His mutant protein and use thereof

Abstract

Provided are a HIS mutant protein and a use thereof in plant breeding. Specifically, provided is a HIS mutant protein. The HIS mutant protein, when overexpressed in a plant, can not only confer herbicide resistance upon the plant, but also reduce the height of the plant and increase yield, showing broad application prospects in the field of biological breeding.

Description

A HIS mutant protein and its application

[0001] This application claims priority to Chinese patent application CN202510052734.2, filed on January 14, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of agricultural genetic engineering, specifically relating to an HIS mutant protein and its application in plant breeding. Background Technology

[0003] Soybeans are a major source of oil and protein for humans, and one of the world's most important economic crops. They are also a primary source of plant oils and plant-based proteins. With rising living standards and improved dietary habits, the demand for high-quality soybean oil is increasing, making the cultivation of high-yield, high-quality soybeans a key objective of soybean breeding.

[0004] HIS1 (HPPD INHIBITOR SENSITIVE 1), with the gene code LOC_Os02g17940 in rice, encodes a 351-amino acid Fe(II) / 2-oxoglutarate(2OG)–dependent oxygenase. A mutation at position 242 of the rice HIS1 protein enhances herbicide resistance, particularly resistance to HPPD-inhibiting herbicides. To further investigate the application of HIS1 and its mutant proteins in herbicide resistance and trait improvement, this invention expresses the OsHIS1 mutant protein in soybean to obtain high-quality soybean varieties. Summary of the Invention

[0005] This invention provides an application of HIS1 mutant protein in plant trait modification. After overexpression of the HIS1 mutant protein in plants, the plant height is reduced and the yield is increased.

[0006] On the one hand, the present invention provides the application of HIS1 (HPPD INHIBITOR SENSITIVE 1) mutant protein in the preparation of plants with improved traits, or in the improvement of plant traits.

[0007] In another preferred embodiment, the HIS1 mutant protein has a mutation at amino acid position 242, corresponding to the amino acid sequence shown in SEQ ID No. 1, compared to the parental HIS1 protein.

[0008] In one embodiment, the parental HIS1 protein is derived from rice.

[0009] In another preferred embodiment, the parental HIS1 protein or the wild-type HIS1 protein is encoded by the HIS1 gene, which in rice is numbered LOC_Os02g17940 and encodes Fe(II) / 2-oxoglutarate(2OG)–dependent oxygenases, with amino acids as shown in SEQ ID No. 1.

[0010] In another preferred embodiment, the mutation is the insertion, deletion, or substitution of an amino acid.

[0011] In another preferred embodiment, the mutant protein is a HIS1 herbicide resistance / tolerance protein, particularly resistance / tolerance to HPPD inhibitor herbicides.

[0012] In one embodiment, the 242nd amino acid site is S.

[0013] In one embodiment, the 242nd amino acid is mutated to a non-S amino acid, such as A, V, G, Q, F, W, Y, D, N, E, K, M, T, C, P, H, R, I, L; preferably, G.

[0014] In one embodiment, the 242nd amino acid is mutated to G.

[0015] In one embodiment, the HIS1 mutant protein is selected from any group I-III below:

[0016] I. HIS1 mutant protein obtained by mutating at amino acid position 242 of the amino acid sequence shown in SEQ ID No. 1;

[0017] II. Compared with the HIS1 mutant protein described in I, it has the mutation site described in I; and compared with the HIS1 mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the HIS1 mutant protein described in I, and retains herbicide resistance activity;

[0018] III. Compared with the HIS1 mutant protein described in I, it has the mutation site described in I; and compared with the HIS1 mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions, or additions, and retains herbicide resistance activity; the one or more amino acids include substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0019] In another preferred embodiment, the HIS1 mutant protein further includes other mutation sites, which are one or more of the following positions corresponding to amino acid sequences 327, 283, 111, 145, 75, 218, 322, 5, 80, and 89 of the amino acid sequence shown in SEQ ID No. 1. These other mutation sites can maintain or enhance the tolerance or resistance of the mutant protein to HPPD inhibitory herbicides or increase the applicability of the HIS1 mutant protein to herbicides.

[0020] In another preferred embodiment, the parental HIS1 protein is derived from monocotyledonous and / or dicotyledonous plants.

[0021] In another preferred embodiment, the parental HIS1 protein is derived from one or more plants selected from the group consisting of: grasses, legumes, gooseberries, and cruciferous plants.

[0022] In another preferred embodiment, the parental HIS1 protein is derived from one or more plants selected from the group consisting of: Arabidopsis thaliana, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0023] In another preferred embodiment, the parental HIS1 protein is derived from rice, including indica rice and japonica rice. Preferably, the amino acid sequence of the parental HIS1 is shown in SEQ ID No. 1.

[0024] In another preferred embodiment, the amino acid sequence of the parent HIS1 protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the amino acid sequence shown in SEQ ID No. 1.

[0025] In another preferred embodiment, the HIS1 mutant protein (herbicide-resistant polypeptide) is formed by mutation of the polypeptide shown in SEQ ID No. 1.

[0026] In another preferred embodiment, the HIS1 mutant protein, except for the mutations described above, has the same or substantially the same amino acid sequence as the sequence shown in SEQ ID No. 1.

[0027] In another preferred embodiment, the basic similarity is that there are at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acid differences, wherein the differences include amino acid substitutions, deletions, or additions, and the mutant protein has herbicide tolerance activity (HPPD inhibitor herbicides).

[0028] In another preferred embodiment, the amino acid sequence of the HIS1 mutant protein is shown in SEQ ID No. 3.

[0029] In this invention, the HIS1 mutant protein is introduced into plants (e.g., by overexpression) to prepare trait-improved plants or to improve the traits of plants.

[0030] In one embodiment, the HIS1 mutant protein is introduced into a plant to overexpress the HIS1 mutant protein in the plant.

[0031] Overexpression in this invention includes increasing gene expression by introducing additional copies of the target gene to increase the copy number of the target gene in the cell, or by modifying or replacing the promoter of the target gene to increase the expression level of the target gene.

