Application of ZmCYP81Q32 gene in improving nicosulfuron resistance in plants

Abstract

This invention belongs to the field of plant genetic engineering and plant stress resistance biotechnology, and discloses... ZmCYP81Q32 Application of genes in improving nicosulfuron resistance in plants. This invention provides... ZmCYP81Q32 The nucleotide sequence of the gene, as shown in SEQ ID NO:1, encodes a cytochrome P450 enzyme that can directly bind to sulfonylurea herbicides and promote their hydroxylation metabolism. This reduces the accumulation of the parent herbicide in the plant, alleviates herbicide-induced growth inhibition, leaf chlorosis, and oxidative damage, and significantly improves plant tolerance to sulfonylurea herbicides, especially enhancing plant resistance to nicosulfuron. This invention can be widely used for crop herbicide resistance improvement, herbicide-tolerant germplasm creation, and related molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and plant stress resistance biotechnology, specifically involving ZmCYP81Q32 Application of genes in improving plant resistance to nicosulfuron. Background Technology

[0002] Herbicides are widely used in agricultural production, playing a crucial role in improving weed control efficiency and ensuring crop yields. Sulfonylurea herbicides, such as nicosulfuron, are highly effective acetolactate synthase (ALS) inhibitors widely used in agriculture. They kill weeds by blocking the synthesis of essential branched-chain amino acids in plants. However, the application of these herbicides is limited by crop sensitivity and soil residue issues. Improving crop tolerance to herbicides through genetic engineering is a fundamental solution to this problem.

[0003] Currently, existing technologies mostly focus on modifying the herbicide target protein ALS to make it insensitive to herbicides. However, this single-target resistance mechanism carries the risk that weeds may also develop resistance. Therefore, developing new genes to enhance plant herbicide resistance and applying them to herbicide-resistant plant improvement and herbicide-tolerant breeding has significant theoretical importance and application prospects. Summary of the Invention

[0004] The purpose of this invention is to solve the problems of easy induction of weed resistance caused by relying on single-target modification in the prior art, and to provide... ZmCYP81Q32 The application of genes in improving plant resistance to sulfonylurea herbicides has enabled plants to exhibit more stable and efficient resistance to sulfonylurea herbicides such as nicosulfuron.

[0005] This invention provides ZmCYP81Q32 The application of genes in improving plant resistance to sulfonylurea herbicides, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] This invention provides ZmCYP81Q32 The application of genes in breeding plants resistant to sulfonylurea herbicides, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] This invention provides ZmCYP81Q32 The application of genes in promoting the metabolism of sulfonylurea herbicides in plants and / or yeast, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] Preferably, the sulfonylurea herbicide is metabolized as a hydroxylation metabolism of the sulfonylurea herbicide.

[0009] Preferably, the yeast is brewer's yeast.

[0010] Preferably, the plant is Arabidopsis thaliana and / or maize; the sulfonylurea herbicide is nicosulfuron.

[0011] This invention provides a recombinant yeast, comprising a base yeast and a yeast into which the base yeast is introduced. ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0012] This invention provides a plant material that, compared to wild-type plants, overexpresses... ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0013] Preferably, the plant is Arabidopsis thaliana and / or maize.

