Application of CYP92C6 gene from Lolium multiflorum in improving herbicide tolerance of rice

Abstract

The application discloses application of a Lolium multiflorum CYP92C6 gene in improving herbicide tolerance of rice and belongs to the technical field of biotechnology. A cytochrome P450 family gene CYP92C6 is identified from Lolium multiflorum resistant to florasulam, the CDS sequence of the CYP92C6 gene is shown as SEQ ID NO:1, and the CYP92C6 protein encoded by the amino acid sequence shown as SEQ ID NO:2. The gene can efficiently metabolize florasulam through in-vitro yeast expression verification; the gene is introduced into rice for overexpression, and the tolerance of the transgenic rice to florasulam and pinoxaden is significantly improved. Molecular docking analysis shows that the CYP92C6 protein has stable binding force with florasulam and pinoxaden. The application provides important gene resources for developing herbicide-resistant rice varieties and provides a new strategy for preventing and controlling weeds in rice fields.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the cytochrome P450 family gene CYP92C6 in improving the herbicide tolerance of rice. Background Technology

[0002] Rice is one of my country's most important food crops. Weeds in paddy fields compete with rice for nutrients, sunlight, and growing space, inhibiting normal rice growth and severely restricting high and stable yields. Currently, weed control in paddy fields mainly relies on herbicide spraying. However, herbicide resistance in paddy field weeds develops rapidly, making weed control increasingly difficult. Developing and applying new herbicides is an important means of weed control in paddy fields; however, the development of new drugs is time-consuming and costly. Therefore, extending the application of herbicides that have shown good results in wheat fields to paddy fields has promising prospects.

[0003] If rice can be made resistant to herbicides used in wheat fields using biotechnology, then weeds in rice paddies can be controlled by spraying the same herbicides. This weed control method is simple, efficient, low-cost, and safe for crops. Crops can be modified to acquire herbicide resistance through genetic engineering. For example, soybeans can be genetically modified to express exogenous 5-enolpyruvate-3-phosphate synthase (EPSPS) to acquire glyphosate resistance. Developing herbicide-resistant transgenic crops is highly useful in production to expand the application of herbicides, develop new herbicide-resistant plant varieties, increase the diversity of resistance genes, and expand the scope of herbicide use.

[0004] Pyrazosulfuron is a sulfonylurea acetyl-lactate synthase (ALS) inhibitor herbicide. Due to its high efficiency, low toxicity, and broad spectrum, it is widely used to control weeds in wheat and barley fields. Clotrimazole is a neophenylpyrazoline acetyl-CoA carboxylase (ACCase) inhibitor herbicide; it features rapid onset of action, high ecological safety, and low risk of resistance, and can effectively control annual grass weeds in wheat fields. Rice is relatively sensitive to both pyrazosulfuron and clotrimazole. If biotechnology can be used to induce resistance in rice to pyrazosulfuron or clotrimazole, then these herbicides could be used for weed control in rice paddies.

[0005] Cytochrome P450 is a large gene family, with over 200 genes typically encoded in plants, participating in various metabolic reactions. It is involved not only in the metabolism of endogenous substances, such as fatty acid metabolism, the biosynthesis and degradation of plant hormones, and the synthesis of secondary metabolites, but also in the degradation of exogenous substances, such as herbicide metabolism and degradation. P450 oxidases can oxidize hydrophobic herbicide molecules into more hydrophilic secondary metabolites, which are then degraded. In recent years, research on the mechanisms by which P450 oxidases participate in herbicide metabolic resistance has attracted widespread attention and made significant progress. A series of P450 family genes have been identified, participating in the detoxification and metabolism of herbicides. Therefore, identifying P450 family genes capable of metabolizing herbicides will provide important genetic resources for the development of herbicide-tolerant crop varieties.

[0006] Oregano ryegrass is one of the most damaging noxious weeds in wheat fields and has evolved resistance to multiple types of herbicides. Studies have found that ryegrass populations from Jiangsu, Henan, and Anhui provinces have developed resistance to ACCase inhibitor herbicides such as clodinafop-propargyl and oxychloride, as well as ALS herbicides such as bispyribac-sodium and mesosulfuron-methyl. Our laboratory has conducted long-term monitoring of herbicide resistance development in ryegrass from wheat fields and found that 50 ryegrass populations from Henan and Jiangsu provinces have developed varying degrees of resistance to acesulfame potassium, with six populations exhibiting high levels of resistance.

[0007] Therefore, identifying the herbicide-resistant P450 gene from resistant ryegrass and applying it to the breeding of herbicide-tolerant rice will provide a new approach to weed control in rice paddies. Summary of the Invention

[0008] To address the technical problems raised in the background section, the present invention aims to provide the application of the CYP92C6 gene from *Lolium perfoliatum* in improving herbicide tolerance in rice. This invention identifies a cytochrome CYP92C6 gene from *Lolium perfoliatum* resistant to acesulfame K. In vitro expression of this gene in yeast can efficiently metabolize acesulfame K. Introducing the CYP92C6 gene into rice significantly improves the rice's tolerance to acesulfame K and cyclohexane. This invention provides an important genetic resource for developing herbicide-tolerant rice varieties and offers more possible strategies for weed control in rice paddies.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] In a first aspect, the present invention seeks protection for the use of the Lolium multiflorum CYP92C6 gene, with the nucleotide sequence shown in SEQ ID NO:1, in at least one of the following (a1) to (a3):

[0011] (a1) Application in improving plant tolerance to pyroxsulam;

[0012] (a2) Application in improving plant tolerance to pinoxaden;

[0013] (a3) Application in the cultivation of transgenic plants tolerant to sulfadiazine and / or cyclohexane;

[0014] The plant in question is rice, wheat, or barley.