[0032] In one embodiment, the "introduction" includes constructing the coding gene of the target protein into an expression vector and transferring the expression vector into a plant to express the target gene. In other embodiments, the "introduction" includes inserting the target gene into the plant genome; preferably, the insertion can be performed using homologous recombination double exchange; in one embodiment, the target gene and homologous arms can be inserted into a vector, and then the vector can be transferred into a plant, utilizing homologous arms to undergo homologous recombination double exchange with the plant genome to insert the target gene into a suitable genomic location; in other embodiments, gene editing methods can also be used, for example, using a CRISPR / Cas system to cut at the desired genomic site, while simultaneously inserting the target gene as a foreign donor into the cutting site.

[0033] In one embodiment, modifying or replacing the promoter of the target gene to increase its expression level includes replacing the promoter of the target gene with a strong promoter to increase its expression level, or modifying the promoter of the target gene to increase its expression level; for example, using gene editing to insert a promoter (e.g., a 35S promoter) into the region of the promoter of the target gene. In one embodiment, overexpression of the HIS1 mutant protein refers to an increase in the expression level of the HIS1 mutant protein or an increase in the expression level of the gene encoding the HIS1 mutant protein.

[0034] In one embodiment, overexpression of the HIS1 mutant protein means that the expression level of the gene encoding the HIS1 mutant protein is increased by at least 1-fold, preferably at least 2-fold, preferably at least 3-fold, preferably at least 4-fold, preferably at least 5-fold, preferably at least 6-fold, preferably at least 10-fold, preferably at least 20-fold, preferably at least 30-fold, preferably at least 50-fold, preferably at least 70-fold, and preferably at least 100-fold compared to the control.

[0035] In one embodiment, the plant overexpressing the HIS1 mutant protein is either homozygous or heterozygous.

[0036] In another preferred embodiment, the improved trait is selected from any one or more of the following groups i-vi:

[0037] i. Increased production;

[0038] ii. The number of pods increases;

[0039] iii. Increased number of grains;

[0040] iv. Increased particle weight;

[0041] v. Plant height decreased;

[0042] vi. Increased resistance to HPPD inhibitory herbicides.

[0043] In another preferred embodiment, the application of the trait improvement includes the step of treating the plants with an HPPD-repressing herbicide. In one embodiment, the treatment is to spray the plants with an HPPD-repressing herbicide.

[0044] In one embodiment, the plants are treated with an HPPD suppressant herbicide 10 days after sowing; for example, 10 to 28 days later; or, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days later.

[0045] In another preferred embodiment, HPPD herbicide is applied when the second trifoliate leaf of the plant (soybean) unfolds.

[0046] In one embodiment, treatment is performed using an HPPD-inhibiting herbicide at a concentration exceeding 50 mg / L; for example, 50 mg / L-2000 mg / L; or, for example, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 6000 mg / L, 650 mg / L. g / L, 700mg / L, 750mg / L, 8000mg / L, 850mg / L, 900mg / L, 950mg / L, 1000mg / L, 1100mg / L, 1200mg / L, 1300mg / L, 1400mg / L, 1500mg / L, 1600mg / L, 1700mg / L, 1800mg / L, 1900mg / L, or 2000mg / L.

[0047] In one embodiment, the reduction in plant height means that the plant containing the overexpressed HIS1 mutant protein (after herbicide treatment) is at least 3% shorter than the parent plant (without herbicide treatment), preferably 5%, preferably 6%, preferably 10%, preferably 15%, preferably 20%, preferably 23%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 100%.

[0048] In one embodiment, the increase in pod number means that the number of pods per plant or the number of harvested pods per unit area of ​​plants containing the overexpressed HIS1 mutant protein (after herbicide treatment) is at least 3% higher than that of the parent plants (without herbicide treatment), preferably 5%, preferably 6%, preferably 10%, preferably 15%, preferably 20%, preferably 23%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 100%.

[0049] In one embodiment, the increase in seed number means that the number of seeds per plant or the number of seeds harvested per unit area is at least 3%, preferably 5%, preferably 6%, preferably 10%, preferably 15%, preferably 20%, preferably 23%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 100%, preferably 100% higher than that of the parental plants (without herbicide treatment).

[0050] In one embodiment, the increase in grain weight refers to an increase of at least 5%, preferably 10%, preferably 12%, preferably 13%, preferably 15%, preferably 20%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, and preferably 100% in the single grain weight, hundred-grain weight, or thousand-grain weight of plants containing the overexpressed HIS1 mutant protein (after herbicide treatment) compared to the parental plants (without herbicide treatment).

[0051] In one embodiment, the yield increase means that the plants containing the overexpressed HIS1 mutant protein (after herbicide treatment) have a yield, yield per plant, or yield per acre that is at least 5% higher than that of the parent plants (without herbicide treatment), preferably 10%, preferably 15%, preferably 20%, preferably 21%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 60%, and preferably 100%.

[0052] In another preferred embodiment, the plants include crops, forestry plants, vegetables, fruits, flowers, and forage grasses (including turfgrass).

[0053] In another preferred embodiment, the plant is a monocotyledonous plant and / or a dicotyledonous plant.

[0054] In another preferred embodiment, the plant is selected from one or more plants from the group consisting of: grasses, legumes, cruciferous plants, solanaceous plants, cucurbitaceous plants, chenopodiaceae plants, polygonaceae plants, sesame plants, asteraceae plants, madder plants, rose family, sesame family, convolvulaceae family, dioscoreaceae family, umbelliferous plants, lily family, ginger family, and palm family.

[0055] In another preferred embodiment, the plant is selected from one or more of the following: rice, soybean, Arabidopsis thaliana, tobacco, tomato, potato, corn, cotton, alfalfa, sorghum, barley, wheat, millet, sweet potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, peanut, watermelon, cabbage, strawberry, cucumber, coconut, or combinations thereof.

[0056] In another preferred embodiment, the plant is selected from one or more of the following groups: rice, soybean, Arabidopsis thaliana, maize, cotton, sorghum, barley, wheat, millet, quinoa, foxtail millet, or combinations thereof.

[0057] In another preferred embodiment, the plant is rice, corn, sorghum, barley, wheat, quinoa, Arabidopsis thaliana, soybean, millet, or a combination thereof.

[0058] In another preferred embodiment, the plant is soybean.

[0059] In another preferred embodiment, the plant is soybean yellow 302.