[0014] This invention provides a method for improving plant resistance to sulfonylurea herbicides, comprising the following steps: [The method involves...] ZmCYP81Q32 The gene was transferred into the target plant, the ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0015] Beneficial effects: The present invention provides ZmCYP81Q32 The gene's nucleotide sequence, as shown in SEQ ID NO:1, encodes a cytochrome P450 enzyme that can directly bind to sulfonylurea herbicides and promote their hydroxylation metabolism. This reduces the accumulation of the parent herbicide in the plant, alleviates herbicide-induced growth inhibition, leaf chlorosis, and oxidative damage, and significantly improves plant tolerance to sulfonylurea herbicides, particularly enhancing resistance to nicosulfuron. Overexpression ZmCYP81Q32 This invention can significantly improve the survival rate, biomass, and overall growth performance of recipient plants. Furthermore, its EMS mutant exhibits stronger sensitivity after nicosulfuron treatment, demonstrating the important role of this gene in plant herbicide tolerance from both positive and negative perspectives. Therefore, this invention can be widely applied to crop herbicide resistance improvement, herbicide-tolerant germplasm creation, and related molecular breeding, possessing significant theoretical value and application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 for ZmCYP81Q32 The expression of information in various tissues of maize; Figure 2 for ZmCYP81Q32 The results of RNA in situ hybridization; Figure 3 for ZmCYP81Q32 Phylogenetic tree; Figure 4 for ZmCYP81Q32 Subcellular localization results; Figure 5 for ZmCYP81Q32 -Fluorescence intensity distribution curves of EGFP and endoplasmic reticulum marker protein CD3-959 (ER-rk) along ROI1 and ROI2; Figure 6 for ZmCYP81Q32 Molecular docking results with nicosulfuron; Figure 7 The results of Coomassie brilliant blue staining and Western blot analysis of recombinant protein ZmCYP81Q32; Figure 8 for ZmCYP81Q32 The dissociation constant of nicosulfuron; Figure 9 For overexpression ZmCYP81Q32 The growth status of yeast strains on nicosulfuron-methyl culture media of different concentrations; Figure 10 For the reason CaMV 35S Startup driver 35S::ZmCYP81Q32 Schematic diagram of the overexpression vector; Figure 11 For wild type and overexpression ZmCYP81Q32 Phenotypic images of Arabidopsis thaliana seeds cultured on MS medium with different concentrations of nicosulfuron for 7 days; Figure 12 For wild type and overexpression ZmCYP81Q32 Phenotypic diagram of homozygous Arabidopsis thaliana lines 12 days after spraying with nicosulfuron; Figure 13 For wild type and overexpression ZmCYP81Q32 Rosette diameter, plant height and fresh weight of homozygous Arabidopsis thaliana lines 12 days after nicosulfuron spraying; Figure 14 Accumulation of hydroxylated metabolites 1 day after spraying wild-type Arabidopsis thaliana with nicosulfuron; Figure 15 Accumulation of hydroxylated metabolites 3 days after spraying wild-type Arabidopsis thaliana with nicosulfuron; Figure 16 For overexpression ZmCYP81Q32 Accumulation of hydroxylated metabolites in Arabidopsis thaliana 1 day after nicosulfuron spraying; Figure 17 For overexpression ZmCYP81Q32 Accumulation of hydroxylated metabolites in Arabidopsis thaliana 3 days after nicosulfuron spraying; Figure 18The results show the identification of nicosulfuron resistance in maize lines; the left figure is the wild-type maize line, and the right figure is the EMS mutant maize line. Detailed Implementation

[0018] This invention provides ZmCYP81Q32 The application of genes in improving plant resistance to sulfonylurea herbicides, the aforementioned ZmCYP81Q32

[0019] This invention provides ZmCYP81Q32 The application of genes in breeding plants resistant to sulfonylurea herbicides, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0020] This invention provides ZmCYP81Q32 The application of genes in promoting the metabolism of sulfonylurea herbicides in plants and / or yeast, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0021] In one embodiment, the sulfonylurea herbicide of the present invention is metabolized as a hydroxylation metabolism of the sulfonylurea herbicide.

[0022] In one embodiment, the yeast described in this invention is brewing yeast.

[0023] In one embodiment, the plant described in this invention is Arabidopsis thaliana and / or maize.

[0024] As one embodiment, the sulfonylurea herbicide of the present invention is nicosulfuron.

[0025] This invention provides a recombinant yeast, comprising a base yeast and a yeast into which the base yeast is introduced. ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0026] This invention provides a plant material that, compared to wild-type plants, overexpresses... ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0027] In one embodiment, the plant described in this invention is Arabidopsis thaliana and / or maize.

[0028] This invention provides a method for improving plant resistance to sulfonylurea herbicides, comprising the following steps: [The method involves...] ZmCYP81Q32 The gene was transferred into the target plant, the ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1. This invention relates to... ZmCYP81Q32 There are no strict rules regarding the method of gene transfer into the target plant; any conventional method in the field is acceptable. For example, one could... ZmCYP81Q32 The gene is inserted into a plant overexpression vector and introduced into the target plant via Agrobacterium transfection.

[0029] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, explains the invention. ZmCYP81Q32The application of genes in enhancing nicosulfuron resistance in plants is described in detail, but should not be construed as limiting the scope of protection of this invention.

