WO2022205974A1 - Hir mutant having resistance to triketone herbicides and application thereof in plant breeding - Google Patents

Abstract

Provided are an iron/ascorbate-dependent oxidoreductase (HIR) mutant having resistance to triketone herbicides and an application thereof in plant breeding. The HIR mutant is obtained by mutating wild-type HIR derived from a plant, and can degrade triketone herbicides, so that plants expressing the mutant are resistant to triketone herbicides.

Description

HIR mutants with resistance to triketone herbicides and their application in plant breeding

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure requires a Chinese patent application with the application number CN 202110345377.0 and the title of "HIR mutant with resistance to triketone herbicides and its application in plant breeding" filed with the China Patent Office on March 31, 2021. Priority, the entire contents of which are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the field of plant proteins, in particular, to a HIR mutant with resistance to triketone herbicides and its application in plant breeding.

Background technique

Weeds in farmland are one of the important factors affecting crop yield, and artificial weeding consumes high human resources and costs, and is not convenient for intensive agricultural production, which seriously restricts the development of crop planting in the direction of high yield, high quality and low cost. process. Therefore, herbicides came into being, and their use played a great role in solving weeds in farmland, promoting the innovation of cultivation methods and increasing production.

The world's herbicide research and development focus on high-efficiency, low-toxicity, broad-spectrum and low-dose varieties. Triketones herbicides are a class of albino (belonging to cyclohexanone) herbicides newly developed by Syngenta, which are used in various (such as soybean, cotton, rapeseed, fruit and beet, etc.) broad-spectrum herbicides. Leaf crop fields control monocotyledonous weeds, sensitive weeds absorb and conduct through young roots, competitively inhibit 4-Hydroxyphenylpyruvate dioxygenase (HPPD), inhibit the synthesis of homogentisic acid, make plastoquinone and The biosynthesis of tocopherols is blocked, causing the plant to develop symptoms such as bleaching, stunted growth, and ultimately killing the plant. Triketone herbicides have a broad herbicidal spectrum and can control a variety of annual grass and broad-leaved weeds, with an efficacy of 3 to 5 days, and are environmentally friendly. Among them, mesotrione and cyclosulfonone are the main herbicides in corn fields, which can effectively control 1-year-old broad-leaved weeds in corn fields, as well as some grass weeds in corn fields, such as barnyardgrass, foxtail, horsetail Tang et al. Mesotrione has been well promoted in domestic corn fields. However, the use of triketone herbicides in other crops is very limited. Because it is harmful to weeds and crops. Even in maize, resistance to triketone herbicides varies among cultivars, and care must be taken when applying them. Although triketone herbicides have been widely used commercially, the use of herbicides is limited in time and space for crops that are generally not herbicide-resistant because these herbicides often kill the crops themselves. There are great restrictions, such as the need to use herbicides some time before crops are sown.

To this end, people research and develop crops with herbicide resistance, so that herbicides can be used during planting to kill weeds without affecting the growth of the plants themselves, thus broadening the scope of herbicide use. In order to expand the herbicidal spectrum of triketone herbicides and use them in other sensitive crops, it is an effective method to construct transgenic plants with resistance to HPPD inhibitors by means of genetic engineering techniques. There are currently two HPPD inhibitor-resistant transgenic soybeans in the USDA approval process. The former expresses a HPPD mutant derived from Pseudomonas fluorescens in soybeans. Overexpression of recombinant HPPD results in maize being resistant to pre-emergence herbicides. 4-fold increase in tolerance and 10-fold increase in soybean tolerance. Made it tolerant to Isoxaflutole; the latter was tolerant to mesotrione and isoxazole using HPPD mutants derived from Avena sativa with lower substrate affinity for the inhibitor oxalone. However, due to the anti-GM wave, the acceptance of GM crops in the world is still low. Even in the Americas, where the area of GM crops is the largest, GM is mainly limited to a few crops such as corn, soybean, and cotton. To this end, people are also devoted to using non-transgenic methods to mutate crops to obtain herbicide-resistant crops, among which the commonly used method is ethyl methanesulfonate (EMS) mutagenesis. Due to the uncertainty of EMS mutagenesis, obtaining herbicide-resistant crops by this method can only rely on the hard work of researchers, especially long-term screening, and can only be obtained by a certain amount of luck.

In view of this, the present disclosure is hereby made.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide a HIR mutant with resistance to triketone herbicides and its application in plant breeding. The HIR mutants provided by the present disclosure are capable of degrading triketone herbicides, thereby rendering plants resistant to triketone herbicides.

