Hir mutants with resistance to triketone herbicides and their use in plant breeding

Abstract

The application discloses an HIR mutant with resistance to triketone herbicides and application of the HIR mutant in plant breeding, and relates to the field of plant proteins. The HIR mutant is obtained by mutating a wild-type HIR derived from a plant. The HIR mutant can degrade triketone herbicides, so that a plant expressing the mutant has resistance to triketone herbicides. The HIR mutant, a coding nucleic acid and the like can be used not only in transgenic crop cultivation, but also in cultivation of non-transgenic plants with resistance to triketone herbicides, and has a wide application prospect.

Description

[0001] This divisional application is a divisional application of Chinese invention patent application No. 2021103453770, filed on March 31, 2021, entitled "HIR mutants with resistance to triketone herbicides and their application in plant breeding". Technical Field

[0002] This invention relates to the field of plant proteins, and more specifically, to an HIR mutant with resistance to triketone herbicides and its application in plant breeding. Background Technology

[0003] Farmland weeds are a significant factor affecting crop yields. Manual weeding is labor-intensive and costly, and it hinders intensive agricultural production, severely restricting the development of high-yield, high-quality, and low-cost crop cultivation. Therefore, herbicides have emerged, playing a crucial role in solving farmland weed problems, promoting innovative cultivation methods, and increasing yields.

[0004] Global herbicide development focuses on high-efficiency, low-toxicity, broad-spectrum, and low-dosage varieties. Triketone herbicides, a recently developed class of albino (belonging to the cyclohexene ketone class) herbicides by Syngenta, are used to control monocotyledonous weeds in various broadleaf crops (such as soybeans, cotton, rapeseed, fruits, and sugar beets). Sensitive weeds absorb and translocate through their young roots, competitively inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPPD), thus inhibiting the synthesis of homogentisic acid. This disrupts the biosynthesis of plastoquinones and tocopherols, leading to symptoms such as whitening and stunted growth, ultimately killing the plant. Triketone herbicides have a broad spectrum of weed control, effective against various annual grasses and broadleaf weeds, with an efficacy period of 3–5 days, and are environmentally friendly. Mesotrione and cyclosulfonamides are major herbicides used in cornfields, effectively controlling annual broadleaf weeds and some grassy weeds such as barnyard grass, foxtail, and crabgrass. Mesotrione has been widely adopted in cornfields in China. However, the use of triketone herbicides in other crops is very limited because they are harmful to both weeds and crops. Even in corn, resistance to triketone herbicides varies among varieties, requiring caution during application. Although triketone herbicides are widely commercially available, their use is severely limited in time and space for crops that do not generally have herbicide resistance, as these herbicides often kill the crops themselves. For example, herbicides may need to be applied some time before sowing.

[0005] Therefore, researchers have been developing herbicide-resistant crops, allowing the use of herbicides during planting to kill weeds without affecting plant growth, thus broadening the scope of herbicide application. To expand the weed-control spectrum of triketone herbicides and their application in other sensitive crops, constructing transgenic plants resistant to HPPD inhibitors through genetic engineering is an effective method. Currently, two HPPD inhibitor-resistant transgenic soybeans are under review by the USDA. The former expresses a large amount of an HPPD mutant derived from *Pseudomonas fluorescens* in soybeans; overexpression of recombinant HPPD increases maize tolerance to pre-emergence herbicides by 4 times and soybean tolerance by 10 times, making it resistant to isoxaflutole. The latter utilizes an HPPD mutant derived from oats (*Avena sativa*) with a lower substrate affinity for inhibitors, thus enabling tolerance to mesotrione and isoxaflutole. However, due to the anti-GMO movement, the acceptance of GMO crops worldwide remains low. Even in the Americas, where GMO crops are grown in the largest area, GMOs are mainly limited to a few crops such as maize, soybeans, and cotton. Therefore, researchers are also working to obtain herbicide-resistant crops by mutating crops using non-GMO methods, among which ethyl methanesulfonate (EMS) mutagenesis is a commonly used method. Due to the uncertainty of EMS mutagenesis, obtaining herbicide-resistant crops through this method relies on arduous research, especially long-term screening, and a certain amount of luck.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an HIR mutant with resistance to triketone herbicides and its application in plant breeding. The HIR mutant provided by this invention can degrade triketone herbicides, thereby giving plants resistance to triketone herbicides.

[0008] This invention is implemented as follows: On the one hand, the present invention provides an HIR mutant with resistance to triketone herbicides, wherein the HIR mutant is obtained by mutation of wild-type HIR derived from plants, wherein the mutation is any one or a combination of the following (1)-(9): (1): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 5 of the reference sequence corresponding to the wild-type HIR to P; (2): Align the wild-type HIR with the reference sequence, and mutate the amino acid at position 75 of the reference sequence corresponding to the wild-type HIR to L; (3): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 80 of the reference sequence corresponding to the wild-type HIR to R; (4): Align the wild-type HIR with the reference sequence, and mutate the amino acid at position 89 of the reference sequence in the wild-type HIR to G; (5): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 111 of the reference sequence to S in the wild-type HIR; (6): Align the wild-type HIR with the reference sequence, and mutate the amino acid at position 145 of the reference sequence in the wild-type HIR to G; (7): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 218 of the reference sequence to N in the wild-type HIR; (8): Align the wild-type HIR with the reference sequence, and mutate the amino acid at position 283 of the reference sequence corresponding to the wild-type HIR to R; (9): Align the wild-type HIR with the reference sequence, and mutate the amino acid at position 322 of the reference sequence in the wild-type HIR to G; The amino acid sequence of the reference sequence is shown in SEQ ID NO.1.