[0015] Secondly, the present invention seeks protection for the use of the CYP92C6 protein encoded by the CYP92C6 gene of *Lycium chinense* with the nucleotide sequence shown in SEQ ID NO:1 in at least one of the following (a1) to (a3):

[0016] (a1) Application in improving plant tolerance to acesulfame potassium;

[0017] (a2) Application in improving plant tolerance to clopyralid;

[0018] (a3) Application in the cultivation of transgenic plants tolerant to sulfadiazine and / or cyclohexane;

[0019] The plant in question is rice, wheat, or barley.

[0020] Thirdly, the present invention seeks protection for the use of biological material containing the CYP92C6 gene of *Lycium pubescens* with the nucleotide sequence shown in SEQ ID NO:1 in at least one of the following (a1) to (a3):

[0021] (a1) Application in improving plant tolerance to acesulfame potassium;

[0022] (a2) Application in improving plant tolerance to clopyralid;

[0023] (a3) Application in the cultivation of transgenic plants tolerant to sulfadiazine and / or cyclohexane;

[0024] The plant in question is rice, wheat, or barley;

[0025] The biomaterial is at least one of the following (c1) to (c9):

[0026] (c1) An expression cassette containing the CYP92C6 gene;

[0027] (c2) A recombinant vector containing the CYP92C6 gene, or a recombinant vector containing the expression cassette (c1);

[0028] (c3) A recombinant microorganism containing the CYP92C6 gene, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2);

[0029] (c4) A transgenic plant cell line containing the CYP92C6 gene, or a transgenic plant cell line containing the expression cassette of (c1), or a transgenic plant cell line containing the recombinant vector of (c2);

[0030] (c5) Transgenic plant tissue containing the CYP92C6 gene, or transgenic plant tissue containing the expression cassette of (c1), or transgenic plant tissue containing the recombinant vector of (c2);

[0031] (c6) A transgenic plant organ containing the CYP92C6 gene, or a transgenic plant organ containing the expression cassette of (c1), or a transgenic plant organ containing the recombinant vector of (c2);

[0032] (c7) A transgenic plant containing the CYP92C6 gene, or a transgenic plant containing the expression cassette (c1), or a transgenic plant containing the recombinant vector (c2);

[0033] (c8) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c7);

[0034] (c9) Propagation material of the transgenic plants as described in (c7).

[0035] Fourthly, this invention claims protection for a method to improve plant tolerance to herbicides or to cultivate herbicide-tolerant transgenic plants, by overexpressing the CYP92C6 gene with the nucleotide sequence shown in SEQ ID NO:1 in a target plant or increasing the activity and / or content of the CYP92C6 protein encoded by the CYP92C6 gene, thereby enabling the plant to acquire the metabolic detoxification ability of acesulfame potassium and / or clodinafop-propargyl, thereby improving the plant's tolerance to acesulfame potassium and / or clodinafop-propargyl or cultivating new transgenic plant germplasm tolerant to acesulfame potassium and / or clodinafop-propargyl; wherein the plant is rice, wheat, or barley; and the herbicide is acesulfame potassium and / or clodinafop-propargyl.

[0036] Fifthly, the present invention claims protection for a method for determining the ability of CYP92C6 protein to metabolize herbicides in vitro, comprising: heterologously expressing the CYP92C6 protein with the amino acid sequence shown in SEQ ID NO:2 in Saccharomyces cerevisiae, adding a herbicide to the culture system, and detecting the residual amount or metabolites of the herbicide; wherein the herbicide is sulfadiazine and / or cyclohexane.

[0037] In a sixth aspect, the present invention seeks protection for the CYP92C6 gene of ryegrass, with a nucleotide sequence as shown in SEQ ID NO:1.

[0038] Seventhly, the present invention seeks protection for the CYP92C6 protein encoded by the above-mentioned CYP92C6 gene of *Lycium chinense*, wherein the CYP92C6 protein is a protein as shown in (b1) or (b2) below:

[0039] (b1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0040] (b2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0041] Eighthly, the present invention seeks protection for biological materials containing the above-mentioned ryegrass CYP92C6 gene, wherein the biological material is at least one of the following (c1) to (c9):

[0042] (c1) An expression cassette containing the CYP92C6 gene;

[0043] (c2) A recombinant vector containing the CYP92C6 gene, or a recombinant vector containing the expression cassette (c1);

[0044] (c3) A recombinant microorganism containing the CYP92C6 gene, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2);

[0045] (c4) A transgenic plant cell line containing the CYP92C6 gene, or a transgenic plant cell line containing the expression cassette of (c1), or a transgenic plant cell line containing the recombinant vector of (c2).