This disclosure is implemented as follows:

In one aspect, the present disclosure provides an HIR mutant with resistance to triketone herbicides, wherein the HIR mutant is obtained by mutating a plant-derived wild-type HIR, wherein the mutation is as follows (1)-( 9) any one or a combination of several:

(1): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 5th position of the reference sequence to P;

(2): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 75th position of the reference sequence to L;

(3): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 80th position of the reference sequence to R;

(4): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 89th position of the reference sequence to G;

(5): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 111th position of the reference sequence to S;

(6): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 145th position of the reference sequence to G;

(7): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 218th position of the reference sequence to N;

(8): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 283rd position of the reference sequence to R;

(9): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 322nd position of the reference sequence to G;

Wherein, the amino acid sequence of the reference sequence is shown in SEQ ID NO.1.

Iron/ascorbate-dependent oxidoreductases (Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases, HIR) are present in most plants. In the present disclosure, by mutating the wild-type HIR, the obtained HIR mutant as described above can degrade triketone herbicides, so that the plants expressing the mutant are resistant to triketone herbicides. The HIR mutant and its encoding nucleic acid can be used not only for the cultivation of transgenic crops, but also for the cultivation of non-transgenic plants with resistance to triketone herbicides, and has broad application prospects.

The reference sequence shown in SEQ ID NO.1 is the wild-type HIR of rice Nipponbare, and the HIR obtained by any plant-derived wild-type HIR through any one or combination of mutations in the above-mentioned mutation modes (1)-(9) The mutants were all resistant to triketone herbicides.

The above-mentioned plants include but are not limited to rice, wheat, corn, barley, oats, sorghum, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato, cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, Carrots, turnips, beets, cabbage, mustard greens, cabbage, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, Green onions, onions, leeks, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grapes, strawberries, beets, sugar cane, tobacco, alfalfa, pasture, lawn grass, tea and cassava, etc.

It should be noted that the alignment method used in the protein sequence alignment involved in the present disclosure is Clustal online alignment, and its website address is: http://www.ebi.ac.uk/Tools/msa/clustalo/. The results obtained by using other sequence alignment tools (such as DNAMAN, the relevant parameter settings are set by default) are basically consistent with the results obtained by Clustal online alignment.

The comparison results of sequence similarity and identity of wild-type HIRs from different plant sources are shown in the following table:

In the present disclosure, an amino acid sequence refers to the type and arrangement of amino acid residues constituting a protein or polypeptide, usually the three-letter format commonly used in the art is used to denote amino acid residues, and the single-letter format commonly used in the art can also be used to denote amino acid residues Basically, those skilled in the art should be able to convert the three-letter amino acid sequence to the one-letter amino acid sequence. It should be clear that no matter which way is used in the present disclosure, those skilled in the art can understand and convert. For example: alanine, a single letter is A, three letters are Ala; arginine, a single letter is R, three letters are Arg; aspartic acid, a single letter is D, three letters are Asp; cysteine, Single letter is C, three letters are Cys; glutamine, single letter is Q, three letters are Gln; glutamic acid, single letter is E, three letters are Glu; histidine, single letter is H, three letters are His; Isoleucine, one letter is I, three letters are Ile; Glycine, one letter is G, three letters are Gly; Asparagine, one letter is N, three letters are Asn; Leucine, one letter is L, three letters are Leu; lysine, one letter is K, three letters are Lys; methionine, one letter is M, three letters are Met; phenylalanine, one letter is F, three letters are Phe ; Proline, single letter is P, three letters are Pro; Serine, single letter is S, three letters are Ser; Threonine, single letter is T, three letters are Thr; Tryptophan, single letter is W, Three letters are Trp; tyrosine, one letter is Y, three letters are Tyr; valine, one letter is V, three letters are Val.

Optionally, in some embodiments of the present disclosure, the amino acid sequence of the wild-type HIR is selected from any one of SEQ ID NOs. 1-2.

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, and the mutant mutated in the above-mentioned mutation mode has resistance to triketone herbicides.

SEQ ID NO.2 is the wild-type HIR of maize, and the mutant after it is mutated according to the above-mentioned mutation mode has resistance to triketone herbicides.

In the present disclosure, positions 5, 75, 80, 89, 111, 145, 218, 283 and 322 on the reference sequence are referred to as reference sites, The sites on the wild-type HIR or HIR mutants corresponding to the above reference sites are referred to as mutation sites. The positions of the wild-type HIRs of different plant sources (take rice and maize as examples) corresponding to the mutation sites of the above-mentioned reference sites on their wild-type HIR sequences are shown in the following table:

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

For example, take corn wild-type HIR as an example to illustrate how it is mutated:

For the above mutation method (1), the maize wild-type HIR is aligned with the reference sequence, and the amino acid of the maize wild-type HIR corresponding to the 5th position of the reference sequence (that is, the 5th position of the maize wild-type HIR) is mutated. is P;

For the above-mentioned mutation mode (2), the maize wild-type HIR is aligned with the reference sequence, and the maize wild-type HIR corresponds to the amino acid at position 75 of the reference sequence (ie, the 76th position of the maize wild-type HIR). mutated to L.