[0030] The plant materials and cultivation conditions used in the embodiments of this invention are as follows: Maize: The nicosulfuron-sensitive inbred line “HB39”, preserved by the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences. Seed culture conditions: Maize seeds were surface-sterilized and sown in a substrate containing a mixture of peat and vermiculite (3:1, v / v). Maize seedlings were cultured in an artificial climate chamber under the following conditions: 16 h light / 8 h dark, temperature 25±2℃, and light intensity 600 μmol·m⁻¹. -2 ·s -1 ; Arabidopsis thaliana: Col-0. Seeds were sown in 1 / 2 MS medium or soil and cultured at 22°C, 60% relative humidity, and under 16 h light / 8 h dark conditions.

[0031] Ben's tobacco ( Nicotiana benthamiana ): Cultivate in a greenhouse at 25 ℃, 60% relative humidity, 16 h light / 8 h dark; select 4-6 week old plants for Agrobacterium-mediated transient transformation experiments.

[0032] Example 1 1. The inventors discovered through qRT-PCR analysis that... ZmCYP81Q32 Widely expressed in various tissues of maize B73 ( Figure 1 RNA in situ hybridization further revealed that its transcriptional signaling was mainly distributed in leaf mesophyll cells. Figure 2 This is consistent with its expected protective function in photosynthetic tissues.

[0033] 2. Phylogenetic analysis In order to analyze ZmCYP81Q32 Phylogenetic relationships with homologous proteins from other Poaceae species were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). ZmCYP81Q32 The amino acid sequences were obtained, and homologous protein sequences from grass species such as rice, wheat, and sorghum were retrieved from the Phytozome database for phylogenetic analysis. Amino acid sequences were aligned using Jalview 2.11.5.1, and a phylogenetic tree was constructed using MEGA 11 software based on the Neighbor-Joining (NJ) method.

[0034] The results show that ZmCYP81Q32 With what has been reported BsCYP81Q32 and OsCYP81A6Key proteins involved in herbicide metabolism and degradation closely clustered within the same conserved branch. Furthermore, multiple sequence alignment revealed high conservation among representative grasses. Figure 3 ).therefore, ZmCYP81Q32 It is an important candidate gene that may be involved in the process by which CSA promotes the metabolic detoxification of nicosulfuron.

[0035] 3. Subcellular localization analysis Amplification ZmCYP81Q32 The CDS sequence of the stop codon was removed, and then inserted into the 35S::pBA002-EGFP vector using the corresponding primers (ZmCYP81Q32-pC1300-F: gacgggccccaattgggatccATGTGCAGCCATGCAGGATG, SEQ ID NO:2; ZmCYP81Q32-pC1300-R: ggcagcagctctagaggatccGAGCTCCAGAAGAAGATGGCG, SEQ ID NO:3) to construct the 35S::pBA002-EGFP vector. ZmCYP81Q32 -EGFP fusion expression vector. This vector was then co-transformed with the endoplasmic reticulum marker protein ER-rk (CD3-959) into tobacco leaves. After 24 h of dark culture followed by 48 h of light culture, the samples were observed using a laser confocal microscope (Leica TCS SP5 II).

[0036] The results show that ZmCYP81Q32 The fluorescence signals of -EGFP and the endoplasmic reticulum marker protein CD3-959 (ER-rk) highly overlapped, and the fluorescence intensity distribution curves along ROI1 and ROI2 showed a consistent trend, indicating that... ZmCYP81Q32 It is mainly located in the endoplasmic reticulum, a core organelle in which P450 enzymes exert their activity. Figure 4 and Figure 5 ).

[0037] 4. Structural modeling and molecular docking Obtain from the Uniprot database (https: / / www.uniprot.org / uniprotkb / ) ZmCYP81Q32 The protein sequences were accession numbers A0A804M4E3. Homology modeling was performed using the SWISS-MODEL online server (https: / / swissmodel.expasy.org / ), where... ZmCYP81Q32A three-dimensional structural model was constructed using PDB 2hi4 as a template. Subsequently, molecular docking of the protein and nicosulfuron was performed using AutoDock Vina 1.2.7, and the binding ability of the protein and ligand was evaluated based on affinity scores. The conformation with the highest affinity score was visualized and analyzed using PyMOL 2.3.0.

[0038] The results showed that nicosulfuron could be embedded in... ZmCYP81Q32 The predicted active pocket was identified, and it formed stable interactions with multiple residues in the active site, with a predicted binding energy of -6.9 kcal / mol. -1 ( Figure 6 ).