[0046] The transgenic plants obtained through the technical solution of this invention, their plant parts, or their seeds are also within the scope of protection of this invention. The transgenic plants have a stable integration of the CYP92C6 gene with a nucleotide sequence as shown in SEQ ID NO:1, or a nucleic acid molecule encoding the CYP92C6 protein with an amino acid sequence as shown in SEQ ID NO:2, into their genome, and exhibit enhanced tolerance to sulfadiazine and / or cyclophosphamide. The plants are rice, wheat, or barley.

[0047] In a specific embodiment of the present invention, the plant is rice (Oryza sativa). The transgenic rice cultivated under treatment with 1 to 8 times the field recommended dose of pyrazosulfuron and / or 0.5 to 4 times the field recommended dose of cyclophosphamide showed a significantly higher survival rate than the wild-type control.

[0048] This invention utilizes molecular docking analysis of the CYP92C6 protein with sulfadiazine and clodinafop-propargyl to demonstrate that the CYP92C6 protein exhibits stable and high binding affinity to both sulfadiazine and clodinafop-propargyl, effectively binding herbicide molecules and playing a crucial role in the detoxification and metabolism of herbicides.

[0049] The room temperature described in the technical solution of this invention is 25±5℃.

[0050] The beneficial effects of this invention are:

[0051] (1) This invention discloses for the first time the CDS sequence of the CYP92C6 gene of ryegrass;

[0052] (2) This invention is the first to discover that expressing the CYP92C6 gene in yeast can enable transgenic yeast to metabolize pyrazosulfuron;

[0053] (3) This invention is the first to discover that transgenic rice expressing CYP92C6 exhibits enhanced resistance to chlorpyrifos and clodinafop-propargyl;

[0054] (4) Molecular docking analysis of CYP92C6 protein with pyrazosulfuron and cyclohexane showed that CYP92C6 protein has stable binding force with both pyrazosulfuron and cyclohexane, which provides important support for the function of CYP92C6. Attached Figure Description

[0055] Figure 1 To identify and verify the metabolic function of the CYP92C6 gene in Lernica polyflora;

[0056] In this table, A represents the determination of the metabolic capacity of chlorpyrifos in sensitive (S) and resistant (R) ryegrass populations; B represents the expression level analysis of the CYP92C6 gene in sensitive (S) and resistant (R) ryegrass populations after chlorpyrifos treatment; the CYP92C6 gene was previously screened by transcriptome sequencing and its differential expression was observed in sensitive (S) and resistant (R) ryegrass populations; C represents the protein expression of empty vector (EV) and CYP92C6 gene-expressing recombinant yeast (CYP92C6); and D represents the metabolic capacity analysis of chlorpyrifos in empty vector (EV) and CYP92C6 gene-expressing recombinant yeast (CYP92C6).

[0057] Figure 2 To determine the sensitivity of rice overexpressing the CYP92C6 gene to chlorpyrifos and clodinafop-propargyl;

[0058] Where A represents twice the recommended dose of acesulfame potassium (the recommended field dose is 14 g ai ha). -1The whole-plant bioassay phenotypes of wild-type rice (WT) and CYP92C6 transgenic rice (CYP92C6) were compared. CK represents no herbicide treatment; B represents the whole-plant bioassay phenotype of wild-type rice and CYP92C6 transgenic rice treated with 4 times the recommended dose of acesulfame potassium; C represents the survival rate of wild-type rice and CYP92C6 transgenic rice after acesulfame potassium treatment (1, 2, 4, and 8 times the field recommended dose); D represents 1 / 2 times the field recommended dose of cyclophosphamide (the field recommended dose is 52.5 g ai ha). -1 The whole-plant bioassay phenotypes of wild-type rice and CYP92C6 transgenic rice treated with cyclohexane were shown in Figure 1. E represents the whole-plant bioassay phenotypes of wild-type rice and CYP92C6 transgenic rice treated with the field recommended dose of cyclohexane. F represents the survival rate of wild-type and CYP92C6 transgenic rice after cyclohexane treatment (1 / 2, 1, 2, and 4 times the field recommended dose).

[0059] Figure 3 A schematic diagram of the molecular docking of the CYP92C6 protein of Lernica polyflora with chlorpyrifos and cyclohexane.

[0060] Where A represents the molecular docking pattern of CYP92C6 protein and pyrazosulfuron, with a binding energy of -8.319 kcal / mol in the first binding posture. -1 The key amino acid residues that play a role in the degradation of cyprodinil by CYP92C6 include Arg-96, Ile-112, and Thr-113; B represents the molecular docking pattern of CYP92C6 protein with cyprodinil, with a binding energy of -7.641 kcal / mol in the first binding posture. -1 Among them, the key amino acid residues that play a role in the degradation of cyclophosphamide by CYP92C6 are Thr-113, Leu-368, Ala-369, Arg-371 and Phe-432. Detailed Implementation

[0061] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0062] Example 1: Determination of the metabolic capacity of sensitive and resistant ryegrass to sulfonamide