Optionally, in some embodiments of the present disclosure, the triketone herbicide is selected from the group consisting of mesotrione, sulfenolone, bicyclosulfuron, sulcotrione, sulcotrione, diflufenazone, and Any one of bispyrazone.

It should be noted that the resistance to triketone herbicides refers to the resistance to herbicides including mesotrione, cyclosulfazone, bicyclosulfuron, furoscotrione, sulcotrione, fenoxadiazone, and diazotrione, etc. The triketone herbicides within are tolerant.

It should be noted that, when the protein sequence is known, those skilled in the art have the ability to change the coding gene sequence of the known protein and introduce it into the expression vector, so as to prepare amino acid residues substituted, added or deleted. Proteins, these methods are recorded in "Molecular Cloning Experiment Guide" (Beijing: Science Press, 2002 edition) and other documents.

In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding a HIR mutant having triketone herbicide resistance as described in any of the above.

In the present disclosure, nucleic acid may be DNA or RNA, preferably DNA. Under the premise of knowing the encoded protein sequence or nucleic acid sequence, through conventional codon correspondence and host expression frequency, using PCR method, DNA recombination method or artificial synthesis method, those skilled in the art have the ability to obtain and optimize the encoding of the present disclosure. Nucleic acids of HIR mutant proteins. Once the nucleic acid is obtained, it can be cloned into a vector, transformed or transfected into corresponding cells, and then propagated by conventional host cells, from which the nucleic acid can be isolated in large quantities.

In another aspect, the present disclosure provides a recombinant vector containing the nucleic acid molecule as described above.

In the present disclosure, vectors refer to bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal cell viruses and various other viral vectors commonly used in the art. Vectors can be divided into cloning vectors, expression vectors and transformation vectors according to the purpose of application, which means that the purpose of use is to clone and verify genes, express corresponding genes and transform corresponding genes respectively. Vectors useful in the present disclosure include, but are not limited to, vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (eg, Pichia vectors), baculovirus vectors for expression in insect cells, Vectors for expression in mammalian cells (retroviral vectors, adenovirus vectors, etc.), plant viral vectors for expression in plants, and various vectors for expression in mammalian mammary glands.

It is easy to understand that a person skilled in the art can select a suitable carrier as needed as a tool for carrying the above-mentioned nucleic acid molecules, which all belong to the protection scope of the present disclosure.

In another aspect, the present disclosure provides a recombinant bacteria or recombinant cell containing the nucleic acid molecule as described above or the recombinant vector as described above.

Recombinant bacteria can be cocci, bacilli such as Escherichia coli or spirochete; it can also be autoxic bacteria or heterooxygen bacteria and the like. Cells can be prokaryotic or eukaryotic. Eukaryotic cells can be animal cells or plant cells. Another preferred cell is a plant cell, preferably a rice cell, more preferably a Nipponbare cell. The above-mentioned nucleic acid molecules contained in the plant cells can be introduced into plant cells by transgenic technology, including introduction into the nucleus, chloroplast and/or plastid of plant cells, or by mutation techniques (such as chemical mutagenesis) conventional in the art. Such as ethyl methanesulfonate (EMS) mutagenesis or radiation mutagenesis, etc.) so that the above-mentioned nucleic acid molecules exist in plant cells.

It is easy to understand that those skilled in the art can select suitable bacteria or cells as the host of the above nucleic acid molecules or recombinant vectors as needed, which all belong to the protection scope of the present disclosure.

In another aspect, the present disclosure provides the HIR mutant as described in any one of the above, the nucleic acid molecule as described above, the recombinant vector as described above, or the recombinant bacteria or recombinant cell as described above, after obtaining a herbicide having triketones Applications in resistant plant varieties.

The means of obtaining plant varieties with resistance to triketone herbicides in the present disclosure include preparing, breeding, producing or otherwise producing, for example, including obtaining by transgenic breeding, such as transforming the above-mentioned nucleic acid molecules into plants and making them Expression of the above-mentioned HIR mutants; also including those obtained by non-transgenic breeding, such as by crossing, backcrossing, selfing or asexual propagation and sorting plant varieties comprising the above-mentioned nucleic acid molecules and expressing the above-mentioned HIR mutants.

Optionally, in some embodiments of the present disclosure, the application comprises: modifying the endogenous HIR gene of the plant of interest to encode the HIR mutant.