[0009] Iron / ascorbic acid-dependent oxidoreductases (HIRs) are present in most plants. This invention, through mutation of wild-type HIR, yields an HIR mutant as described above that can degrade triketone herbicides, thereby terminating triketone herbicide resistance in plants expressing this mutant. This HIR mutant and its encoded nucleic acid can be used not only for transgenic crop breeding but also for cultivating non-transgenic plants resistant to triketone herbicides, demonstrating broad application prospects.

[0010] The reference sequence shown in SEQ ID NO.1 is the wild-type HIR of rice Nipponbare. Any HIR mutant obtained by combining any one or more of the above mutation methods (1)-(9) from wild-type HIR of any plant source has resistance to triketone herbicides.

[0011] The aforementioned plants include, but are not limited to, rice, wheat, corn, barley, oats, sorghum, buckwheat, millet, mung beans, broad beans, peas, lentils, sweet potatoes, potatoes, cotton, soybeans, rapeseed, sesame, peanuts, sunflowers, radishes, carrots, turnips, beets, cabbage, mustard greens, kale, cauliflower, Chinese kale, cucumbers, zucchini, pumpkins, winter melons, bitter melons, loofahs, cucumbers, watermelons, cantaloupes, tomatoes, eggplants, peppers, green beans, cowpeas, edamame, leeks, scallions, onions, leeks, spinach, celery, amaranth, lettuce, garland chrysanthemum, daylilies, grapes, strawberries, sugarcane, tobacco, alfalfa, pasture grass, turfgrass, tea, and cassava, etc.

[0012] It should be noted that the protein sequence alignment used in this invention is Clustal online alignment, the website address of which is: http: / / www.ebi.ac.uk / Tools / msa / clustalo / . Results obtained using other sequence alignment tools (such as DNAMAN, with relevant parameter settings at default) are basically consistent with those obtained using Clustal online alignment.

[0013] The results of the comparison of sequence similarity and identity of wild-type HIRs from different plant sources are shown in Table 1: Table 1. Comparison of sequence similarity and identity among wild-type HIRs from different plant sources.

[0014] In this invention, the amino acid sequence refers to the types and arrangement of amino acid residues that constitute a protein or polypeptide. Amino acid residues are typically represented using a three-letter notation commonly used in the art, but a single-letter notation also commonly used in the art can be used. Those skilled in the art should be able to convert between three-letter and single-letter amino acid sequences. It should be clear that regardless of the notation used in this invention, those skilled in the art can understand and convert it. For example: alanine, single letter A, three letters Ala; arginine, single letter R, three letters Arg; aspartic acid, single letter D, three letters Asp; cysteine, single letter C, three letters Cys; glutamine, single letter Q, three letters Gln; glutamic acid, single letter E, three letters Glu; histidine, single letter H, three letters His; isoleucine, single letter I, three letters Ile; glycine, single letter G, three letters Gly; asparagine, single letter N, three letters A sn; Leucine (L, Leu); Lysine (K, Lys); Methionine (M, Met); Phenylalanine (F, Phe); Proline (P, Pro); Serine (S, Ser); Threonine (T, Thr); Tryptophan (W, Trp); Tyrosine (Y, Tyr); Valine (V, Val).

[0015] Optionally, in some embodiments of the present invention, the amino acid sequence of the wild-type HIR is selected from any one of SEQ ID NO. 1-2.

[0016] SEQ ID NO.1 is the wild-type HIR of rice (Oryza sativa) Nipponbare, which is both a reference sequence and a wild-type HIR. The mutant mutated according to the above mutation method has resistance to triketone herbicides.

[0017] SEQ ID NO.2 is a wild-type HIR of maize, and the mutant produced by the above mutation method has resistance to triketone herbicides.

[0018] In this invention, positions 5, 75, 80, 89, 111, 145, 218, 283, and 322 on the reference sequence are referred to as reference sites, and the sites on wild-type HIRs or HIR mutants corresponding to the above reference sites are referred to as mutation sites. Table 2 shows the positions of the mutation sites corresponding to the above reference sites on the wild-type HIR sequences of different plant sources (taking rice and maize as examples). Table 2. Site correspondence between wild-type HIRs and reference sequences from different plant sources.