[0063] Sow approximately 15 seeds of ryegrass evenly in each small pot (7 cm × 7 cm × 7 cm), cover with soil, and place the plants in a light incubator. The incubator temperature is set to 20 / 15℃, 12 / 12 h, light intensity to 8000 lux, and relative humidity to 65%. When the ryegrass seedlings reach the 2-3 leaf stage, thin them to 10 seedlings per pot, ensuring uniform growth. Continue cultivation until the seedlings reach the 3-4 leaf stage, then apply a foliar spray. Simulating field application of sulfadiazine, apply the recommended dose of sulfadiazine (14 g ai ha) to the leaves of the 3-4 leaf stage ryegrass seedlings. -1 The specific procedure involves applying 2 μL of the active ingredient (0.343 μg / μL) to the leaves of each plant. -1 The dosage of the acesulfame potassium formulation is 20 μL per pot (ten plants per pot), totaling 6.86 μg of the active ingredient acesulfame potassium (pot area 0.0049 m²). 2 After treatment with the agent, the plants were moved back to the light incubator for further cultivation. The aboveground parts were sampled 24 hours and 72 hours after treatment for subsequent experiments. Three replicates were obtained for each treatment (each replicate sample included a pool of 10 plants per pot).

[0064] The collected sample was placed in a 50.0 mL stoppered centrifuge tube, and 10 mL of acetonitrile (HPLC grade, 99.9% purity) was added and stirred to obtain the residual agent on the plant surface. The sample was then removed and flash-frozen in liquid nitrogen, transferred to a mortar and mortar and ground into powder, transferred to a 50 mL stoppered plastic centrifuge tube, and 10 mL of acetonitrile was added. The mixture was vortexed for 5 min, then 4 g of anhydrous magnesium sulfate and 2 g of sodium chloride were added and vortexed for 1 min. The mixture was centrifuged at 8000 rpm for 5 min at room temperature. 1 mL of the supernatant was transferred to a purification tube containing 150 mg of anhydrous magnesium sulfate, 25 mg of PSA, and 2.5 mg of GCB. The tube was vortexed for 1 min to ensure complete purification. The tube was centrifuged at 8000 rpm for 5 min at room temperature. 1 mL of the supernatant was transferred through a 0.22 μm organic filter membrane to a vial and stored at -20℃ for detection by high-performance liquid chromatography-tandem triple quadrupole mass spectrometry. The detection conditions are as follows:

[0065] Chromatographic conditions: The mobile phase consisted of acetonitrile and 0.1% (v / v) formic acid aqueous solution. Mobile phase A consisted of 0.1% formic acid aqueous solution, and mobile phase B consisted of acetonitrile. A Waters BEH C18 column (50 mm × 2.1 mm, 1.7 μm) was used for linear gradient elution. The gradient elution program was: 0–2.0 min, 5% B phase; 2.0–4.0 min, 95% B phase; 4.0–5.0 min, 5% B phase. The flow rate was 0.4 mL / min. The column temperature was 25 °C, and the injection volume was 1 μL. Mass spectrometry conditions: Electrospray positive ion mode (ESI+); multiple reaction monitoring (MRM); curtain gas pressure: 30 psi; collision gas pressure: Medium; electrospray voltage: 5500 V; ion source temperature: 500℃; sprayer pressure: 50 psi; auxiliary heater pressure: 50 psi; residence time: 120 ms; MRM ion pairs: m / z 435.1>195.1, collision energy 15 eV, de-clustering voltage 30 V; m / z 435.1>194.0, collision energy 27 eV, de-clustering voltage 30 V; among which the quantitative ion pair is m / z 435.1>195.1. The results showed that the resistant ryegrass population (R) had a significantly higher metabolic capacity for sulfadiazine than the susceptible population (S). Figure 1 (A in the middle).

[0066] Example 2: Identification of upregulated expression of cytochrome P450 gene in resistant ryegrass

[0067] Sow sensitive and resistant ryegrass seeds evenly in each small pot (7 cm × 7 cm × 7 cm). Place the plants in a light incubator with a temperature of 20 / 15℃, a 12 / 12 h interval, a light intensity of 8000 lux, and a relative humidity of 65%. When the plants reach the 3-4 leaf stage, spray with acesulfame potassium (at the field recommended concentration of 14 g aiha). -1 Samples were taken 12 and 24 hours after treatment and sent for transcriptome analysis (without herbicide treatment as a control). Based on the transcriptome results, the P450 gene with significantly higher expression levels in the resistant population than in the susceptible population was screened. Combined with qPCR, a P450 gene CYP92C6 with upregulated expression was successfully identified. Figure 1 (B in the middle).