Optionally, in some embodiments of the present disclosure, the application comprises: mutagenizing, screening plant cells, tissues, individuals or populations to encode the HIR mutant.

Plant varieties described in the present disclosure include, but are not limited to, individual plants, groups of plants or their propagation material, plant events, plant progeny, plant seeds, or other reproducible parts of plants. Among them, the plant progeny itself is the plant, including the plant progeny produced by transgenic technology, the plant progeny produced by crossing with other plant varieties, and the plant progeny produced by backcrossing or self-crossing.

The plant species described in the present disclosure 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 , potato, cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard greens, cabbage, cauliflower, kale, cucumber, zucchini, pumpkin, winter squash, bitter gourd, loofah, vegetable melon, watermelon , melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, pasture , lawn grass, tea and cassava, etc.

Optionally, in some embodiments of the present disclosure, the triketone herbicide is selected from the group consisting of mesotrione, sulfenolone, bicyclosulfuron, sulcotrione, sulcotrione, diflufenazone, and Any one of bispyrazone.

In the present disclosure, a plant refers to a single plant, group of plants, or reproductive material thereof, including plants, plant varieties, plants, plant events, plant progeny, plant seeds, or other reproductive parts of plants. Among them, the plant progeny itself is the plant, including the plant progeny produced by transgenic technology, the plant progeny produced by crossing with other plant varieties, and the plant progeny produced by backcrossing or self-crossing.

Optionally, in some embodiments of the present disclosure, the applying comprises the steps of:

(1) causing the target plant to express the above-mentioned nucleic acid molecule; and/or, (2) causing the target plant to express the above-mentioned HIR mutant.

The target plant is made to contain the above-mentioned nucleic acid molecule, or the target plant is made to express the above-mentioned HIR mutant, thereby obtaining a plant variety having resistance to triketone herbicides.

Those skilled in the art can use gene editing technology, breeding technology, transgenic technology, etc. well-known in the art to make the target plant contain the above-mentioned nucleic acid molecule and make the target plant express the above-mentioned HIR mutant, and these methods include but are not limited to the following methods:

(a): Introducing the nucleic acid molecule encoding the above-mentioned HIR mutant into the cells of the target plant, and culturing the cells to differentiate and develop into plants with resistance to triketone herbicides to obtain glufosinate resistance sexual plant species;

(b): Editing the endogenous HIR gene of the target plant by gene editing technology to encode the above-mentioned HIR mutant to obtain a plant variety with resistance to triketone herbicides;

(c): using mutagenesis technology, mutagenize the cells or tissues of the target plant, or the individual or population of the target plant, and screen out the cells, tissues or individuals encoding the above-mentioned HIR mutants in vivo to obtain three ketone herbicide-resistant plant varieties;

(d): Using the plant species with resistance to triketone herbicides obtained in the above (a), (b) or (c) as the parent, the plants encoding the above-mentioned HIR mutants in vivo are obtained by sexual or asexual hybridization, and Plant varieties that acquire resistance to triketone herbicides.

In another aspect, the present disclosure provides a method for breeding a plant having resistance to a triketone herbicide, comprising: sexually or vegetatively a plant variety obtained by the application as described above.

Among them, the methods of sexual reproduction include crossbreeding, backcrossing and selfing.

Based on well-known breeding techniques in the art, after obtaining plant varieties with resistance to triketone herbicides through the above application, those skilled in the art can easily breed more triketone herbicides through sexual or asexual reproduction. Herbicide-resistant plant varieties, therefore, such breeding methods are also within the scope of the present disclosure.

In another aspect, the present disclosure provides a method for identifying a plant with resistance to triketone herbicides, comprising: determining whether the plant to be tested expresses the HIR mutant described in any one of the above; and/or determining the plant to be tested Whether it contains nucleic acid molecules as described above.

Those skilled in the art can realize the above identification method by conventional methods in the art, such as protein sequencing, nucleic acid sequencing, PCR detection, probe hybridization detection, and the like.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present disclosure more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 shows the growth of rice seedlings on different days after spraying mesotrione.

Fig. 2 shows the growth of rice seedlings after spraying of bisoxazone.

Figure 3 shows the growth of rice seedlings after spraying with cyclopentanone.

Figure 4 shows the amino acid sequence alignment results of wild-type rice OsHIR (OsWT) and OsB, OsC, OsD, OsE, OsI and OsJ.

Figure 5 shows the growth of rice mutant seeds at different seeding densities.

Figure 6 shows the growth of Kasalath seeds 10 days after sowing.

Figure 7 shows the growth of Kasalath seeds 20 days after sowing.

Figure 8 shows the alignment results of the amino acid sequences of wild-type rice OsHIR (OsWT) and wild-type maize ZmHIR (ZmWT).