[0019] For example, taking the wild-type HIR of maize as an example, the 5th bit of the wild-type HIR of maize corresponds to the 5th bit of the reference sequence, the 76th bit corresponds to the 75th bit of the reference sequence, the 81st bit corresponds to the 80th bit of the reference sequence, the 90th bit corresponds to the 89th bit of the reference sequence, the 112th bit corresponds to the 111th bit of the reference sequence, the 146th bit corresponds to the 145th bit of the reference sequence, the 220th bit corresponds to the 218th bit of the reference sequence, the 285th bit corresponds to the 283rd bit of the reference sequence, and the 324th bit corresponds to the 322nd bit of the reference sequence.

[0020] For example, let's take wild-type maize HIR as an example to illustrate its mutation patterns: For the above mutation method (1), the wild-type maize HIR is compared with the reference sequence, and the amino acid at the position corresponding to the 5th position of the reference sequence (i.e. the 5th position of the wild-type maize HIR) is mutated to P. For the above mutation method (2), the wild-type maize HIR is compared with the reference sequence, and the amino acid at the position corresponding to the 75th position of the reference sequence (i.e. the 76th position of the wild-type maize HIR) is mutated to L.

[0021] Optionally, in some embodiments of the present invention, the triketone herbicide is selected from any one of nicosulfuron, cyclosulfuron, dicyclosulfuron, furazolidone, sulfuron, flupyrazole, and bispyribac-sodium.

[0022] It should be noted that triketone herbicide resistance refers to tolerance to triketone herbicides, including mesotrione, cyclosulfonyl, dicyclosulfonyl, furazolidone, sulfonyl, flupyrazole, and bispyribac-sodium.

[0023] It should be noted that, given a known protein sequence, those skilled in the art are able to prepare proteins with substituted, added, or missing amino acid residues by altering the coding gene sequence of a known protein and introducing it into an expression vector. These methods are documented in literature such as Molecular Cloning: A Laboratory Manual (Beijing: Science Press, 2002).

[0024] On the other hand, the present invention provides an isolated nucleic acid molecule that encodes an HIR mutant with triketone herbicide resistance as described in any of the preceding claims.

[0025] In this invention, the nucleic acid can be DNA or RNA, preferably DNA. Given knowledge of the encoded protein or nucleic acid sequence, and using conventional codon correspondences and host expression frequencies, those skilled in the art can obtain and optimize the nucleic acid encoding the HIR mutant protein of this invention through PCR, DNA recombination, or artificial synthesis. Once the nucleic acid is obtained, it can be cloned into a vector, transformed or transfected into appropriate cells, and then proliferated using conventional host cells to isolate a large quantity of nucleic acid.

[0026] On the other hand, the present invention provides a recombinant vector containing nucleic acid molecules as described above.

[0027] In this invention, vectors refer to bacterial plasmids, phage particles, phage particles, yeast plasmids, plant cell viruses, animal cell viruses, and various other viral vectors commonly used in the field. Depending on the intended use, vectors can be categorized into cloning vectors, expression vectors, and transformation vectors, referring to their respective purposes of cloning and validating genes, expressing corresponding genes, and transforming corresponding genes. Vectors usable in this invention include, but are not limited to: vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (such as Pichia pastoris vectors), baculovirus vectors for expression in insect cells, vectors for expression in mammalian cells (retroviral vectors, adenovirus vectors, etc.), plant virus vectors for expression in plants, and various vectors for expression in mammalian mammary glands.

[0028] It is readily understood that those skilled in the art can choose suitable carriers as tools for transporting the aforementioned nucleic acid molecules, and all of these fall within the scope of protection of this invention.

[0029] On the other hand, the present invention provides a recombinant bacterium or recombinant cell containing the nucleic acid molecule as described above or the recombinant vector as described above.

[0030] The recombinant bacteria can be cocci, bacilli such as Escherichia coli, or spirilla; they can also be autoaerobic or heteroaerobic bacteria. The cells can be prokaryotic or eukaryotic. Eukaryotic cells can be animal or plant cells. Plant cells are preferred, rice cells are preferred, and Nipponbare cells are more preferred. The aforementioned nucleic acid molecules contained in the plant cells can be introduced into the plant cells via transgenic technology, including introduction into the nucleus, chloroplasts, and / or plastids of the plant cells, or the aforementioned nucleic acid molecules can be present in the plant cells through conventional mutagenesis techniques (e.g., chemical mutagenesis such as ethyl methanesulfonate (EMS) mutagenesis or radiation mutagenesis).

[0031] It is readily understood that those skilled in the art can select suitable bacteria or cells as hosts for the aforementioned nucleic acid molecules or recombinant vectors, and all of these fall within the scope of protection of this invention.

[0032] On the other hand, the present invention provides the use of the HIR mutant as described in any of the preceding claims, the nucleic acid molecule as described above, the recombinant vector as described above, or the recombinant bacteria or recombinant cells as described above in obtaining plant varieties resistant to triketone herbicides.