[0068] The specific steps for qPCR are as follows: qPCR primers for CYP92C6 are designed using the qPCR primer design software Primer3 web (https: / / primer3.ut). The primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0069] SEQ ID NO:3: ATCCATGCGCTCTCCAACAA

[0070] SEQ ID NO:4: GGAAGAACCTGGCCATCTCC

[0071] The RGTP gene was selected as the internal reference gene, and the primer sequences are shown in SEQ ID NO:5 and SEQ ID NO:6:

[0072] SEQ ID NO:5:GATGTGACTGACCAAGAGAGCTTCA

[0073] SEQ ID NO:6: CTCAGCTAAGTCGCATTTGTTCCCC

[0074] RNA was extracted from *Lycium rubrum* according to the instructions of the Tiangen Plant Total RNA Extraction Kit (DP-419). Reverse transcription of the RNA was performed using the Nanjing Novizan HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (R323-01) kit. Gene expression levels were measured using an Applied Biosystems QS1 real-time quantitative PCR instrument with the SYBR program selected. The reaction system was prepared according to the Nanjing Novizan ChamQ SYBR qPCR Master Mix (Q311-02) kit, and the reaction program was as follows: 95℃ for 30 sec; 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles.

[0075] Example 3: Cloning the CYP92C6 gene of Lorraine polyflora

[0076] Combining transcriptome data and previously reported ryegrass genome information, the full-length CDS of the CYP92C6 gene sequence of ryegrass was cloned. The specific cloning steps are as follows: the CDS sequence of the CYP92C6 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0077] The primer sequences for amplifying the CYP92C6 gene are shown in SEQ ID NO:7 and SEQ ID NO:8:

[0078] SEQ ID NO:7: ATGGAGCTACCACCATGGGC

[0079] SEQ ID NO:8: TCAAGCTTGGGTGTAGAGGCG

[0080] Approximately 15 seeds of Lernaea perfoliata were evenly sown in each small flowerpot (7 cm × 7 cm × 7 cm), covered with soil, and placed in a light incubator. The incubator temperature was set to 20 / 15℃, 12 / 12 h, light intensity to 8000 lux, and relative humidity to 65%. Samples were taken at the 3-4 leaf stage. Lernaea perfoliata RNA was extracted according to the instructions of the Plant Total RNA Extraction Kit (DP-419) from Tiangen Plant Resources Co., Ltd. Reverse transcription was performed using the HiScript III 1st Strand cDNASynthesis Kit (+gDNA wiper) (R312-01) from Nanjing Novizan Pharmaceutical Co., Ltd. to obtain cDNA.

[0081] Using the obtained cDNA as a template and the aforementioned primers, PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (P505-d1). The total volume of the amplification reaction system was 50 μL, including 25 μL 2 × Phanta Max Buffer, 1 μL dNTP Mix, 2 μL upstream primer, 2 μL downstream primer, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 2 μL template DNA, and 17 μL ddH2O. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 90 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0082] After the PCR reaction, 5 μL of the PCR product was subjected to 1% agarose gel electrophoresis (120 V, 25 min) to detect the target band. The correct PCR product was purified according to the EasyPure® PCRPurification Kit (EP101-01) of Beijing TransGen Biotech Co., Ltd. for subsequent construction of cloning vectors.

[0083] The gene was cloned using pClone007 Vector (TSV-007VS) from Beijing Qingke Biotechnology Co., Ltd., and the specific steps are as follows:

[0084] The reaction system consisted of 8 µL of the target gene and 2 µL of 5×pClone007 Versatile Simple VectorMix (5× pClone007 universal simple vector premix), and was placed in a PCR instrument and reacted at 25°C for 15 min.

[0085] DH5α chemocompetent cells (C502-02, Nanjing Novizan Biotechnology Co., Ltd.) were thawed on ice. After the above reaction was completed, the cells were immediately added to the thawed competent cells and transformed according to the instructions. After transformation, 100 µL of bacterial culture was evenly spread onto an LB agar plate containing Amp antibiotic. The remaining bacterial culture was centrifuged at 5000 rpm for 3 min, and 600 µL of supernatant was removed. 100 µL of the precipitate was evenly spread onto an LB agar plate containing Amp. The plate was then inverted and incubated in the dark at 37℃ for 12 h.

[0086] Remove the cultured agar plates and select single colonies with consistent growth in a sterile laminar flow hood. Use an inoculation stick to pick up a single colony and inoculate it into 5 mL of LB liquid medium containing Amp antibiotic. Incubate overnight at 37ºC with shaking at 200 rpm to extract plasmids.

[0087] Plasmid extraction was performed using a high-purity plasmid DNA mini-extraction kit (TSP501-50, Beijing Qingke Biotechnology Co., Ltd.). Plasmids were extracted according to the instructions, and plasmid concentration and mass were determined using a MIULAB spectrophotometer (ND-100C).

[0088] The PCR reaction system consisted of 5 μL 2 × Taq Master Mix, 0.5 μL each of forward and reverse primers, 0.5 μL template, and 3.5 μL ddH2O. The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 90 s, 35 cycles, and a final extension at 72°C for 5 min.

[0089] After the PCR reaction, 5 μL of the PCR product was subjected to 1% agarose gel electrophoresis (120 V, 25 min) to detect the target band. The plasmid with the correct band size was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed using NCBI online BLAST. The comparison and analysis with the NCBI database sequence showed that the CYP92C6 gene of Lernaea perlatum was successfully cloned.