Figure 9 is a map of the pPAH vector.

Figure 10 shows the color reaction results of rice HIR mutants.

Figure 11 shows the color response results of the maize ZmHIR mutant.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The features and properties of the present disclosure will be further described in detail below with reference to the embodiments.

Example 1

Mutagenesis screening and mutant gene isolation of F1 rice variety resistant to triketone herbicides

1. Induction of rice callus

Sterilize the seeds of the rice (Oryza sativa) variety F1 of the seed industry company: take the mature seeds, manually dehull them, select the seeds with full sterile spots, put them into a 50ml sterile centrifuge tube, and add 70% alcohol for sterilization for 30 seconds. Pour off the alcohol and wash it once with sterile water; add an appropriate amount of 2.6% sodium hypochlorite solution, soak and disinfect for 15 minutes. Pour off the sodium hypochlorite solution, soak and wash with sterile water 6-7 times, 3 minutes each time.

Induction and subculture: The seeds were blotted dry on sterile filter paper, placed in induction medium, 12 per dish; after the operation, the petri dish was sealed with parafilm, and cultured at 30°C in the dark for 21-28 days, the more The wounded tissue was transferred to fresh medium and cultured for about 7 days, and the spherical callus of 1-2 mm in size was taken as the culture material.

Induction medium: NB+hydrolyzed casein 0.3g/L+L-proline 2.8g/L+sucrose 30g/L+2,4-D 4mg/L+agar 8g/L, pH5.8. Autoclave.

2. Suspension Culture and EMS Treatment

Prepare liquid medium for suspension culture: S0 (without agar) + cephalosporin (100mg/L) + 50-100mg/L mesotrione + 0.05%-0.5% ethyl methosulfate (EMS) (only add a liquid medium).

5 g of subcultured callus and 50 ml of liquid medium were added to a 150 ml Erlenmeyer flask, and cultured on a shaker for 4 weeks (120 rpm, 28° C., dark). Change the medium every week (EMS only add the first liquid medium). Transfer to differentiation medium after 3-5 weeks.

S0 medium: NB+L-proline 2.8g/L+sucrose 30g/L+2,4-D 2mg/L, pH5.8, autoclaved.

3. Induction of Differentiation of Resistant Callus

The callus of suspension culture was blotted dry on sterile filter paper, irradiated with UV light for 5 minutes at the same time, transferred to differentiation medium (containing 0.1-5.0 mg/L mesotrione), 30°C, light, and cultured 21 days or so. Subculture 1 time. Select the new young green shoots and move them to a new differentiation medium at 30°C, light, and continue to culture for about 21 days.

Differentiation medium: MS+sucrose 30g/L+agar 8g/L, pH5.8, autoclaved, plus sorbitol 30g/L+KT 2mg/L+NAA0.02mg/L+mesotrione 0.1-5.0mg/L.

4. Rooting

When the new seedlings grow to about 2cm, they are transferred to the rooting medium, illuminated at 30°C, and cultivated for 3 to 4 weeks. medium, and moved to soil.

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

5. Field spraying

The transplanted rice seedlings were evenly arranged in the same experimental area (to avoid overlapping leaves). Calculate the area occupied by the experimental group and the control group, and according to the area, 105 grams per hectare (10.5 mg per square meter) of the active ingredient measured by 1 time of mesotrione was sprayed with mesotrione. Mesotrione 6 times the dose is 630 g/ha. The actual spraying is according to 40ml per square meter, and the drug content is 6 times of mesotrione. Photographs were taken 4 days, 10 days and 21 days after spraying. Some representative plants were photographed, and the results are shown in Figure 1.

It can be seen from Figure 1 that after spraying mesotrione for 4 days, the leaves of the control group (WT) turned white, but the experimental group (Mutant) was not affected. Ten days after spraying mesotrione, the WT group, Nipponbare group and Kasalath group started to wither obviously, and the Mutant group remained unaffected and grew well. After 21 days of mesotrione spraying, WT, Nipponbare and Kasalath had died, and Mutant was still unaffected and growing well. Mutant was shown to be resistant to at least 6 times the field dose of mesotrione.

Mutants and new WTs that survived mesotrione spraying were re-sprayed with fenoxazone and sulfazone herbicides. According to the area of the area, it is 60 grams per hectare (6 mg per square meter) of the active ingredient in 1 times the measurement of dioxazone, and 92 grams per hectare (9.2 mg per square meter) of the active ingredient in cyclosulfonone. Spray herbicides . Bifenazone 6 times is measured at 360 g/ha, and cyclosulfonone 6 times is measured at 552 g/ha. The actual spraying is based on 40ml per square meter, and the drug content is 6 times the herbicide. After spraying, some surviving plants were photographed, and the results are shown in Figures 2 and 3.