[0033] The present invention provides methods for obtaining plant varieties resistant to triketone herbicides, including preparation, cultivation, production, or other methods, such as obtaining them through transgenic breeding, such as transforming the above-mentioned nucleic acid molecules into plants to express the above-mentioned HIR mutant; and obtaining them through non-transgenic breeding, such as through hybridization, backcrossing, self-pollination, or asexual reproduction and selecting plant varieties containing the above-mentioned nucleic acid molecules and expressing the above-mentioned HIR mutant.

[0034] Optionally, in some embodiments of the present invention, the application includes modifying the endogenous HIR gene of the target plant to encode the HIR mutant.

[0035] Optionally, in some embodiments of the present invention, the application includes: mutagenesis and screening of plant cells, tissues, individuals or populations to encode the HIR mutant.

[0036] The plant varieties described in this invention include, but are not limited to, individual plants, plant populations or their propagation material, plant events, plant offspring, plant seeds or other reproductive parts of plants. Plant offspring are themselves plants, including plant offspring produced through transgenic technology, plant offspring produced by hybridization with other plant varieties, and plant offspring produced by backcrossing or self-pollination.

[0037] The plant varieties described in this invention can be dicotyledonous plants or monocotyledonous plants, including but not limited to rice, wheat, corn, barley, oats, sorghum, buckwheat, millet, mung beans, broad beans, peas, lentils, sweet potatoes, potatoes, cotton, soybeans, rapeseed, sesame, peanuts, sunflowers, radishes, carrots, turnips, beets, cabbage, mustard greens, kale, cauliflower, Chinese kale, cucumbers, zucchini, pumpkins, winter melons, bitter melons, loofahs, cucumbers, watermelons, cantaloupes, tomatoes, eggplants, peppers, green beans, cowpeas, edamame, leeks, scallions, onions, leeks, spinach, celery, amaranth, lettuce, garland chrysanthemum, daylilies, grapes, strawberries, sugarcane, tobacco, alfalfa, pasture grass, turfgrass, tea, and cassava, etc.

[0038] Optionally, in some embodiments of the present invention, the triketone herbicide is selected from any one of nicosulfuron, cyclosulfuron, dicyclosulfuron, furazolidone, sulfuron, flupyrazole, and bispyribac-sodium.

[0039] In this invention, "plant" refers to a single plant, a group of plants, or its propagation material, including plants, plant varieties, plantlets, plant events, plant offspring, plant seeds, or other reproductive parts of plants. Plant offspring are themselves plants, including plant offspring produced through transgenic technology, plant offspring produced by hybridization with other plant varieties, and plant offspring produced by backcrossing or self-pollination.

[0040] Optionally, in some embodiments of the present invention, the application includes the following steps: (1) To induce the target plant to express the above-mentioned nucleic acid molecules; and / or, (2) To induce the target plant to express the above-mentioned HIR mutant.

[0041] The target plant is made to contain the above-mentioned nucleic acid molecules, or to express the above-mentioned HIR mutant, thereby obtaining plant varieties resistant to triketone herbicides.

[0042] Those skilled in the art can use gene editing techniques, breeding techniques, transgenic techniques, etc., well-known in the art to enable target plants to contain the above-mentioned nucleic acid molecules and to express the above-mentioned HIR mutants. These methods include, but are not limited to, the following: (a): The nucleic acid molecule encoding the above HIR mutant is introduced into the cells of the target plant, and the cells are cultured to differentiate and develop into plants resistant to triketone herbicides, so as to obtain a plant variety resistant to glufosinate. (b): The endogenous HIR gene of the target plant is edited using gene editing technology to encode the above-mentioned HIR mutant in order to obtain plant varieties resistant to triketone herbicides; (c): Using mutagenesis technology, cells or tissues of the target plant, or individuals or populations of the target plant, are mutated to screen out cells, tissues or individuals that encode the above-mentioned HIR mutants in vivo, so as to obtain plant varieties with resistance to triketone herbicides. (d): Using the plant varieties with triketone herbicide resistance obtained in (a), (b) or (c) above as parents, plants encoding the above HIR mutants are obtained through sexual or asexual hybridization to obtain plant varieties with triketone herbicide resistance.

[0043] On the other hand, the present invention provides a method for breeding plants resistant to triketone herbicides, comprising: sexual or asexual reproduction of plant varieties obtained by the application as described above.

[0044] Sexual reproduction methods include hybridization, backcrossing, and self-fertilization.

[0045] Based on breeding techniques well-known in the art, those skilled in the art can easily breed more plant varieties resistant to triketone herbicides through sexual or asexual reproduction after obtaining plant varieties resistant to triketone herbicides through the above-mentioned applications. Therefore, such breeding methods also fall within the protection scope of this invention.

[0046] On the other hand, the present invention provides a method for identifying plants resistant to triketone herbicides, comprising: determining whether the plant to be tested expresses the HIR mutant as described in any of the preceding claims; and / or determining whether the plant to be tested contains nucleic acid molecules as described above.

[0047] Those skilled in the art can implement the above identification methods using conventional methods in the field, such as protein sequencing, nucleic acid sequencing, PCR detection, and probe hybridization detection. Attached Figure Description

[0048] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 The growth of rice seedlings at different days after spraying mesotrione.