[0090] Example 4: Construction of a yeast expression vector for the CYP92C6 gene of Lorrae polyflora

[0091] 1. Plasmid construction

[0092] Primers for ligating the pYeDp60 vector were designed based on the CYP92C6 sequence above. A 6×His tag was fused upstream of the stop codon during primer design. The primer sequences are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0093] SEQ ID NO:9: TAAATTACCGGATCCATGGAGCTACCACCATGGGC

[0094] SEQ ID NO:10:

[0095] CCCCGCGAATTCTCAATGGTGATGGTGATGATGAGCTTGGGTGTAGAGGCG

[0096] Using a monoclonal plasmid of the CYP92C6 gene as a template, PCR amplification was performed using the primers designed above and a high-fidelity enzyme. The total volume of the amplification reaction system was 50 μL, including 25 μL 2×PhantaMax Buffer, 1 μL dNTPMix, 2 μL upstream primer, 2 μL downstream primer, 1 μL PhantaMax Super-Fidelity DNA Polymerase, 2 μL template DNA, and 17 μL ddH2O. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 90 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0097] After the PCR reaction, 5 μL of the PCR product was subjected to 1% agarose gel electrophoresis (120 v, 25 min) to detect the target band. The correct PCR product was purified for subsequent homologous recombination.

[0098] The pYeDp60 vector (BioVector NTCC Inc.) was digested with enzymes in a 50 μL volume, including 1 ng of pYeDp60 vector, 5 μL of 10×rCutsmart Buffer, 1 μL of BamHI-HF (NEB, R3136), 1 μL of LEcoRI-HF (NEB, R3101), and ddH2O to bring the volume to 50 μL. Digestion was performed at 37°C for 90 min. The digested product was purified, and homologous recombination was performed using the Novizan ClonExpress II One Step Cloning Kit (C112). The reaction mixture included 120 ng of linearized vector, 60 ng of fragment, 2 μL of 5×CE II Buffer, and 1 μL of Exnase II. The mixture was gently mixed, briefly centrifuged, and the reaction solution was collected at the bottom of the tube. The reaction was incubated at 37°C for 30 min, then cooled to 4°C or immediately placed on ice.

[0099] The ligation product was transferred into DH5α competent cells, placed on ice for 30 min, heat-shocked in a water bath at 42°C for 45 s, and immediately placed on ice for 2-3 min. 900 μL of antibiotic-free LB liquid medium was added, and the cells were transferred to a shaker and cultured at 37°C and 200 rpm for 1 h to revive them. After centrifugation at 5000 rpm for 1.5 min, 900 μL of supernatant was discarded, the precipitate was mixed by pipetting, and spread onto LB solid medium containing Amp antibiotic. The plates were placed in a constant temperature biochemical incubator and inverted for 12 h.

[0100] Single colonies were picked and cultured in LB broth containing Amp antibiotic at 37°C and 200 rpm for 12 h with shaking. A portion of the bacterial culture was then used for colony PCR to verify successful transformation. The PCR reaction system consisted of 5 μL 2×Taq Master Mix, 0.5 μL each of forward and reverse primers, 0.5 μL template, and 3.5 μL ddH2O. The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 90 s, 35 cycles, and a final extension at 72°C for 5 min.

[0101] Transform the successfully validated plasmid into the WAT11 strain as follows: Heat the carrier DNA at 95-100℃ for 5 min, then quickly place it on ice; repeat once; take 100 μL of WAT11 competent cells and thaw them on ice, then add 2-5 μg of pre-chilled target plasmid, 10 μL of carrier DNA, and 500 μL of PEG / LiAc and mix thoroughly by pipetting; incubate at 30℃ for 30 min (twisting 6-8 times at 15 min to mix); place the tube in a 42℃ water bath for 15 min (twisting 6-8 times at 7.5 min to mix); centrifuge at 5000 rpm for 40 s and discard the supernatant; resuspend the bacterial pellet in 400 μL of ddH2O, centrifuge for 30 s and discard the supernatant; resuspend the bacterial pellet in 50 μL of ddH2O, plate it onto a plasmid selection plate (SD / -Ura medium), and incubate in the dark at 29℃ for 48-96 h.

[0102] Single colonies were picked and transferred to SD-U medium containing glucose at a final glucose concentration of 20 g / L (glucose was sterilized using a sterile filter membrane). The culture was incubated overnight at 29°C and 200 rpm. Then, the culture was transferred to SD-U medium containing galactose at a volume ratio of 1:100, with a final galactose concentration of 20 g / L (galactose was sterilized using a sterile filter membrane). The culture was incubated at 29°C and 200 rpm for 40 h. The bacterial culture was then centrifuged to extract DNA for verification.

[0103] The PCR system and procedure are as described above. After the PCR reaction, take 5 μL of the PCR product and perform 1% agarose gel electrophoresis to detect the target band. Proceed to the next experiment with the correct bacterial culture.

[0104] 2. Protein expression validation

[0105] Take 50 mL of the correctly induced bacterial culture and centrifuge at 4000 rpm for 10 min at room temperature. Resuspend the bacterial cells in 2 mL of 0.1 M sodium phosphate buffer (pH=8.0), add 20 μL of PMSF, and sonicate the cells for 60 min (power: 35w; disruption time: 1 h; pulse-on time: 5 s; pulse-off time: 5 s). Centrifuge the disrupted bacterial culture at 4000 rpm for 15 min at 4℃, and collect the supernatant as the crude protein extract. Add 80 μL of the crude protein extract to 20 μL of 5×SDS-PAGE Protein Loading Buffer (Yisheng Biotechnology, 20315ES), incubate at 100℃ for 5 min, and detect protein expression by Western blot. Figure 1 (C in the middle).