[0039] Example 2 1. Recombinant protein expression and purification Will ZmCYP81Q32 The full-length coding sequence was cloned into the pET28a-MBP-His vector to construct... ZmCYP81Q32 -MBP-His fusion expression vector. The recombinant vector was expressed in *E. coli* Rosetta (DE3) competent cells and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 ℃ for 12 h. Subsequently, it was purified using MagneHis™ Ni-Particles (Promega). 16 μL of the purified protein was mixed with 4 μL of loading buffer and heated at 100 ℃ for 8 min. 10 μL of the sample was separated in a 12% SDS-PAGE gel (120 V) and then subjected to Coomassie brilliant blue staining and Western blotting analysis. His-tagged proteins were detected using an anti-His monoclonal antibody (1:5000), and the secondary antibody was HRP-labeled goat anti-mouse IgG (H+L) antibody (1:10000). The Omni-ECL™ Pico chemiluminescence assay kit (Shanghai Yamei Biomedical Technology Co., Ltd.) was used. Results showed that the ZmCYP81Q32 protein was successfully purified. Figure 7 ) 2. Surface plasmon resonance measurement The concentrated and desalted ZmCYP81Q32 protein was diluted to 25 μg / mL with 10 mM sodium acetate buffer (pH 4.0) and immobilized on a CM5 sensor chip via amino-coupling at a fixation level of approximately 8000 RU. Different concentrations of the compound were injected at a flow rate of 30 μL / min. The run buffer contained 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P2O, and 5% DMSO. Both binding and dissociation times were set to 60 s. The equilibrium dissociation constant (KD) was calculated using Biacore8K evaluation software.

[0040] The results show that ZmCYP81Q32 A detectable direct binding exists between it and nicosulfuron, with a dissociation constant (KD) of 2.90 × 10⁻⁶. -5 M ( Figure 8 ).

[0041] 3. Yeast heterologous expression and functional verification The effects of candidate genes on herbicide tolerance were assessed using a *Saccharomyces cerevisiae* heterologous expression system. ZmCYP81Q32 The full-length coding sequence was cloned into the yeast expression vector pYeDP60. The resulting recombinant plasmid was transformed into *Saccharomyces cerevisiae* strain WAT11 (Coolaber, Beijing, China). The empty pYeDP60 vector was used as a negative control. Figure 10 Positive single colonies were first incubated in 2 mL SG / -Ura liquid medium at 28 °C for 12 h, then transferred to 10 mL of the same medium and incubated for another 8 h. The bacterial suspension was collected by centrifugation, washed three times with sterile water, resuspended, and adjusted to OD. 600 = 0.6, followed by 10-fold serial dilutions. 5 μL of each gradient bacterial suspension was dropped onto SG / -Ura solid medium containing different concentrations of herbicide, and the growth differences of the strains were recorded after incubation at 28 ℃ for 72-96 h.

[0042] The results showed that, compared with WAT11 yeast carrying the empty vector (pYeDP60), the expression ZmCYP81Q32 The yeast strains showed better growth on media containing different concentrations of nicosulfuron. Figure 9 These results indicate that... ZmCYP81Q32 It can significantly improve yeast tolerance to nicosulfuron, supporting its role as a CSA-induced candidate detoxification-related P450 in maize's response to nicosulfuron.

[0043] Example 3 1. Plasmid construction and plant transformation Translation-free stop codons were amplified from maize B73. ZmCYP81Q32The CDS sequence was cloned into the pC1300 vector to construct the fusion expression vector 35S:: driven by the cauliflower mosaic virus 35S promoter. ZmCYP81Q32 ( Figure 10 After sequencing verification, the recombinant vector was transformed into Agrobacterium GV3101 and then transformed into wild-type Arabidopsis thaliana (Col-0) using the inflorescence immersion method. Transformed seeds were sown on a selection medium containing hygromycin to obtain resistant plants, and positive overexpression lines (OE) were further identified and screened by RT-PCR.

[0044] 2. Tolerance test of nicosulfuron in Arabidopsis thaliana (1) In order to evaluate ZmCYP81Q32 The effect of overexpression on nicosulfuron tolerance in Arabidopsis thaliana was investigated by sowing Col-0, OE1, and OE2 seedlings on MS medium containing different concentrations of nicosulfuron (0-10 nM) and observing seedling growth after 7 days of culture.