[0060] In another preferred embodiment, the herbicide is an HPPD inhibitor herbicide (or, referred to as an HPPD-suppressing herbicide). HPPD inhibitor herbicides mainly include triketones, pyrazolones, isoxazolones, diketonitriles, and benzophenones. Triketone herbicides are preferably one or more of mesotrione, cyclosulfonamide, triazolylsulfonamide, furazolylsulfonamide, dicyclosulfonamide, mesotrione, sulfonamide, flupyrazole, quinalazine, or methylquinalazine; pyrazolone herbicides are preferably one or more of benzosulfonamide, sulfonylurea, benzylazine, pyrazol, pyrazosulfuron, pyrasulfotole, or tolpyralate; and isoxazolone herbicides are preferably one or more of isoxazol, isoxazolylchlor, or isoxazol.

[0061] In another preferred embodiment, the HPPD suppressive herbicide is preferably one or any combination of mesotrione, isoxaflutole, cyclosulfonone, methylquinallon, benzoxazine, or sulfonylurea.

[0062] In another preferred embodiment, the HPPD inhibitory herbicide is a triketone herbicide.

[0063] In another preferred embodiment, the HPPD suppressive herbicide is mesotrione and / or cyclosulfonyl.

[0064] In another preferred embodiment, the HPPD-inhibiting herbicide is mesotrione.

[0065] In another preferred embodiment, after overexpression of the HIS1 mutant protein, the maximum tolerance concentration of the plant to the herbicide is increased by at least 1.5 times, preferably at least 2 times, preferably at least 3 times, preferably at least 4 times, preferably at least 5 times, preferably at least 6 times, preferably at least 10 times, preferably at least 20 times, preferably at least 30 times, preferably at least 50 times, preferably at least 100 times, and preferably at least 200 times compared to the parent plant.

[0066] In another preferred embodiment, the plant containing the HIS1 mutant protein overexpressing the herbicide has at least a 2-fold increase in maximum tolerance concentration to the herbicide compared to the parent plant, preferably a 3-fold increase, preferably a 4-fold increase, preferably a 5-fold increase, preferably a 6-fold increase, preferably a 7-fold increase, preferably an 8-fold increase, preferably a 10-fold increase, preferably a 12-fold increase, preferably a 14-fold increase, preferably a 16-fold increase, preferably a 20-fold increase, preferably a 30-fold increase, preferably a 50-fold increase, preferably a 100-fold increase, and preferably a 200-fold increase.

[0067] In another aspect, the present invention provides an isolated nucleic acid molecule that encodes the aforementioned HIS1 mutant protein.

[0068] In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: genomic sequences, cDNA sequences, RNA sequences, or combinations thereof.

[0069] In another preferred embodiment, the nucleic acid molecule is preferably single-stranded or double-stranded.

[0070] In another preferred embodiment, the nucleic acid molecule further comprises an operatively linked promoter.

[0071] In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue-specific promoters, inducible promoters, or strong promoters.

[0072] In another aspect, the present invention provides a carrier comprising the aforementioned nucleic acid molecule.

[0073] In another preferred embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, or an integration vector.

[0074] The vector can be of the following types: plasmid, virus, granule, bacteriophage, etc., which are well known to those skilled in the art.

[0075] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0076] In one embodiment, the host cell is introduced into the cell by means selected from the group consisting of: Agrobacterium-mediated transformation, gene gun method, microinjection method, electrocautery method, ultrasound method, and polyethylene glycol (PEG) mediated method.

[0077] In another aspect, the present invention provides the use of the aforementioned nucleic acid molecules, or biological materials containing the aforementioned nucleic acid molecules (e.g., the aforementioned carriers or host cells), in the preparation of plants with improved traits, or in the improvement of plant traits.

[0078] On the other hand, the present invention provides a transgenic reagent or gene editing reagent that can edit plants to overexpress the HIS1 mutant protein.

[0079] This invention also provides the application of the above-mentioned gene editing reagents or transgenic reagents in the preparation of plants with improved traits, or in the improvement of plant traits.

[0080] In another aspect, the present invention provides a plant cell, plant tissue, plant part, or plant, wherein the plant cell, plant tissue, plant part, or plant includes overexpressed HIS1 mutant protein, or the nucleic acid molecule, or the vector, or the host cell.

[0081] In another aspect, the present invention provides a method for improving the traits of a plant, or a method for preparing a plant with improved traits, the method comprising the step of overexpressing the HIS1 mutant protein in the plant.

[0082] In another preferred embodiment, the method includes the step of overexpressing the HIS1 mutant protein in the plant cells, plant seeds, plant tissues, plant parts, or plant.

[0083] The improved trait is selected from any one or more of the following groups i-vi:

[0084] i. Increased production;

[0085] ii. The number of pods increases;

[0086] iii. Increased number of grains;

[0087] iv. Increased particle weight;

[0088] v. Plant height decreased;

[0089] vi. Increased resistance to HPPD inhibitory herbicides.

[0090] In one embodiment, the method includes the steps of obtaining a plant overexpressing the HIS1 mutant protein and cultivating the plant.

[0091] In another preferred embodiment, the method further includes the step of treating the plants with an HPPD-suppressing herbicide. In one embodiment, the treatment is to spray the plants with an HPPD-suppressing herbicide.

[0092] In one embodiment, the plants are treated with an HPPD suppressant herbicide 10 days after sowing; for example, 10 to 28 days later; or, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days later.

[0093] In another preferred embodiment, HPPD herbicide is applied when the second trifoliate leaf of the plant (soybean) unfolds.

[0094] In one embodiment, treatment is performed using an HPPD-inhibiting herbicide at a concentration exceeding 50 mg / L; for example, 50 mg / L-2000 mg / L; or, for example, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 6000 mg / L, 650 mg / L. g / L, 700mg / L, 750mg / L, 8000mg / L, 850mg / L, 900mg / L, 950mg / L, 1000mg / L, 1100mg / L, 1200mg / L, 1300mg / L, 1400mg / L, 1500mg / L, 1600mg / L, 1700mg / L, 1800mg / L, 1900mg / L, or 2000mg / L.

[0095] In another preferred embodiment, the above method includes the following steps:

[0096] (1) Provides Agrobacterium carrying an expression vector, wherein the expression vector comprises the above-mentioned nucleic acid molecule;

[0097] (2) Contacting plant cells, plant tissues, and plant parts with Agrobacterium in step (1) to overexpress the HIS1 mutant protein and integrate it into the chromosome of the plant cells; and

[0098] (3) Select plant cells, plant tissues, or plant parts that overexpress the HIS1 mutant protein.

