enzyme Function Detection of HOWUSG4593100 Protein

[0076] 1. Experimental Methods

[0077] 1.1. HOVUSG4593100 was transfected into yeast to express the protein.

[0078] After converting the yeast culture with HOVUSG4593100, 5 mL of diluted culture was taken, and 1 µg of JA-Ile substrate was added. The reaction was carried out at 28 °C for 16 h. After the reaction, the culture was sonicated under ice bath conditions (20 cycles, 5 s sonication / 5 s interval). The pH was adjusted to 4.0 with concentrated HCl, and the product was then extracted with ethyl acetate. The acidic ethyl acetate phase was collected and dried under nitrogen. The residue was redissolved in methanol and analyzed by LC-MS / MS. Data acquisition and processing were performed using MassLynx software. The ion pairs monitored in negative ion mode were: JA m / z 209→59, 12-OH-JA m / z 227→163, 12-COOH-JA m / z 211→167, JA-Ile m / z 322→130, 12-OH-JA-Ile m / z 338→130, and 12-COOH-JA-Ile m / z 352→130, and were confirmed by comparison with the corresponding standards.

[0079] 2. Experimental Results

[0080] After the above catalytic reaction, the product was analyzed by LC-MS / MS. The synthesis amounts of 12-OH-JA-Ile and 12-COOH-JA-Ile, especially the latter, were significantly increased when HAVUSG4593100 was transformed into yeast compared with the wild-type control.

[0081] The results of liquid chromatography-tandem mass spectrometry (LC-MS / MS) showed that ( Figure 2 In wild-type WT yeast that does not express HvCYP94C1, substrate and trace amounts of 12-OH-JA-Ile were detected. In the microsomal reaction system of yeast expressing HvCYP94C1, residual substrate JA-Ile and its oxidation products 12-OH-JA-Ile and 12-COOH-JA-Ile were detected. The products were identified by multiple reaction monitoring (MRM) mode, with characteristic ion pairs as follows: JA-Ile m / z 324.4→129.7, 12OH-JA-Ile m / z 338.2→130.1, and 12COOH-JA-Ile m / z 352.2→130.1, with retention times consistent with the standards.

[0082] Experimental Example 2: The resistance to powdery mildew in the silenced barley lines was significantly increased.

[0083] 1. Experimental Methods

[0084] 1.1 Construction of Silent Barley Lines

[0085] Refer to the construction method in Example 2.

[0086] 1.2 Material Collection

[0087] After inoculation with powdery mildew, leaves from the silent strain and wild-type control were cut and immediately placed in pre-weighed EP tubes containing steel beads, labeled, and then rapidly frozen in liquid nitrogen for lyophilization. The lyophilized samples were ground using a grinder (MM 400, Retsch) at 30 Hz for 60 s, and the powder was transferred to 2 mL EP tubes and weighed. 30-60 mg of powder was accurately weighed into a new EP tube, and after calculating the net weight, 90% methanol (v / w) pre-cooled to 4 ℃ was added at 12 μL / mg. The mixture was vortexed for 15 s at 4 ℃ on an ice bath, repeated every 30 min for a total of 4 times, followed by static extraction at 4 ℃ for ≥12 h. After extraction, the mixture was centrifuged at 12,000 rpm for 10 min at 4 ℃, and the supernatant was filtered through a 0.22 μm microporous membrane. The filtrate was collected in a sample vial for UPLC-MS analysis.

[0088] 1.3 Detection of the target product

[0089] 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. The chromatographic column was an Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) and its guard column. The mobile phases were: Phase A: 0.1% formic acid in water; Phase B: 0.1% formic acid in methanol. The gradient program was: 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. The column temperature was 35 °C; the flow rate was 0.35 mL / min. - ¹; Injection volume 3 µL. Ion source parameters: Nitrogen gas is also used as the nebulizer (50 L h⁻¹). - ¹) with 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; 12-COOH-JA-Ile 352.2 → 130.1 • Positive ion mode: JA-Ile 322.4 → 129.7, data acquisition and processing are performed using MassLynx software.

[0090] 2. Experimental Results

[0091] This invention found that HvCYP94C1 expression was significantly inhibited in resistant materials infected with powdery mildew, while no significant change was observed in susceptible materials. Figure 1 ).

[0092] To clarify the functional differences of HvCYP94C1 in the JA-Ile inactivation cascade of highland barley, this invention compared the expression of HvCYP94C1 and the metabolomic profiles of JA-related substances in the silenced strain (Hvcyp94c1) and wild-type (WT) 7 days after powdery mildew infection. The results showed that, 7 days after powdery mildew infection, the transcriptional level of HvCYP94C1 in the leaves of both the wild-type (WT) and Hvcyp94c1 strains was significantly downregulated in the silenced strain compared to the wild-type WT (p<0.01). Figure 3 A). Corresponding to the decreased expression, the proportion of leaf lesions on Hvcyp94c1 leaves was only 42.3% of the total leaf area of ​​WT ( Figure 3 B). Metabolic analysis showed that decreased HvCYP94C1 expression led to a sharp 71.9% decrease in 12-COOH-JA-Ile content (p<0.01), while 12-OH-JA-Ile only decreased by 32.2% (p<0.05), and active JA-Ile significantly accumulated. Figure 3 (CE). This difference indicates that HvCYP94C1 exhibits a significant step-biased catalysis of JA-Ile: in the JA-Ile→12-OH-JA-Ile→12-COOH-JA-Ile cascade, its main function is concentrated in the second step, namely, efficiently promoting the further oxidation of 12-OH-JA-Ile to 12-COOH-JA-Ile; its catalytic efficiency for the first step of hydroxylation is relatively low. Therefore, the absence of HvCYP94C1 significantly blocks the final inactivation of JA-Ile, leading to the continuous activation of defense signals, which is manifested in the reduced lesion area and enhanced disease resistance in HvCYP94C1-silenced lines. Figure 3 B, Figure 4 ).

[0093] In summary, this invention provides a cytochrome P450 gene HvCYP94C1 and its application in improving the resistance of highland barley to powdery mildew. The cytochrome P450 gene HvCYP94C1 of this invention can hydroxylate JA and JA-Ile to 12-OH-JA-Ile and 12-COOH-JA-Ile, respectively. Silencing this gene in powdery mildew-sensitive materials can significantly increase the jasmonic acid content, thereby enhancing the disease resistance of the material, and shows excellent application prospects.

Claims

1. The use of the cytochrome P450 gene HvCYP94C1, a recombinant plasmid containing the cytochrome P450 gene HvCYP94C1, or a recombinant bacterium containing the cytochrome P450 gene HvCYP94C1 in constructing powdery mildew-resistant highland barley, characterized in that, Resistance to powdery mildew in barley can be enhanced by silencing the gene described herein, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. A method for constructing highland barley resistant to powdery mildew, characterized in that, Includes the following steps: (a) Construct a recombinant viral vector containing the cytochrome P450 gene HvCYP94C1; the nucleotide sequence of the gene is shown in SEQ ID NO.1; (b) The recombinant viral vector was transcribed in vitro to obtain viral RNA; (c) Inoculate the viral RNA onto the leaves of barley seedlings; (d) Cultivate and obtain barley resistant to powdery mildew with reduced HvCYP94C1 gene expression.

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