It can be seen from Figure 2 that after 21 days of spraying bisoxazone, the two WT plants also withered obviously, while the two Mutant plants had yellowed leaf tips, but the overall growth was still good, and it was not affected by the herbicide. These two strains of Mutant were shown to be resistant to at least 6 times the field dose of diflufenazone.

As can be seen from Figure 3, 21 days after spraying cyclopentanone, WT obviously also withered, and although Mutant has yellowed leaf tips, the overall growth is still good, and it is not affected by herbicides. This strain of Mutant was shown to be resistant to at least 6 times the field dose of cyclopentanone.

These well-growing green seedlings (Mutants), which are F1 mutant plants that are resistant to triketone herbicides, were retained and propagated.

Example 2

Validation of the rice HIR mutant in Example 1 for degradation of mesotrione

1. Propagation of mutant plants resistant to triketone herbicides in Example 1 and harvesting seeds, and then sterilizing the seeds: get mature seeds (mixed seeds of mutant plants), artificially dehull, and select seeds with full sterile spots , put it into a 50ml sterile centrifuge tube, add 70% alcohol to sterilize for 30 seconds. Pour off the alcohol and wash it once with sterile water; add an appropriate amount of 2.6% sodium hypochlorite solution, soak and disinfect for 15 minutes. Pour off the sodium hypochlorite solution, soak and wash with sterile water 6-7 times, 3 minutes each time.

2. Prepare a solid medium containing 1 mg/L of mesotrione, which is then sterilized and dispensed into culture flasks, each of which contains 100 mL of solid culture medium.

3. Take different seed densities and cultivate them on the medium containing mesotrione, that is, take 0, 10, 30, 100 and 300 sterilized mutant plant seeds, respectively, and place them in a medium containing step 2. The flasks were marked as M0, M10, M30, M100, and M300, and each experimental group was repeated 5 times.

4. After 9 days of culture, observe the color of the seedlings in different experimental groups and take pictures. The results are shown in Figure 4.

5. All seedlings and grains on the medium were then removed and 10 dehulled mesotrione sensitive rice Kasalath seeds were placed in each culture flask.

6. Observe the color of the seedlings and take pictures on the 10th day and the 20th day respectively. The results are shown in Figures 5 and 6.

It can be seen from Figure 4 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 are all affected by mesotrione Impact.

As can be seen from Figure 5, on the 10th day, the Kasalath seedlings in the M0 and M10 culture flasks were all albino seedlings, indicating that the growth of the seedlings was affected by mesotrione in the medium, in other words, the mesotrione in the medium The oxalone was not degraded or not much degraded by the rice mutants sown in the previous round. In the M30, M100 and M300 flasks, green Kasalath seedlings were evidently growing, indicating that the mesotrione concentration in these mediums was low or not and would not cause damage to the seedlings, which means that the nitrates in these mediums Sulcotrione has been degraded to below inhibitory levels for sensitive varieties by numerous rice mutant seedlings planted in the previous round. As can be seen from Figure 6, on the 20th day, the condition of the seedlings was not much different from that on the 10th day, and the green seedlings that had grown were still alive. These results all show that the mutant plants of Example 1 can degrade mesotrione, and their degradation has a colony effect. The more colonies, the better the degradation effect.

Get the leaves of the mutant plants in the above-mentioned embodiment 1, extract their genomic DNAs respectively, according to the existing research, design the gene primers relevant to the triketone herbicides, and use the extracted mutant plant genomic DNA as a template to amplify, After ligating the pMD19-T vector, it 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 it was found that:

Compared with the OsHIR gene (SEQ ID NO.3) of wild-type Nipponbare and the wild-type OsHIR protein (SEQ ID NO.1) encoded by it, the OsHIR gene and OsHIR protein of F1 mutant plant 1 to F1 mutant plant 6 each have The mutations are shown in Table 1 below:

Table 1 OsHIR gene and OsHIR protein mutations in F1 mutant plants

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

Example 3

This example provides HIR mutants derived from maize, and the HIR mutants are obtained by the following mutation methods:

The wild-type ZmHIR protein (SEQ ID NO.2) encoded by the ZmHIR gene shown in SEQ ID NO.4 of wild-type maize (Zea mays) Zheng 58 is compared with the reference sequence SEQ ID NO.1, the result is shown in the figure 8, the wild-type ZmHIR protein corresponds to one or more of the reference sites (positions 111, 75, 218, 322, 5, 80, and 89) of the reference sequence. 1 site (as indicated by the arrow in Figure 8), the mutation site is mutated, and the mutant obtained after the mutation and the mutation it has are shown in Table 4 below:

Table 4. Mutants obtained from maize wild-type ZmHIR protein mutation

The genes encoding the maize ZmHIR mutants and the maize ZmHIR mutants provided in the embodiments of the present disclosure can be obtained by chemical synthesis.