[0099] In another preferred embodiment, the method further includes the step of regenerating the plant cells, plant tissues, and plant parts into a plant.

[0100] Another aspect of the present invention provides a method for controlling unwanted plants in a plant cultivation site, the method comprising:

[0101] (1) Provide the plant prepared by the method described;

[0102] (2) Cultivate the plants and apply an effective amount of HPPD suppressive herbicide at the cultivation site.

[0103] In one implementation, the unwanted plant is a weed.

[0104] On the other hand, the present invention also provides a method for controlling the growth of weeds near plants, comprising:

[0105] a) Provide the above-mentioned herbicide-resistant plants;

[0106] b) Apply an effective amount of herbicide to the plant and the weeds nearby to control the weeds near the plant.

[0107] On the other hand, the present invention also provides plants with improved traits obtained by the above method.

[0108] On the other hand, the present invention also provides a method for preparing hybrid plants, the method comprising hybridizing a first plant with a second plant to obtain the hybrid plant, wherein the first plant is a plant with improved traits prepared by the method of the present invention.

[0109] General definition

[0110] Unless otherwise defined in this application, the scientific terms or technical terms used in this invention have the meanings understood by those skilled in the art. In the event of a conflict between the meanings understood by those skilled in the art and the meanings defined in this application, the meanings defined in this application shall prevail.

[0111] As used herein, the term "HPPD" refers to 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), which is present in various organisms and is a key enzyme catalyzing the oxygenation of 4-hydroxyphenylpyruvate (HPP), a degradation product of tyrosine, to homogenates (HGA). Inhibition of HPPD leads to photosynthetic uncoupling in plant cells, a lack of auxiliary light-harvesting pigments, and, due to the absence of photoprotection normally provided by carotenoids, the destruction of chlorophyll by reactive oxygen species intermediates and photo-oxidation. This results in chlorosis symptoms in photosynthetic tissues, inhibited growth, and ultimately, death. HPPD-inhibiting herbicides have proven to be highly effective selective herbicides with broad-spectrum herbicidal activity. They can be applied pre- and post-emergence, and are characterized by high activity, low residue, safety for mammals, and environmental friendliness.

[0112] As used herein, the term "herbicide" refers to a substance that has herbicidal activity on its own or in combination with other herbicides and / or additives that can alter its effect, and is manifested as an inhibitor of plant growth or even plant death.

[0113] As used herein, the terms "HPPD inhibitor," "HPPD herbicide," "HPPD-inhibiting herbicide," and "HPPD-inhibiting herbicide" are used interchangeably to refer to substances that have herbicidal activity themselves or, when used in combination with other herbicides and / or additives that can alter their effects, exert their effects by inhibiting HPPD, manifesting as inhibition of plant growth or even plant death. Substances that exert their herbicidal effect by inhibiting HPPD are well known in the art and include many types, such as: 1) triketones, for example, sulcotrione (CAS No.: 99105-77-8); mesotrione (CAS No.: 104206-82-8); bicyclopyrone (CAS No.: 352010-68-5); tembotrione (CAS No.: 335104-84-2); tefuryltrione (CAS No.: 473278- 76-1); Benzobicyclon (CAS No.: 156963-66-5); Quinalazine (CAS No.: 1639426-14-4); Methylquinalazine (CAS No.), 6-(2-hydroxy-6-oxocyclohexane-1-ene-1); 2) Diketone nitriles, for example, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)prop-1,3-dione (CAS No.: 143701-75-1); 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-3,4-dichlorophenyl)prop-1,3-dione (CAS No.: 21282) 9-55-5); 2-cyano-1-[4-(methanesulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)prop-1,3-dione (CAS No.: 143659-52-3); 3) isoxazolones, for example, isoxaflutole (also known as isoxazolone, CAS No.: 141112-29-0); isoxachlortole (CAS No.: 141112-06-3); clomazone (CAS No.: 81777-89-1); 4) pyrazolones, for example, benzoxazolone (to Pramezone (CAS No.: 210631-68-8); Pyrasulfotole (CAS No.: 365400-11-9); Pyrazoxyfen (CAS No.: 71561-11-0); Pyrazolate (CAS No.: 58011-68-0); Benzofenap (CAS No.: 82692-44-2); Bisoxane (CAS No.: 1622908-18-2); Tolpyralate (CAS No.: 1101132-67-5);Benzyl-flufenican (CAS No.: 1992017-55-6); Cycloflufenican (CAS No.: 1855929-45-1); Triazolesulfuron (CAS No.: 1911613-97-2); 5) Benzophenones; 6) Others: lancotrione (CAS No.: 1486617-21-3); fenquinotrione (CAS No.: 1342891-70-6). The herbicides described can comprehensively consider the type of crop or weed to control unwanted plants (such as weeds) before emergence, after emergence, before planting, and at planting time.

[0114] The terms "effective amount" or "effective concentration" refer to an amount or concentration sufficient to kill or inhibit the growth of similar parental (or wild-type) plants, plant tissues, plant cells, or host cells, but not to kill or severely inhibit the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention. Generally, the effective amount of a herbicide is the amount routinely used to kill target weeds in an agricultural production system. This amount is known to those skilled in the art. The herbicides described in this invention exhibit weed-controlling activity when applied directly to plants or to the site of application at any growth stage or before planting or emergence. The observed effects depend on the plant species to be controlled, the plant growth stage, the application parameters of the diluent and the spray droplet size, the particle size of the solid component, the environmental conditions at the time of application, the specific compound used, the specific adjuvants and carriers used, the soil type, and the amount of chemical applied. As is known in the art, these and other factors can be adjusted to promote non-selective or selective weed control.

[0115] The term "parental nucleotide or polypeptide" refers to nucleic acid molecules or polypeptides (proteins) that can be found in nature. This includes wild-type nucleic acid molecules or proteins (polypeptides) that have not been artificially modified, and may also include artificially modified nucleic acid molecules or proteins (polypeptides) that do not contain the content of this invention. The nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and their amino acid sequences can be deduced from the nucleotide sequences. The "parental plant" refers to a plant containing the parental nucleotide or polypeptide. The "parental nucleotide or polypeptide" can be extracted from the parental plant using techniques well known to those skilled in the art, or it can be obtained through chemical synthesis.