[0050] Figure 2 The growth of rice seedlings after spraying with cyprodinil.

[0051] Figure 3 The growth of rice seedlings after spraying with cyclophosphamide.

[0052] Figure 4 The growth of rice mutant seeds at different sowing densities.

[0053] Figure 5 The growth of Kasalath seeds 10 days after sowing.

[0054] Figure 6 The growth of Kasalath seeds 20 days after sowing.

[0055] Figure 7 The amino acid sequence alignment results for wild-type rice OsHIR (OsWT) and OsB, OsC, OsD, OsE, OsI and OsJ.

[0056] Figure 8The amino acid sequence alignment results of wild-type rice OsHIR (OsWT) and wild-type maize ZmHIR (ZmWT).

[0057] Figure 9 The pPAH vector spectrum is shown.

[0058] Figure 10 The results show the color reaction of the HIR mutant rice.

[0059] Figure 11 The results show the color reaction of the ZmHIR mutant in maize. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0061] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0062] Example 1 Screening for F1 rice varieties resistant to triketone herbicides and isolation of mutant genes. 1. Induction of rice callus Disinfect the F1 rice variety (Oryza sativa) seeds from the seed company as follows: Take mature seeds, manually remove the husks, select plump seeds without sterile spots, place them in a 50ml sterile centrifuge tube, and add 70% alcohol for 30 seconds. Discard the alcohol and rinse once with sterile water; add an appropriate amount of 2.6% sodium hypochlorite solution and soak for 15 minutes. Discard the sodium hypochlorite solution and soak and rinse 6-7 times with sterile water, 3 minutes each time.

[0063] Induction and subculture: Place the seeds on sterile filter paper to absorb the dryness, and place them in induction culture medium, 12 seeds per dish; after the operation, seal the culture dish with sealing film and culture in the dark at 30℃ for 21-28 days. Transfer the callus tissue to fresh culture medium and continue to culture for about 7 days. Take spherical callus tissue with a size of 1-2 mm as culture material.

[0064] Induction medium: NB + hydrolyzed casein 0.3 g / L + L-proline 2.8 g / L + sucrose 30 g / L + 2,4-D 4 mg / L + agar 8 g / L, pH 5.8. Autoclave.

[0065] 2. Suspension culture and EMS treatment Prepare the liquid culture medium for suspension culture: S0 (without agar) + cephalosporin (100mg / L) + 50-100mg / L mesotrione + 0.05%-0.5% ethyl methyl sulfate (EMS) (only add the first liquid culture medium).

[0066] Take 5g of callus tissue that has been subcultured for 7-21 days and add it to 50ml of liquid culture medium in a 150ml Erlenmeyer flask. Incubate on a shaker for 4 weeks (120 rpm, 28℃, in the dark). Change the culture medium once a week (for EMS, only add the first batch of liquid culture medium). After 3-5 weeks, transfer to differentiation medium.

[0067] S0 medium: NB + L-proline 2.8 g / L + sucrose 30 g / L + 2,4-D 2 mg / L, pH 5.8, autoclaved.

[0068] 3. Induction of differentiation of resistant callus Blot the suspension-cultured callus tissue dry on sterile filter paper and irradiate it with UV light for 5 minutes. Transfer it to differentiation medium (containing 0.1-5.0 mg / L mesotrione) and culture at 30°C under light for approximately 21 days. Subculture once. Select newly formed green shoots and transfer them to a fresh differentiation medium. Culture at 30°C under light for another 21 days.

[0069] Differentiation medium: MS medium + sucrose 30 g / L + agar 8 g / L, pH 5.8, autoclaved, with the addition of sorbitol 30 g / L + KT 2 mg / L + NAA 0.02 mg / L + mesotrione 0.1-5.0 mg / L.

[0070] 4. Rooting When the new seedlings grow to about 2cm, they are transferred to a rooting medium and cultured at 30℃ for 3-4 weeks. When roots are induced and the seedlings grow to 7-10cm, they are removed from the medium, the medium is washed off the roots, and they are transferred to the soil.

[0071] Rooting medium: 1 / 2 MS + inositol 0.1 g / L + sucrose 20 g / L, pH 5.8, autoclaved, with NAA 0.2 mg / L + mesotrione 0.1-5 mg / L.

[0072] 5. Field spraying Transplanted rice seedlings were evenly placed in the same experimental area (avoiding leaf overlap). The area occupied by the experimental group and the control group was calculated. Based on the area, 105 grams of nicosulfuron (10.5 mg per square meter) of active ingredient was sprayed at a dosage of 1x nicosulfuron. A 6x nicosulfuron dosage was 630 grams per hectare. Actual spraying was performed at 40 ml per square meter, containing 6x nicosulfuron. Photos were taken at 4, 10, and 21 days after spraying. Representative plants were photographed, and the results are shown below. Figure 1 .