Example 4

Color response of rice OsHIR mutants

The mesotrione resistance of OsB, OsC, OsD, OsE, OsI and OsJ of rice OsHIR mutants provided in Example 1 was detected, as follows:

According to the rice nucleotide sequence provided in the above Example 1, PCR primers were designed, and enzyme cleavage sites (Pac1 and Sbf1) were introduced at both ends of the gene. DH5α Escherichia coli is transformed into DH5α E. coli.

After verification, positive clones were picked, inoculated on LTM liquid medium containing different concentrations of mesotrione for growth, and the color change of the medium was observed, which was a color reaction system. In this detection system, in the presence of tyrosine (Tyr), PaHPPD on the pPAH carrier can decompose tyrosine to form homogentisic acid (HGA), and HGA is further oxidized to turn brown. In the presence of ketone, PaHPPD activity was inhibited, and the medium could not show a brown color change. When the HIR mutant degraded mesotrione, the PaHPPD activity recovered, and the medium appeared brown color change, so it can be observed by observing the color reaction of the medium. The ability of HIR to degrade mesotrione was determined.

Taking wild-type rice OsHIR as the control CK1, and replacing the OsHIR on the pPAH vector with a gene OsGS (Glutamine synthetase, GS) that has no ability to degrade mesotrione, another control CK2 was constructed. Color change in the control group. The results are shown in Figure 10.

Among them, the preparation method of LTM (LB+1‰Tyr+kan+mesotrione) medium is as follows: LTM0 (1L):

Tryptone	10g
NaCl	10g
Yeast Extract	5g
ddH 2 O	Dilute to 250ml and sterilize for later use.

1‰Tyr: Dissolve 1g Tyr in 750ml ddH ₂ O, add an appropriate amount of NaOH solution to promote dissolution, and place it on a magnetic stirrer to heat and dissolve. After it is completely dissolved, adjust PH=7.0, and after filtration and sterilization, mix it with the solution sterilized by high temperature as soon as possible (high concentration of Tyr is easy to precipitate) to form LTM0.

LTM25: LTM0 + 25 μM mesotrione;

LTM50: LTM0 + 50 μM mesotrione;

LTM100: LTM0 + 100 μM mesotrione.

According to the results in Figure 10, it can be seen that in LTM0, the 6 rice mutants and the control CK1 and CK2 have obvious brown color, indicating that they can grow normally in LTM0, and PaHPPD exhibits normal enzymatic activity. In LTM25, there was no brown reaction between CK1 and CK2, indicating that OsHIR in CK1 and OsGS in CK2 did not degrade mesotrione, and PaHPPD in the vector was affected by mesotrione and could not be expressed normally, while OsB, OsC, OsD, OsE, OsI, and OsJ showed brown reactions, indicating that mesotrione in these media had been degraded by rice OsHIR mutants to a level below the inhibitory level of PaHPPD, and PaHPPD could be expressed, resulting in a color reaction. It indicated that the mutations in OsB, OsC, OsD, OsE, OsI and OsJ of rice OsHIR mutants endowed these mutants with the ability to degrade mesotrione, and the ability was significantly better than that of wild-type OsHIR. It shows that these OsB, OsC, OsD, OsE, OsI, OsJ can at least degrade to a certain extent at the concentration of 25μM mesotrione.

Therefore, the mutations S5P, F75L, H80R, S89G, L111S, E145G, D218N, H283R and/or R322G mutations can confer or enhance mesotrione resistance in rice OsHIR mutants.

Example 5

Maize ZmHIR mutant color response

With reference to the detection method of Example 4, the mesotrione resistance of the maize ZmHIR mutants ZmC, ZmE, ZmI and ZmJ provided in Example 3 was verified. The results are shown in Figure 11. Among them, wild-type maize ZmHIR was used as control CK3.

According to the results in Figure 11, it can be seen that in LTMO, the 4 maize mutants and the control CK2 and CK3 have obvious brown color, indicating that they can grow normally in LTMO, and PaHPPD exhibits normal enzymatic activity. In LTM25, CK2 had no brown reaction, indicating that OsGS in CK2 did not degrade mesotrione, PaHPPD in the vector was affected by mesotrione and could not be expressed normally, while ZmC, ZmE, ZmI, ZmJ and CK3 appeared Brown reaction, indicating that mesotrione in these media has been degraded by wild-type maize ZmHIR and its mutants to below the inhibitory level of PaHPPD, PaHPPD can be expressed, resulting in a color reaction. However, the colors of ZmC, ZmE, ZmI, and ZmJ were significantly darker than those of CK3. Especially in LTM50, the colors of ZmC, ZmE, ZmI, and ZmJ were still darker than those of CK3. These mutants were endowed with the ability to degrade mesotrione and the ability was significantly better than that of wild-type maize ZmHIR. It shows that these ZmC, ZmE, ZmI, ZmJ can at least play a certain role in the degradation of mesotrione at the concentration of 25uM.