[0116] The "tolerance" or "resistance" described in this invention refers to the ability of a plant to withstand herbicides under certain growing conditions, and is generally characterized by parameters such as the amount or concentration of herbicide used. Furthermore, plants that are "conferred herbicide resistance" or "enhanced herbicide resistance" are those whose tolerance or resistance to the herbicide is increased compared to the parent plant, with a tolerance concentration at least 1.5 to 200 times higher than that of the parent plant. The optimal degree of increased "tolerance" or "resistance" described in this invention is such that, at the same herbicide dosage or concentration, it can reduce, inhibit, or kill unwanted plants without affecting the growth or survival ability of plants containing the mutant protein described in this invention.

[0117] The "conferring herbicide resistance" described in this invention includes, for parent plants that do not have resistance or tolerance to herbicides, or parent plants that have a certain or low tolerance to herbicides (at the same herbicide concentration), overexpressing the HIS1 mutant protein in the plant to give non-resistant plants a certain degree of herbicide resistance or tolerance, thereby improving the tolerance of plants with a certain or low tolerance to herbicides.

[0118] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated by recombinant synthesis or by chemical synthesis. The term “mutant protein” or “mutant protein” refers to a protein that, compared to the amino acid sequence of a parent protein, has one or more substitutions, insertions, deletions, and / or additions of amino acid residues. As used herein, the terms “herbicide-resistant polypeptide,” “mutant HIS1 polypeptide,” “mutant HIS1 polypeptide,” “mutant HIS1 protein,” “mutant HIS1 enzyme,” “mutant protein,” “mutant polypeptide,” “polypeptide of this invention,” etc., are used interchangeably.

[0119] The term "homology" or "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Therefore, the compositions and methods of the present invention also comprise homologs of the nucleotide and polypeptide sequences of the present invention. Homology can be calculated using known methods including, but not limited to, the following: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.), Stockton. Stockton Press, New York (1991).

[0120] The specific amino acid positions (numbers) within the protein described in this invention are determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO.1. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, with the sequence considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein nick opening penalty = 10.0; protein nick extension penalty = 0.2; protein matrix = Gonnet; protein / DNA terminal gap = -1; protein / DNA GAPDIST = 4. The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID No. 1.

[0121] The term "encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes that have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological characteristics they produce. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein.

[0122] The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

[0123] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0124] As used herein, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "S242G" indicates that amino acid S at position 242 is mutated to G, and so on. For multiple mutation types at the same site, the types are separated by " / ", for example, S242G / L indicates relative to the amino acid sequence of SEQ ID No. 1.

[0125] In this invention, the parental HIS1 can be derived from any plant, particularly the aforementioned monocotyledonous or dicotyledonous plants. Prior art literature has disclosed the HIS1 protein sequences and coding sequences of several parental sources (such as wild-type), which are incorporated herein by reference.

[0126] Preferably, the parental HIS1 protein of the present invention is derived from the genus *Oryza*, particularly *Rhizophora*. In this invention, HIS1 (HPPD INHIBITOR SENSITIVE 1), gene number LOC_Os02g17940, encodes a 351-amino acid Fe(II) / 2-oxoglutarate(2OG)–dependent oxygenase. More preferably, the parental HIS1 has the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0127] Based on the teachings of this invention, those skilled in the art can also obtain other HIS1s in rice or other plants by sequence alignment according to the HIS1 shown in SEQ ID No. 1, and obtain the amino acid site corresponding to the 242nd position of the amino acid sequence shown in SEQ ID No. 1 according to the amino acid site corresponding to SEQ ID No. 1, and perform corresponding mutations to obtain HIS1 mutant polypeptides with resistance / tolerance to herbicides.

[0128] The HIS1 protein of the present invention also includes its active fragments, variants, derivatives and analogs, including substances resulting from any substitution, mutation or modification of the HIS1 protein.

[0129] Those skilled in the art will understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., replacing another nonpolar amino acid residue with a nonpolar amino acid residue, replacing another polar uncharged amino acid residue with a polar uncharged amino acid residue, replacing another basic amino acid residue with a basic amino acid residue, and replacing another acidic amino acid residue with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where an amino acid is replaced by another amino acid belonging to the same group, fall within the scope of this invention, provided that the substitution does not lead to the inactivation of the protein's biological activity. Therefore, the proteins of this invention can contain one or more conserved substitutions in their amino acid sequence, preferably generated by substitutions according to Table 1. Furthermore, this invention also covers proteins that also contain one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the proteins of this invention. Conserved amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing their biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in non-conserved regions of a protein. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants can have fewer conserved or non-conserved alterations in conserved regions.

[0130] As is well known in the art, one or more amino acid residues can be altered (replaced, deleted, truncated, or inserted) from the N and / or C ends of a protein while retaining its functional activity. Therefore, proteins that have one or more amino acid residues altered from their N and / or C ends while retaining their desired functional activity are also within the scope of this invention. These alterations can include those introduced by modern molecular methods such as PCR, which includes PCR amplification that alters or lengthens the protein-coding sequence by means of oligonucleotides containing amino acid-coding sequences used in the PCR amplification.

[0131] It should be recognized that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such operations are generally known in the art. For example, amino acid sequence variants of proteins can be prepared by mutating DNA. This can also be accomplished through other forms of mutagenesis and / or directed evolution, for example, using known mutagenesis, recombination, and / or shuffling methods, combined with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0132] Those skilled in the art will understand that these minor amino acid changes in the HIS1 protein of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, a smaller impact can be expected.

[0133] Those skilled in the art can identify the essential amino acids of the HIS1 protein using methods known in the art, such as localized mutagenesis, protein evolution, or bioinformatics analysis. The protein's catalytic domains, active sites, or other functional domains can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key site amino acids.

[0134] Table 1

[0135] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0136] As used herein, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0137] The term "regulatory element," as used herein, is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), for detailed description in Goeddel, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct expression of that nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a time-dependent manner (e.g., cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0138] As used herein, the term "promoter" has the meaning known to those skilled in the art, referring to a non-coding nucleotide sequence located upstream of a gene that initiates the expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to that promoter.

[0139] The term "vector" refers to an element that allows the vector to integrate into the host cell's genome or to replicate autonomously within the cell independently of the genome. The vector may contain any element that guarantees self-replication. It typically carries a gene that is not part of the cell's central metabolism and is usually in the form of double-stranded DNA. The choice of vector generally depends on its compatibility with the host cell to which it is to be introduced. If a vector is used, the choice of vector depends on methods well-known to those skilled in the art for transforming host cells. For example, plasmid vectors may be used.