[0073] from Figure 1 It can be seen that 4 days after spraying nicosulfuron, the control group (WT) showed leaf whitening, while the experimental group (Mutant) was unaffected. 10 days after spraying nicosulfuron, the WT, Nipponbare, and Kasalath groups began to wilt significantly, while the Mutant group remained unaffected and grew well. 21 days after spraying nicosulfuron, the WT, Nipponbare, and Kasalath groups had died, while the Mutant group remained unaffected and grew well. This indicates that Mutant is resistant to at least 6 times the field dose of nicosulfuron.

[0074] Mutant and new WT plants that survived the initial spraying of mesotrione were re-sprayed with biazole and sulfadiazine herbicides. Based on the area, biazole was applied at a 1x dosage of 60 grams of active ingredient per hectare (6 mg per square meter), and sulfadiazine at a 1x dosage of 92 grams of active ingredient per hectare (9.2 mg per square meter). A 6x dosage of biazole was 360 grams per hectare, and sulfadiazine at a 6x dosage of 552 grams per hectare. Actual spraying was performed at 40 ml per square meter, containing 6x the herbicide dosage. Photos were taken of some surviving plants after spraying; results are shown below. Figure 2 and Figure 3 .

[0075] from Figure 2 It can be seen that 21 days after spraying with bifenthrin, the two WT plants also withered and died, while the two Mutant plants, although showing yellowing of leaf tips, were still growing well overall and were not significantly affected by the herbicide. This indicates that the two Mutant plants are resistant to at least 6 times the field dose of bifenthrin.

[0076] from Figure 3 It can be seen that WT also died visibly 21 days after spraying with cyclosulfonamide, while Mutant, although showing yellowing of leaf tips, still grew well overall and was not significantly affected by the herbicide. This indicates that this Mutant plant can resist at least 6 times the field dose of cyclosulfonamide.

[0077] These healthy green seedlings (Mutant) were preserved and propagated; they were F1 mutant plants resistant to triketone herbicides.

[0078] Example 2 Verification of the degradation of nicosulfuron by the rice HIR mutant in Example 1 1. Propagate the triketone-resistant mutant plants from Example 1 and harvest their seeds. Then, sterilize the seeds: Take mature seeds (a mixture of mutant seeds), manually remove the husks, select plump seeds without sterile spots, and place them in a 50ml sterile centrifuge tube. Add 70% alcohol for 30 seconds. Discard the alcohol and rinse once with sterile water. Add an appropriate amount of 2.6% sodium hypochlorite solution and soak for 15 minutes. Discard the sodium hypochlorite solution and soak and rinse 6-7 times with sterile water, 3 minutes each time.

[0079] 2. Prepare a solid culture medium containing 1 mg / L of mesotrione. After sterilization, dispense the medium into culture bottles, with each bottle containing 100 mL of solid culture medium.

[0080] 3. Different seed densities were cultured on a medium containing mesotrione. Specifically, 0, 10, 30, 100, and 300 sterilized mutant plant seeds were placed in a culture bottle containing the medium from step 2 and labeled as M0, M10, M30, M100, and M300, respectively. Each experimental group was replicated 5 times.

[0081] 4. After 9 days of cultivation, observe and photograph the color of the seedlings in different experimental groups. The results are shown below. Figure 4 .

[0082] 5. Then remove all seedlings and grains from the culture medium and place 10 hulled Kasalath rice seeds sensitive to mesotrione in each culture bottle.

[0083] 6. Observe and photograph the color of the seedlings on days 10 and 20 respectively. The results are shown in [the table below]. Figure 5 and Figure 6 .

[0084] from Figure 4 It can be seen that the seedlings grown on the medium containing 1 mg / L mesotrione, regardless of the seed density, are all albino seedlings, indicating that these seedlings were affected by mesotrione.

[0085] from Figure 5As can be seen, on day 10, the Kasalath seedlings in culture bottles M0 and M10 were all albino seedlings, indicating that the seedling growth was affected by nicosulfuron in the culture medium. In other words, the nicosulfuron in the culture medium was not degraded or only minimally degraded by the rice mutants from the previous planting. However, in culture bottles M30, M100, and M300, green Kasalath seedlings were clearly observed growing, indicating that the concentration of nicosulfuron in these media was low or absent, and would not harm the seedlings. This means that the nicosulfuron in these media had been degraded to below the inhibitory level for sensitive varieties by the numerous rice mutant seedlings from the previous planting. Figure 6 As can be seen, on day 20, the condition of the seedlings was not much different from that on day 10, and the green seedlings that had already emerged were still alive. These results all indicate that the mutant plants of Example 1 have a degradation effect on mesotrione, and their degradation effect has a population effect; the larger the population, the better the degradation effect.

[0086] Leaves from the mutant plants in Example 1 were taken, and their genomic DNA was extracted. Based on existing research, primers for genes related to triketone herbicides were designed, and the extracted mutant plant genomic DNA was used as a template for amplification. After ligation into the pMD19-T vector, the DNA was sent to Chengdu Qingke Weiye Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the corresponding rice gene sequences on NCBI, and the following findings were observed: Compared with the OsHIR gene (SEQ ID NO.3) and its encoded wild-type OsHIR protein (SEQ ID NO.1) of wild-type Nipponbare, the mutations of the OsHIR gene and OsHIR protein in F1 mutant plants 1 to F1 mutant plants 6 are shown in Table 3 below: Table 3. Mutations in the OsHIR gene and OsHIR protein of F1 mutant plants.