[0045] The results showed that the wild-type (Col-0) exhibited significant growth inhibition and leaf yellowing under 2 nM nicosulfuron treatment, and its growth was severely inhibited under 3 nM treatment. In contrast, ZmCYP81Q32 Overexpression (OE) lines exhibited significant tolerance: only slight inhibition was observed at a concentration of 6 nM, and even at 10 nM treatment, some seedlings maintained relatively normal growth. Figure 11 ).

[0046] (2) For whole-plant treatment, 4-week-old plants were sprayed with 0, 3.75, 7.5, 15, and 30 g ai / ha nicosulfuron, and phenotypes were recorded at 4, 8, and 12 days after treatment. Rosette diameter, plant height, and aboveground fresh weight were measured 12 days after treatment. Each treatment had 3 biological replicates, and differences between groups were analyzed using Student's t-test.

[0047] The results showed that after spraying 3.75 g ai / ha nicosulfuron for 12 days, Col-0 plants exhibited severe wilting and chlorosis, with growth severely inhibited; while the OE line maintained good growth even under a 4-fold increase (15 g ai / ha) treatment, and even under an 8-fold increase (30 g ai / ha) treatment, although growth was somewhat inhibited, the leaves remained green, and the overall damage was significantly lower than that of the wild type. Figure 12 and Figure 13 ). ZmCYP81Q32 Overexpression significantly improves the tolerance of Arabidopsis seedlings to nicosulfuron.

[0048] (3) To detect the metabolism of nicosulfuron in the plants, 30 g ai ha was sprayed. -1Whole plant samples were taken 1 day and 3 days after nicosulfuron administration. The contents of nicosulfuron and its hydroxylated metabolites were determined by LC-MS / MS.

[0049] The results showed that after spraying 30 g ai / ha nicosulfuron, the nicosulfuron content in Col-0 cells did not change significantly between 1 and 3 days, and the accumulation of hydroxylated metabolites was slow. Figure 14 and Figure 15 In stark contrast, the nicosulfuron content in the OE strain decreased significantly by approximately 50% within 3 days, while the content of its hydroxylated metabolites more than doubled. Figure 16 and 17 This indicates that, ZmCYP81Q32 Overexpression can promote the hydroxylation metabolism of nicosulfuron in Arabidopsis thaliana, thereby reducing the accumulation of the parent herbicide and enhancing plant tolerance.

[0050] Example 4 Purchased from the Maize EMS Mutant Library (https: / / elabcaas.cn / memd / public / index.html# / ). ZmCYP81Q32 EMS mutant maize with gene mutation (MutantID: EMS5-3413a2).

[0051] In a greenhouse, EMS mutant maize lines and wild-type maize lines at the three-leaf stage were treated with nicosulfuron at dose gradients (0, 120, 240, 360, 480, and 600 g ai / ha), and observed after 14 days. Results are as follows: Figure 18 As shown, the results revealed that, compared to the wild type, the EMS mutant maize lines exhibited chlorosis and yellowing of leaves, and their growth was significantly inhibited. The EMS mutant also showed greater sensitivity to nicosulfuron treatment.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. ZmCYP81Q32 The application of genes in improving plant resistance to sulfonylurea herbicides, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. ZmCYP81Q32 The application of genes in breeding plants resistant to sulfonylurea herbicides, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. ZmCYP81Q32 The application of genes in promoting the metabolism of sulfonylurea herbicides in plants and / or yeast, the aforementioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The metabolism of the sulfonylurea herbicides is the hydroxylation metabolism of sulfonylurea herbicides.

5. The application according to claim 3, characterized in that, The yeast mentioned is brewer's yeast.

6. The application according to any one of claims 1 to 5, characterized in that, The plant is Arabidopsis thaliana and / or maize; the sulfonylurea herbicide is nicosulfuron.

7. A recombinant yeast, characterized in that, Includes base yeast and the base yeast introduced into it. ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

8. A plant material, characterized in that, Compared to wild-type plants, the plant materials overexpressed ZmCYP81Q32 Genes, the ones mentioned ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

9. The plant material according to claim 8, characterized in that, The plants mentioned are Arabidopsis thaliana and / or maize.

10. A method for improving plant resistance to sulfonylurea herbicides, characterized in that, Includes the following steps: ZmCYP81Q32 The gene was transferred into the target plant, the ZmCYP81Q32 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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