Therefore, the mutations S5P, F75L, H80R, D89G, L111S, D218N and/or R322G mutations can confer or enhance the mesotrione resistance of maize ZmHIR mutants.

Based on the above results, it can be seen that the mutants obtained by mutating wild-type HIR derived from plants can degrade triketone herbicides and have resistance to triketone herbicides. Plants expressing such mutants also have For resistance to triketone herbicides, the aforementioned mutation mode can be 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 of the wild-type HIR corresponding to the 5th position of the reference sequence to P;

(2): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 75th position of the reference sequence to L;

(3): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 80th position of the reference sequence to R;

(4): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 89th position of the reference sequence to G;

(5): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 111th position of the reference sequence to S;

(6): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 145th position of the reference sequence to G;

(7): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 218th position of the reference sequence to N;

(8): align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 283rd position of the reference sequence to R;

(9): Align the wild-type HIR with the reference sequence, and mutate the amino acid of the wild-type HIR corresponding to the 322nd position of the reference sequence to G.

The above descriptions are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims (12)

1. An HIR mutant with resistance to triketone herbicides, characterized in that the HIR mutant is obtained by mutating a wild-type HIR derived from a plant, wherein the mutation method is as follows (1)-(9) Any one or a combination of:

   (1): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 5th position of the reference sequence to P;

   (2): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 75th position of the reference sequence to L;

   (3): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 80th position of the reference sequence to R;

   (4): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 89th position of the reference sequence to G;

   (5): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 111th position of the reference sequence to S;

   (6): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 145th position of the reference sequence to G;

   (7): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 218th position of the reference sequence to N;

   (8): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 283rd position of the reference sequence to R;

   (9): aligning the wild-type HIR with the reference sequence, and mutating the amino acid of the wild-type HIR corresponding to the 322nd position of the reference sequence to G;

   Wherein, the amino acid sequence of the reference sequence is shown in SEQ ID NO.1.
2. The HIR mutant according to claim 1, wherein the plant is selected from the group consisting of rice, wheat, corn, barley, oat, sorghum, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato, cotton , soybean, rapeseed, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard greens, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon, tomato , eggplant, peppers, kidney beans, cowpeas, edamame, leeks, green onions, onions, leeks, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grapes, strawberries, beets, sugarcane, tobacco, alfalfa, pasture, turf grass, Either tea or tapioca.
3. The HIR mutant according to claim 1 or 2, wherein the amino acid sequence of the wild-type HIR is selected from any one of SEQ ID NO.1-2;

   Preferably, the triketone herbicides are selected from any one of mesotrione, cyclosulfazone, bicyclosulfuron, sulcotrione, sulcotrione, diflufenazone and pyraclotrione.
4. An isolated nucleic acid molecule, characterized in that it encodes the HIR mutant with resistance to triketone herbicides according to any one of claims 1-3.
5. A recombinant vector, characterized in that it contains the nucleic acid molecule of claim 4 .
6. A recombinant bacteria or recombinant cell, characterized in that it contains the nucleic acid molecule of claim 4 or the recombinant vector of claim 5.
7. The HIR mutant according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, or the recombinant bacteria or recombinant cell according to claim 6 are obtained after obtaining a triketone. Use in herbicide-resistant plant varieties.
8. The application according to claim 7, wherein the application comprises: modifying the endogenous HIR gene of the target plant to encode the HIR mutant.
9. The application according to claim 7, characterized in that, the application comprises: mutagenizing and screening plant cells, tissues, individuals or populations to encode the HIR mutant.
10. The application according to claim 7, wherein the triketone herbicide is selected from the group consisting of mesotrione, cyclosulfonone, bicyclosulcotrione, sulcotrione, sulcotrione, diflufenazone and Any one of bispyrazone.
11. A method for breeding a plant with resistance to triketone herbicides, characterized in that it comprises: sexually or vegetatively the plant variety obtained by the application according to claim 7 or 8.
12. A method for identifying a plant with resistance to triketone herbicides, comprising: determining whether the plant to be tested expresses the HIR mutant described in any one of claims 1-3; and/or determining whether the plant to be tested expresses the HIR mutant of any one of claims 1-3; Whether the plant contains the nucleic acid molecule of claim 4.

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