[0087] The amino acid sequence alignment results of wild-type rice OsHIR and rice HIR mutants OsB, OsC, OsD, OsE, OsI, and OsJ are as follows: Figure 7 As shown in the figure: the position indicated by the arrow is the mutation site, and OsWT represents wild-type rice OsHIR.

[0088] Example 3 This embodiment provides a HIR mutant derived from maize, which is obtained through the following mutation method: wild-type corn ( Zea mays The wild-type ZmHIR protein (SEQ ID NO.2) encoded by the ZmHIR gene shown in SEQ ID NO.4 of Zheng 58 was compared with the reference sequence SEQ ID NO.1, and the results are as follows: Figure 8 As shown, the wild-type ZmHIR protein corresponds to one or more of the reference sites in the reference sequence (positions 111, 75, 218, 322, 5, 80, and 89, as shown in the figure). Figure 8 Mutations were performed at the mutation sites indicated by the middle arrows. The resulting mutants and their mutations are shown in Table 4 below: Table 4. Mutants obtained from the mutation of wild-type ZmHIR protein in maize

[0089] The coding gene for the maize ZmHIR mutant and the maize ZmHIR mutant provided in this embodiment of the invention can both be obtained by chemical synthesis.

[0090] Example 4 Color reaction of rice OsHIR mutant The method for detecting the nicosulfuron resistance of the rice OsHIR mutants OsB, OsC, OsD, OsE, OsI, and OsJ provided in Example 1 is as follows: Based on the rice nucleotide sequence provided in Example 1 above, PCR primers were designed, and restriction enzyme sites (Pac1 and Sbf1) were introduced at both ends of the gene. After restriction enzyme digestion, the gene was ligated into an expression vector that had undergone the same restriction enzyme digestion treatment (e.g., pPAH vector, which carries PaHPPD). Pseudomonas aeruginosa ) gene, its structure is as follows Figure 9 (As shown in the image) and then transformed into DH5α Escherichia coli.

[0091] After verification, positive clones were selected and inoculated onto LTM liquid medium containing different concentrations of nicosulfuron-methyl for growth. The color change of the medium was observed; this is the color reaction system. In this detection system, in the presence of tyrosine (Tyr), PaHPPD on the pPAH vector can decompose tyrosine to form homogentisic acid (HGA). HGA is further oxidized to brown. In the presence of nicosulfuron-methyl, PaHPPD activity is inhibited, and the medium does not show a brown color change. When the HIR mutant degrades nicosulfuron-methyl, PaHPPD activity is restored, and the medium shows a brown color change. Therefore, the ability of HIR to degrade nicosulfuron-methyl can be determined by observing the color reaction of the medium.

[0092] Wild-type rice OsHIR was used as the control CK1. Simultaneously, another control CK2 was constructed by replacing OsHIR on the pPAH vector with OsGS (Glutamine synthetase, GS), a gene that does not have the ability to degrade nicotinamide. Color changes in the experimental and control groups were observed in a color reaction system. Results are as follows: Figure 10 As shown.

[0093] The preparation method of LTM (LB + 1‰ Tyr + Kan + mesotrione) medium is as follows: LTM0 (1L):

[0094] 1‰ Tyr: Dissolve 1g of Tyr in 750ml of ddH2O, then add an appropriate amount of NaOH solution to promote dissolution. Heat on a magnetic stirrer until completely dissolved. Adjust the pH to 7.0, filter and sterilize, then mix with the high-temperature sterilized solution as soon as possible (high concentrations of Tyr precipitate easily) to form LTM0.

[0095] LTM25: LTM0+25μM nicosulfuron; LTM50: LTM0 + 50μM nicosulfuron; LTM100: LTM0+100μM nicosulfuron.

[0096] according to Figure 10 The results showed that in LTM0, all six rice mutants and the controls CK1 and CK2 exhibited a distinct brown color, indicating that they could grow normally in LTM0 and that PaHPPD showed normal enzyme activity. In LTM25, CK1 and CK2 did not show a brown reaction, indicating that OsHIR in CK1 and OsGS in CK2 did not degrade nicosulfuron, and that PaHPPD in the vector was affected by nicosulfuron and could not be expressed normally. However, OsB, OsC, OsD, OsE, OsI, and OsJ showed a brown reaction, indicating that nicosulfuron in these media had been degraded by the rice OsHIR mutants to a level below the inhibitory level of PaHPPD, and PaHPPD could be expressed, producing a color reaction. This demonstrates that the mutations in the rice OsHIR mutants OsB, OsC, OsD, OsE, OsI, and OsJ endowed these mutants with the ability to degrade nicosulfuron, and this ability was significantly superior to that of wild-type OsHIR. This indicates that these OsB, OsC, OsD, OsE, OsI, and OsJ can play a certain role in degradation at a concentration of at least 25 μM nicosulfuron.