[0106] Example 5: Detection of the ability of recombinant CYP92C6 to metabolize sulfadiazine in vitro

[0107] A single yeast colony successfully expressing CYP92C6 protein was inoculated into 5 mL of SD-U medium with a final glucose concentration of 20 g / L (glucose sterilized via sterile filter membrane) and cultured overnight at 29°C and 200 rpm. Then, it was transferred to SD-U medium containing galactose (galactose sterilized via sterile filter membrane) and cultured at 29°C and 200 rpm for 40 h. A final concentration of 200 μM sulfadiazine was added to the bacterial culture, and the culture was returned to a shaker and cultured at 29°C and 200 rpm. Samples were taken at 12 and 24 hours, 20 mL each time, centrifuged at 4000 rpm for 5 min, filtered through a 0.22 μm filter membrane, and stored at -20°C for analysis. The residual amount of sulfadiazine in the samples was detected using HPLC / MS-MS, as described in Example 1. The results showed that the recombinant CYP92C6 yeast could effectively metabolize sulfadiazine, with a metabolic efficiency significantly higher than the empty vector control. Figure 1 (D in the middle).

[0108] Example 6: Evaluation of herbicide tolerance in rice overexpressing the CYP92C6 gene

[0109] The CDS sequence of CYP92C6 was homologously recombined into the pBI121 expression vector, and the primer sequences are shown in SEQ ID NO:11 and SEQ ID NO:12:

[0110] SEQ ID NO.11: ACGGGGGACTCTAGAGATGGAGCTACCACCATGGGC

[0111] SEQ ID NO.12: CGATCGGGGAAATTCGTCAATGGTGATGGTGATGATGAGCTTGGGTTGTAGAGGCG

[0112] A 6×His tag was fused upstream of the stop codon in primer design. The plasmid construction steps were the same as described in Example 4, with the fragment inserted between the BamHI and SacI sites in the vector. The successfully constructed plasmid pBI121-CYP92C6-His was sent to Weimi Biotechnology Co., Ltd. for genetic transformation of rice (Nipponbare) to obtain CYP92C6 overexpression lines. Western blotting was performed on leaf samples collected during F1 generation culture to verify successful expression of the target protein, and T2 generation seeds were harvested.

[0113] Sensitivity tests were conducted using the whole-plant bioassay method after planting and harvesting. The treatment doses of acesulfame potassium were 1, 2, 4, and 8 times the field recommended dose (the field recommended dose is 14 g ai ha). -1The treatment doses of clodinafop-propargyl were 1 / 2, 1, 2, and 4 times the recommended field dose (the recommended field dose is 52.5 g ai ha). -1 Fourteen days after treatment with the herbicides, the fresh weight of the aboveground parts was measured, and the results were statistically analyzed to calculate the fresh weight inhibition rate. The results showed that the survival rate of CYP92C6 overexpressing rice was significantly higher than that of wild-type (WT) under treatment with both cypermethrin and clodinafop-propargyl. The GR (growth inhibition rate) of transgenic rice after cypermethrin treatment was significantly higher. 50 The value is 33.00 g ai ha -1 Wild-type GR 50 The value is 13.38 g ai ha -1 The RI value was 2.47; the GR of transgenic rice treated with clopyralid was... 50 The value is 90.51 g ai ha -1 Wild-type GR 50 The value is 32.28 ga.i. ha -1 The RI value was 2.80. Therefore, overexpression of CYP92C6 leads to higher herbicide resistance in rice. Figure 2 ).

[0114] Example 7: Molecular docking of CYP92C6 protein with pyrazosulfuron and clodinafop-propargyl

[0115] A protein structure model of CYP92C6 was constructed using homology modeling prediction via Swiss-model (https: / / swissmodel.expasy.org / ), and the protein structure was validated using SVAESv6 (https: / / saves.mbi.ucla.edu / ), and saved in pdb format. The structures of the ligands sulfadiazine (Compound CID: 11571555) and cyclophosphamide (Compound CID: 49867066) were obtained from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and converted to pdb format. Receptor protein preprocessing: The pdb protein was imported into AutoDock Tools for visualization, water molecules were removed, hydrogen was added, and the protein was exported and saved in pdbqt format. The ligands were also imported into AutoDock Tools, hydrogen was added, gasteiger charges were calculated, root detection was performed, and torsion was selected, and the protein was exported and saved in pdbqt format. Dosing of the receptor protein to the herbicide was performed using a Visa database. Potential binding regions for the CYP92C6 protein were identified using InterPro (https: / / www.ebi.ac.uk / interpro / ), and the position and size of the docking box were designed in a grid for docking. The docking results were analyzed using Discovery Studio and visualized using PyMOL. The results showed that the CYP92C6 protein has a high affinity for acesulfame potassium, with a binding energy of -8.319 kcal·mol⁻¹ in the first binding posture. -1 The key amino acid residues that play a functional role in the degradation of pyridaben by CYP92C6 include Arg-96, Ile-112, and Thr-113. CYP92C6 also exhibits a high affinity for pyridaben, with a binding energy of -7.641 kcal·mol⁻¹ in the first binding state. -1 Among them, the key amino acid residues that play a role in the degradation of pyridaben by CYP92C6 are Thr-113, Leu-368, Ala-369, Arg-371, and Phe-432. Figure 3 The molecular docking results provide theoretical support for the involvement of the CYP92C6 protein in herbicide metabolism.