[0140] Vectors can be of various types, such as plasmids, viruses, granules, and bacteriophages, which are well known to those skilled in the art and are extensively described in the field. Preferably, the expression vector in this invention is a plasmid. The expression vector may contain a promoter, a ribosome-binding site for translation initiation, a polyadenylation site, a transcription terminator, an enhancer, etc. The expression vector may also contain one or more selectable marker genes for selecting host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase, genes conferring neomycin resistance, genes conferring tetracycline or ampicillin resistance, etc.

[0141] The vector of the present invention may contain elements that allow the vector to integrate into the host cell genome or to replicate autonomously within the cell independent of the genome. For integration into the host cell genome, the vector may rely on a polynucleotide sequence encoding a polypeptide or any other element of a vector suitable for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences for guiding integration into the host cell genome via homologous recombination at a precise location on the chromosome. To increase the likelihood of integration at a precise location, the integrating element preferably contains a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, more preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to increase the probability of homologous recombination. The integrating element may be any sequence homologous to the target sequence within the host cell genome. Furthermore, the integrating element may be a non-coding or coding nucleotide sequence. On the other hand, the vector may integrate into the host cell genome via non-homologous recombination. For autonomous replication, the vector may further contain a replication origin enabling autonomous replication within the host cell. An origin of replication can be any plasmid replicon that mediates autonomous replication within a cell. The terms "origin of replication" or "plasmid replicon" are defined herein as nucleotide sequences that enable plasmids or vectors to replicate in vivo.

[0142] One or more copies of the polynucleotide of the present invention can be inserted into host cells to increase the yield of gene products. The number of polynucleotide copies can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide. In the latter case, cells containing an amplified copy of the selectable marker gene and the resulting additional copy of the polynucleotide can be selected by culturing the cells artificially in the presence of a suitable selectable agent.

[0143] Methods well known to those skilled in the art can be used to construct vectors containing DNA sequences encoding herbicide-resistant peptides and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the vector to guide mRNA synthesis. The vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0144] The vectors applicable in this invention include plasmids that are available from commercial sources, such as, but not limited to: pBR322 (ATCC37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA), pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), etc.

[0145] The term "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, particularly monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, longleaf lettuce), bok choy, taro, cucurbits (e.g., melons, watermelons, crenshaw, cantaloupes, Roman melons), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, headless cabbage, Chinese cabbage, bok choy), artichokes, carrots, napa cabbage, okra, onions, celery, parsley, chickpeas, parsnip, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, baby zucchini, squash, pumpkin), radishes, dried artichokes, etc. Onions, turnips, purple eggplant (also known as eggplant), ginseng, lettuce, scallions, chicory, garlic, spinach, green onions, squash, leafy greens, beets (sugar beets and fodder beets), sweet potatoes, romaine lettuce, wasabi, tomatoes, turnips, and spices; fruits and / or vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus fruits, blueberries, boysenberry. y), cranberries, currants, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pears, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, miscanthus, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans). Oil-bearing plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, hemp, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugarcane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0146] The term "unwanted plants" is understood to refer to plants that affect the normal growth of desired plants (such as crops) and have no practical or applied value. This can include weeds, such as dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds from the following genera: *Sinapis*, *Lepidium*, *Galium*, *Stellaria*, *Matricaria*, *Anthemis*, *Galinsoga*, *Chenopodium*, *Urtica*, *Senecio*, *Amaranthus*, *Portulaca*, *Xanthium*, *Convolvulus*, *Ipomoea*, *Polygonum*, *Sesbania*, and *Agrimonia*. The genera *mbrosia*, *Cirsium*, *Carduus*, *Sonchus*, *Solanum*, *Rorippa*, *Rotala*, *Lindernia*, *Lamium*, *Veronica*, *Abutilon*, *Emex*, *Datura*, *Viola*, *Galeopsis*, *Papaver*, *Centaurea*, *Trifolium*, *Ranunculus*, and *Taraxacum*.Monocotyledonous weeds include, but are not limited to, weeds from the following genera: *Echinochloa*, *Setaria*, *Panicum*, *Digitaria*, *Phleum*, *Poa*, *Festuca*, *Eleusine*, *Brachiaria*, *Lolium*, *Bromus*, *Avena*, *Cyperus*, *Sorghum*, and *Agropyron*. The genera *Cynodon*, *Monochoria*, *Fimbristyslis*, *Sagittaria*, *Eleocharis*, *Scirpus*, *Paspalum*, *Ischaemum*, *Sphenoclea*, *Dactyloctenium*, *Agrostis*, *Alopecurus*, and *Apera* are included. Unwanted plants may also include other plants different from those to be cultivated, such as portions or small amounts of crops like soybeans that grow naturally in rice paddies.

[0147] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0148] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0149] In this invention, the term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. Gene editing tools in CRISPR technology include guide RNA and Cas proteins (such as Cas9, Cpf1, Cas12b, etc.). Gene editing tools in TALEN technology are restriction enzymes capable of cleaving specific DNA sequences, comprising a TAL effector DNA-binding domain and a DNA-cleaving domain. Gene editing tools in ZFN technology are also restriction enzymes capable of cleaving specific DNA sequences, comprising a zinc finger DNA-binding domain and a DNA-cleaving domain. Those skilled in the art know that by constructing nucleotides and other regulatory elements encoding gene editing tools into suitable vectors and then transforming them into cells, editing of the intracellular genome can be achieved, including gene knockout, insertion, and base editing.

[0150] In this invention, the term "cultivation" includes the site for cultivating the plants of this invention, such as soil, and also includes, for example, plant seeds, seedlings, and mature plants. The term "controlling unwanted plants" refers to a herbicide amount sufficient to affect the growth or development of unwanted plants, such as weeds, for example, by preventing or inhibiting the growth or development of unwanted plants, or by killing said unwanted plants. Advantageously, the effective amount for controlling unwanted plants does not significantly affect the growth and / or development of the plant seeds, seedlings, or plants of this invention. Those skilled in the art can determine such effective amounts for controlling unwanted plants through routine experiments.