[0097] This demonstrates that the mutations S5P, F75L, H80R, S89G, L111S, E145G, D218N, H283R, and / or R322G can confer or enhance the nicosulfuron resistance of rice OsHIR mutants.

[0098] Example 5 Color reaction of maize ZmHIR mutant Referring to the detection method in Example 4, the nicosulfuron resistance of the maize ZmHIR mutants ZmC, ZmE, ZmI, and ZmJ provided in Example 3 was verified. The results are as follows: Figure 11 As shown in the figure. Wild-type maize ZmHIR was used as the control (CK3).

[0099] according to Figure 11 The results showed that in LTM0, all four maize mutants and the controls CK2 and CK3 exhibited a distinct brown color, indicating that they could grow normally in LTM0 and that PaHPPD showed normal enzyme activity. In LTM25, CK2 showed no brown reaction, indicating that OsGS in CK2 did not degrade nicosulfuron, and PaHPPD in the vector was affected by nicosulfuron and could not be expressed normally. However, ZmC, ZmE, ZmI, ZmJ, and CK3 showed a brown reaction, indicating that nicosulfuron in these media had been degraded to below the inhibitory level of PaHPPD by wild-type maize ZmHIR and its mutants, allowing PaHPPD to be expressed and produce a color reaction. However, the colors of ZmC, ZmE, ZmI, and ZmJ were significantly darker than CK3, especially in LTM50, where the colors of ZmC, ZmE, ZmI, and ZmJ remained darker than CK3. This indicates that the mutations in the maize ZmHIR mutants ZmC, ZmE, ZmI, and ZmJ endowed these mutants with the ability to degrade nicosulfuron, and this ability was significantly superior to that of wild-type maize ZmHIR. This suggests that these ZmC, ZmE, ZmI, and ZmJ can play a certain role in the degradation of nicosulfuron at a concentration of at least 25 μM.

[0100] This demonstrates that the mutations S5P, F75L, H80R, D89G, L111S, D218N, and / or R322G can confer or enhance nicotinamide resistance in maize ZmHIR mutants.

[0101] Based on the above results, it can be seen that mutants obtained by mutating wild-type HIR derived from plants can degrade triketone herbicides and have triketone herbicide resistance. Plants expressing these mutants also have triketone herbicide resistance. The aforementioned mutation methods can be any one or a combination of several of the following (1)-(9): (1): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 5 of the reference sequence corresponding to the wild-type HIR to P; (2): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 75 of the reference sequence in the wild-type HIR to L; (3): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 80 of the reference sequence corresponding to the wild-type HIR to R; (4): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 89 of the reference sequence in the wild-type HIR to G; (5): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 111 of the reference sequence in the wild-type HIR to S; (6): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 145 of the reference sequence in the wild-type HIR to G; (7): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 218 of the reference sequence in the wild-type HIR to N; (8): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 283 of the reference sequence to R in the wild-type HIR; (9): Align the wild-type HIR with the reference sequence and mutate the amino acid at position 322 of the reference sequence to G in the wild-type HIR.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An HIR mutant with resistance to triketone herbicides, characterized in that, The HIR mutant is obtained by mutation of wild-type HIR derived from plants. The HIR mutant is rice mutant OsB or rice mutant OsD. Among them, the mutation mode of rice mutant OsB is: compared with the rice reference sequence, the amino acid H at position 283 is mutated to R; The mutation mode of rice mutant OsD is as follows: compared with the rice reference sequence, amino acid E at position 145 is mutated to G; The amino acid sequence of the rice reference sequence is shown in SEQ ID NO.

1.

2. An isolated nucleic acid molecule, characterized in that, It encodes the HIR mutant with triketone herbicide resistance as described in claim 1.

3. A recombinant vector, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. A recombinant bacterium, characterized in that, It contains the nucleic acid molecule as described in claim 2 or the recombinant vector as described in claim 3.

5. The application of the HIR mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, or the recombinant bacteria of claim 4 in obtaining plant varieties resistant to triketone herbicides; The plant in question is rice.

6. The application according to claim 5, characterized in that, The application includes modifying the endogenous HIR gene of the target plant to encode the HIR mutant.

7. The application according to claim 5, characterized in that, The triketone herbicides are selected from any one of nicosulfuron, cyclosulfuron, dicyclosulfuron, furazolidone, sulfuron, flupyrazole, and bispyribac-sodium.

8. A method for propagating plants resistant to triketone herbicides, characterized in that, It includes: The plant varieties obtained by the application described in claim 5 or 6 shall be reproduced sexually or asexually. The plant in question is rice.

9. A method for identifying plants resistant to triketone herbicides, characterized in that, It includes: Determine whether the plant to be tested expresses the HIR mutant of claim 1; and / or determine whether the plant to be tested contains the nucleic acid molecule of claim 2; The plant in question is rice.

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