[0116] Therefore, this invention discloses for the first time the CDS sequence of the CYP92C6 gene of Lorrae polyantha, and for the first time discovers that expressing the CYP92C6 gene in rice can enhance its tolerance to herbicides such as chlorpyrifos and clodinafop-propargyl, providing an important gene resource for the development of herbicide-tolerant rice varieties.

[0117] sequence list

[0118] Lolium multiflorum CYP92C6 gene (SEQ ID NO:1):

[0119]

[0120] Lolium multiflorum (Lolium multiflorum) CYP92C6(SEQ ID NO:2):

[0121] MELPPWAPFLAIVLFLATILRRGRRAYRLPPGPKPWPIIGNLIGSLPHRSIHALSKRYGPLMYLQFGSIPVVVGSSAEMAKFFLKTQDIVFIDRPKTAAGKYTAYHYSDITWSSYGAYWRQARK MCLTELFSAKRLESYIRSEEIRVLLRDHGASGRVVLLKDHLFTLSLNVITRMVLGKKYLHKEATTTQEEFRWMVDEWFLLNGVFNIGDSIPWLDWMDLQGYIKRMKKLSKKFDRFLHVVDEH NGRRHLEGESFVAKDMVDVLLEITSDPNLEVPIHRDGAKAFILDLIVAGTESSAITVEWAMSEILKKPEVFAKATEELDRVVGRDRWVTEEDMTSLPYMEAIVKETMRLHPVAPMLAPRLSRQDAS VGGYDIPVGTRVLVNVWSIGRDPALWDAPEQFLPERFIGSKIDVKGQDFELLPFGSGRRMCPPGYSLGLKVIQMSLANLLHGFTWRLPSGVTKEALSMEEIYGMTTPRKFPLEAIPEPPLQARLYTQA

[0122] SEQ ID NO:3: ATCCATGCGCTCTCCAACAA

[0123] SEQ ID NO:4: GGAAGAACCTGGCCATCTCC

[0124] SEQ ID NO:5: GATGTGACTGACCAAGAGAGCTTCA

[0125] SEQ ID NO:6: CTCAGCTAAGTCGCATTTGTTCCCC

[0126] SEQ ID NO:7: ATGGAGCTACCACCATGGGC

[0127] SEQ ID NO:8: TCAAGCTTGGGTGTAGAGGCG

[0128] SEQ ID NO:9: TAAATTACCGGATCCATGGAGCTACCACCATGGGC

[0129] SEQ ID NO:10: CCCCGCGAATTCTCAATGGTGATGGTGATGATGAGCTTGGGTGTAGAGGCG

[0130] SEQ ID NO:11: ACGGGGGACTCTAGAGATGGAGCTACCACCATGGGC

[0131] SEQ ID NO:12: CGATCGGGGAAATTCGTCAATGGTGATGGTGATGATGAGCTTGGGTGTAGAGGCG。

Claims

1. Overexpression of *Lycium oryzae* with the nucleotide sequence shown in SEQ ID NO:1 CYP92C6 The use of genes in at least one of the following (a1) to (a3): (a1) Application in improving rice tolerance to acesulfame potassium; (a2) Application in improving rice tolerance to clopyralid; (a3) Application in the development of transgenic rice tolerant to pyrazosulfuron and / or cyclohexane.

2. Ryegrass containing the nucleotide sequence shown in SEQ ID NO:1 CYP92C6 The application of genetic biomaterials in at least one of the following (a1) to (a3): (a1) Application in improving rice tolerance to acesulfame potassium; (a2) Application in improving rice tolerance to clopyralid; (a3) Application in the development of transgenic rice tolerant to pyrazosulfuron and / or cyclohexane; The biomaterial is at least one of the following (c1) to (c3): (c1) contains the above CYP92C6 Gene expression cassettes; (c2) contains the above CYP92C6 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in (c1); (c3) contains the above CYP92C6 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in (c1), or recombinant microorganisms containing the recombinant vector described in (c2).

3. A method for improving the herbicide tolerance of rice or cultivating herbicide-tolerant transgenic rice, characterized in that, The herbicide is chlorpyrifos and / or clodinafop-methyl, expressed in target rice via overexpression of the nucleotide sequence shown in SEQ ID NO:

1. CYP92C6 Genes that enable rice to metabolize and detoxify pyrazosulfuron and / or cyclohexane, thereby improving rice tolerance to pyrazosulfuron and / or cyclohexane or developing new transgenic rice germplasm tolerant to pyrazosulfuron and / or cyclohexane.

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