[0151] The main advantages of this invention are:

[0152] This invention provides an application of HIS1 mutant protein in the preparation of plants with improved traits. After overexpression of the HIS1 mutant protein, the plant height is reduced and the yield is increased. Attached Figure Description

[0153] Figure 1. PCR electrophoresis results, where M refers to marker, 13 refers to positive seedlings, positive refers to plasmid positive control, WT refers to wild-type Zhonghuang 302 soybean, and water refers to blank control-water.

[0154] Figure 2. Resistance of wild-type soybean plants and soybean plants overexpressing OsHIS1(S242G) to the herbicides mesotrione and cyclosulfonamide. CK represents wild-type soybean, and OsHIS1m represents soybean overexpressing OsHIS1(S242G).

[0155] Figure 3. Field performance of wild-type soybean plants and soybean plants overexpressing OsHIS1(S242G) after herbicide application. Among them, Zhonghuang 302 control is wild-type soybean, and OsHIS1m transgenic soybean is soybean overexpressing OsHIS1(S242G). Detailed Implementation

[0156] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0157] The following experimental descriptions, combined with examples, further illustrate the present invention. All methods and operations described in these examples are provided by way of example and should not be construed as limiting. Methods for manipulating DNA can be found in Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel FM Greene Publishing Associates and Wiley Interscience, 1989; Molecular Cloning, T. Maniatis et al., 1982; or Sambrook J. and Russell D., 2001; Molecular Cloning: a laboratory manual, version 3.

[0158] Example 1: Obtaining and phenotypic characteristics of transgenic soybeans

[0159] 1. Carrier Construction

[0160] The rice endogenous HIS1 gene (LOC_Os02g17940) encodes a Fe(II) / 2-oxoglutarate-dependent oxygenase protein, the amino acid sequence of which is shown in SEQ ID No. 1, and the nucleotide sequence of which is shown in SEQ ID No. 2. Patent CN115927222B discloses that the S242G mutation in the rice HIS1 protein confers resistance to HPPD inhibitor herbicides (especially the triketone herbicide mesotrione). The amino acid sequence of the rice HIS1 protein after the S242G mutation is shown in SEQ ID No. 3, and the nucleotide sequence of which is shown in SEQ ID No. 4.

[0161] The amino acid sequence of wild-type OsHIS1 protein (SEQ ID No. 1):

[0162] Nucleotide sequence of wild-type OsHIS1 gene (SEQ ID No. 2):

[0163] The amino acid sequence of the OsHIS1(S242G) mutant protein (SEQ ID No. 3):

[0164] The nucleotide sequence of the OsHIS1(S242G) mutant gene (SEQ ID No. 4):

[0165] In this embodiment, the 5' end sequence of OsHIS1 was amplified using primers OsHIS1-S242G-F1 / OsHIS1-S242G-R1; the 3' end sequence of OsHIS1 was amplified using primers OsHIS1-S242G-F2 / OsHIS1-S242G-R2. The backbone vector P3333 was linearized by BamHI / PtsHI restriction enzyme digestion and then homologously recombinated with the 5' and 3' end fragments of OsHIS1 to form vector P4017. The primer sequences are shown in the table below:

[0166] 2. Genetic transformation

[0167] The above-mentioned vector was introduced into Agrobacterium tumefaciens using the Agrobacterium-mediated transformation method to obtain soybean transformants. The soybean variety was Zhonghuang 302 (ZH302).

[0168] The soybean cotyledonary node transformation method (half-grain method) mediated by Agrobacterium tumefaciens was adopted: the Agrobacterium strain was EHA105. After soybean seeds were sterilized, explants were prepared. The explants were infected and co-cultured with Agrobacterium, followed by shoot induction and shoot elongation. Resistant shoots were screened, and rooting culture was carried out to regenerate plants.

[0169] Seed sterilization and explant preparation: Select plump, healthy soybean seeds without disease spots and sterilize them with chlorine for 1-1.5 hours. Soak the sterilized soybean seeds in sterile water overnight. Use a sterile scalpel to remove the roots and hypocotyl, leaving 2-3 mm of the hypocotyl. After removing the upper 1 / 3 of the soybean cotyledons, peel off the seed coat and divide the seed into two halves along the midline of the hypocotyl, with each cotyledon attached to half of the hypocotyl. Remove the plumule and gently score the cotyledon node to obtain the cotyledon node explant.

[0170] Preparation of Agrobacterium: (1) Plating. Take Agrobacterium out of the -80°C freezer, dip a small amount of bacterial solution into the YEP bacterial solution with a pipette tip, and streak it in an S-shape on the YEP bacterial solution. Generally, 3-4 streaks are sufficient. Seal with 3M tape and incubate in a 28°C incubator for 1-2 days. After the bacteria have grown well, place it in a 4°C freezer. (2) Gentle shaking. Place 5mL YEP in a 50mL centrifuge tube, add Kan and Rif, pick a single spot on the bacterial plaque with a pipette tip, and then insert the pipette tip into the centrifuge tube. Place it in a 28°C, 200r shaker overnight. (3) Large shaking. Place 50mL YEP in a 250mL Erlenmeyer flask, add Kan and Rif, add a small amount of the shaken bacterial solution, and incubate in a 28°C, 200r shaker overnight.

[0171] Explant treatment and inoculation: First, cut off part of the hypocotyl, leaving only 2-3 mm. Peel back the seed coat, gently separate the two cotyledons, and hold the half of the cotyledon with the remaining cotyledon node. Observe the location of the growing point, remove the two true leaves, and gently make 2-3 cuts towards the growing point with the tip of a knife. Place the cut beans into 25 mL of inoculation solution without hormone. Pour the shaken inoculation solution into the cut beans, and add hormone to a total of 50 mL of inoculation solution. Inoculate on a horizontal shaker at 100-110 rpm for 3 hours. After that, remove the beans and place them on sterile filter paper to blow until the surface is dry, with the cotyledon plane facing upwards.

[0172] Co-culture: Cotyledons that have been blown until their surface is free of liquid are inoculated onto a co-culture medium. Incubate in the dark at 22°C for 5 days.

[0173] Recovery culture: Remove the cotyledons after co-culture and observe whether they meet the standards. Cut off the excess hypocotyl 3-5 mm away from the cotyledon. Insert the cotyledons obliquely into the culture medium to recover for 5-7 days. Inoculate 17 grains per dish. Observe the growth after 5 to 7 days. When the cotyledons have grown out of the culture medium, transfer them to the selection medium. Under light at 28℃.

[0174] Selection culture was performed in two rounds, for a total of 20 days. The hypocotyl of the recovered cotyledons was removed 3-5 mm from the leaf node, and then the explants were inserted into the selection medium. Selection was carried out for 10 days, with seven explants per dish. The cotyledonary nodes and buds were immersed in the medium as completely as possible under light at 28°C.

[0175] Elongation culture: Remove yellow leaves from the screened transformed explants, gently tap off the surface culture medium and rotten leaves, cut off the bottom surface of the cotyledon node, insert the treated cotyledon node into the elongation culture medium, and incubate for 60-75 days at 28℃ under light.

[0176] Rooting: During the elongation period, when the seedlings grow to 3-4cm, cut them off and place them in nutrient soil to root. Develop at least one root system of about 1cm and three green leaves, then transfer them to a climate chamber (28℃ under light).

[0177] Positive screening: Positive seedlings were detected and screened by PCR and sequencing in the T0 generation transformant seedlings to obtain soybeans overexpressing OsHIS1 (S242G). Figure 1 shows the PCR electrophoresis results of positive seedlings. After 2-3 generations of self-pollination, the population was increased.

[0178] 3. Phenotypic characteristics

[0179] To test the resistance of transgenic positive soybeans (overexpressing the OsHIS1(S242G) gene) to HPPD inhibitor herbicides, wild-type soybean (Zhonghuang 302) plants and soybean plants overexpressing OsHIS1(S242G) (i.e., transgenic positive soybeans) were sprayed with 200 mg / L mesotrione herbicide and 200 mg / L cyclosulfonamide herbicide, respectively, when the second trifoliate compound leaf of the soybeans unfolded (approximately 3 weeks after sowing). Seven days after spraying the mesotrione or cyclosulfonamide herbicides, the leaves of wild-type soybean plants turned yellow and withered, and the plant growth slowed down, showing obvious herbicide damage; the transgenic soybean plants overexpressing OsHIS1(S242G) grew normally and showed no herbicide damage; as shown in Figure 2.

[0180] To test the phenotype of soybeans, wild-type soybean (Zhonghuang 302) plants and transgenic soybean plants overexpressing OsHIS1 (S242G) were sprayed with a herbicide (800 mg / L mesotrione) when the second trifoliate compound leaf unfolded after field sowing. A control group was set up without herbicide spraying, namely wild-type soybean (Zhonghuang 302) plants without herbicide spraying and transgenic soybean plants overexpressing OsHIS1 (S242G) without herbicide spraying. A small plot experiment was conducted.

[0181] Figure 3 shows the field performance of each group 14 days after herbicide application. Wild-type soybean (Zhonghuang 302) plants without herbicide application grew normally; wild-type soybean (Zhonghuang 302) plants with herbicide application died due to herbicide damage after 2-3 weeks; transgenic soybean plants overexpressing OsHIS1 (S242G) without herbicide application and transgenic soybean plants overexpressing OsHIS1 (S242G) with herbicide application grew normally.

[0182] The plant height, yield, and other data of wild-type soybeans without herbicide application and transgenic soybeans overexpressing OsHIS1 (S242G) with herbicide application in a small-plot trial are as follows: (There were no significant differences in plant height, number of pods, number of seeds per plant, yield per plant, and 100-seed weight between wild-type soybeans overexpressing OsHIS1 (S242G) without herbicide application and wild-type soybeans without herbicide application.)

[0183] The above results indicate that overexpression of the rice HIS1(S242G) mutant gene (amino acid sequence shown in SEQ ID No. 3) in soybeans can help increase the plant's resistance to β-triketone herbicides (especially mesotrione and cyclosulfonamides); and, under the condition of herbicide application, the transgenic soybeans overexpressing rice HIS1(S242G) have reduced plant height, significantly increased number of pods, number of seeds per plant, yield per plant, and 100-seed weight, resulting in increased soybean yield.

[0184] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The application of HIS1 (HPPD INHIBITOR SENSITIVE 1) mutant protein in the preparation of plants with improved traits, or in the improvement of plant traits, characterized in that, The HIS1 mutant protein has a mutation at amino acid position 242 corresponding to the amino acid sequence shown in SEQ ID No. 1, compared to the parental HIS1 protein; the modified trait is selected from any one or more of the following groups i-vi: i. Increase production; ii. The number of pods increases; iii. Increased number of grains; iv. Increased particle weight; v. Plant height decreased; vi. Increased resistance to HPPD inhibitory herbicides.

2. The application according to claim 1, characterized in that, The HIS1 mutant protein is introduced into plants to prepare plants with improved traits or to improve the traits of plants.

3. The application according to claim 2, characterized in that, The HIS1 mutant protein is introduced into plants to overexpress the HIS1 mutant protein in plants.

4. The application according to claim 1, characterized in that, The application includes the step of treating plants with HPPD inhibitory herbicides.

5. The application according to claim 4, characterized in that, The treatment involves spraying the plants with HPPD suppressive herbicide.

6. The application according to any one of claims 1-5, characterized in that, The plant in question is soybean.

7. Use of a nucleic acid molecule encoding the HIS1 mutant protein of any one of claims 1-6, or of a biological material containing said nucleic acid molecule, in the preparation of plants with improved traits, or in the improvement of plant traits.

8. A method for improving the traits of plants, or a method for preparing plants with improved traits, characterized in that, The method includes the step of introducing the HIS1 mutant protein of any one of claims 1-6 into the plant; the modified trait is selected from any one or more of the following groups i-vi: i. Increase production; ii. The number of pods increases; iii. Increased number of grains; iv. Increased particle weight; v. Plant height decreased; vi. Increased resistance to HPPD inhibitory herbicides.

9. The method according to claim 8, characterized in that, The method includes the step of overexpressing the HIS1 mutant protein in plant cells, plant seeds, plant tissues, or plant parts of the plant.

10. The method according to any one of claims 8-9, characterized in that, The method includes the steps of obtaining a plant overexpressing the HIS1 mutant protein and cultivating the plant.

11. The method according to claim 10, characterized in that, The method also includes the step of treating the plants with HPPD inhibitory herbicides.

12. The method according to claim 11, characterized in that, Ten days after sowing, the plants were treated with HPPD herbicide.

13. A method for preparing a hybrid plant, the method comprising hybridizing a first plant with a second plant to obtain the hybrid plant, characterized in that, The first plant is a plant prepared by the method according to any one of claims 8-12.

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