Rice ALS protein mutant and application thereof

Abstract

The invention relates to the technical field of plant breeding, in particular to a mutant of rice ALS protein and application of the mutant. The mutant is obtained by mutating an amino acid sequence of a rice wild-type ALS protein as follows: serine at the 627th site is mutated into threonine, glycine at the 628th site is mutated into histidine, and amino acid at the 629th site is deleted. The application comprises the following steps: (1) enabling plants to have ALS inhibitor herbicide resistance; (2) the resistance of plants to ALS inhibitor herbicides is improved; (3) the phytotoxicity of ALS inhibitor herbicides to plants is reduced; (4) weed control; (5) plant genetic breeding or seed production; and (6) serving as a plant transgenic selection marker. The rice ALS protein mutant is obtained through resistance stress screening, the mutant is reported for the first time, rice can have high resistance to more than two ALS inhibitor herbicides, and the rice ALS protein mutant has high market value and social benefits.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a mutant of rice ALS protein and its application. Background Technology

[0002] Acetolactate synthase (ALS; EC 2.2.1.6) is the first key enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway in plants. Because this enzyme exists only in plants and microorganisms and not in animals, herbicides that target and inhibit ALS are highly safe for humans and animals. By inhibiting ALS activity, the synthesis of essential amino acids in plants can be specifically blocked, thereby achieving efficient weed control. Based on this excellent target, various ALS inhibitor herbicides have been successfully developed and widely used, mainly including sulfonylureas (SU), imidazolinones (IMI), triazolopyrimidines (TP), and pyrimidine thiobenzoic acids (PTB). However, with the long-term use of herbicides, field weeds have also evolved coping strategies. Mutations at key sites in the ALS gene can cause changes in the spatial structure of the encoded protein, thereby reducing or hindering the binding of herbicides to the target site, ultimately leading to weed resistance to the corresponding herbicides.

[0003] This survival mechanism of weeds has also brought new insights into crop genetic improvement. Specifically, similar ALS mutant genes can be actively introduced into crops. Herbicide-resistant crops bred in this way can survive herbicide spraying without damage, while weeds are effectively eliminated. However, currently reported ALS mutant genes generally suffer from low resistance levels and insufficiently broad resistance spectrums. There is an urgent need to create novel ALS mutant genes with both high-level and broad-spectrum resistance and apply them to crop breeding. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a mutant of rice ALS protein and its application.

[0005] In a first aspect, the present invention provides a mutant of rice ALS protein, wherein the mutant is obtained by the following mutations in the amino acid sequence of wild-type rice ALS protein: serine at position 627 is mutated to threonine, glycine at position 628 is mutated to histidine, and amino acid at position 629 is deleted.

[0006] The amino acid sequence of the wild-type ALS protein in rice is shown in SEQ ID NO.3, and includes: .

[0007] Furthermore, the mutant comprises an amino acid sequence as shown in SED ID NO.1.

[0008] The amino acid sequence shown in SEQ ID NO.1: .

[0009] Secondly, the present invention provides a nucleic acid molecule for encoding the aforementioned mutant.

[0010] Furthermore, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0011] The nucleotide sequence shown in SEQ ID NO.2:

[0012] Thirdly, the present invention provides a biological material, the biological material comprising the aforementioned mutant or the aforementioned nucleic acid molecule; the biological material being an expression cassette, a vector, or a transgenic cell.

[0013] The expression cassette of this invention includes a promoter, a coding sequence (corresponding to the nucleotide sequence of the aforementioned nucleic acid molecule), and a termination signal for terminating the transcription process (e.g., including a terminator and a polyadenylation signal). It can also guide the cell to add a poly(A) tail to the end of the mRNA to increase mRNA stability and translation efficiency. Common examples include SV40polyA and BGH polyA. Furthermore, various elements can be added to the expression cassette, such as enhancers, introns, Kozak sequences, Shine-Dalgarno sequences, or selectable marker genes.

[0014] The vectors described in this invention include: plasmid vectors (extrachromosomal circular DNA molecules derived from bacteria or yeast), viral vectors (modified viruses that have had their pathogenicity and self-replication capabilities removed, but retain their ability to efficiently infect cells and deliver genetic material into cells), bacteriophage vectors, or artificial chromosome vectors (e.g., bacterial artificial chromosome BAC or yeast artificial chromosome YAC).

[0015] The transgenic cells described in this invention are cells whose genetic material has undergone stable artificial alterations, such as the introduction of the nucleic acid molecules provided in this application. The transgenic cells described in this invention include animal cells, plant cells, or microbial cells, wherein the animal and plant cells do not have the potential to develop into a complete individual (and do not belong to any animal or plant species).

[0016] In some embodiments of the present invention, the microbial cells are Escherichia coli cells or Agrobacterium cells.

[0017] Those skilled in the art, having access to the nucleic acid molecules disclosed in this application, are fully aware of the preparation methods of the aforementioned expression cassettes, vectors, transgenic cells, and recombinant viral particles based on existing technology. There are no technical obstacles involved, and therefore, expression cassettes, vectors, transgenic cells, and recombinant viral particles containing the aforementioned gene mutants are also within the scope of this invention.

[0018] Fourthly, the present invention provides the use of the aforementioned mutant rice ALS protein, or the aforementioned nucleic acid molecule, or the aforementioned biological material in any of the following: (1) To make plants resistant to ALS inhibitor herbicides; (2) Improve plant resistance to ALS inhibitor herbicides; (3) Reduce the phytotoxicity of ALS inhibitor herbicides to plants; (4) Weed control; (5) Plant genetic breeding or seed production; (6) As a marker for plant transgenic selection.

[0019] The applications described in (1), (2), and (3) can enable plants to develop resistance to ALS inhibitor herbicides, improve plant resistance to ALS inhibitor herbicides, or reduce the phytotoxicity of ALS inhibitor herbicides by expressing mutants of the rice ALS protein or introducing the nucleic acid molecule or an expression cassette or vector containing the nucleic acid molecule.

[0020] The application described in (4) includes: making the plant contain the nucleic acid molecule or expressing the mutant, and applying an effective dose of an ALS inhibitor herbicide during the plant cultivation process.

[0021] Further, the ALS inhibitor herbicides include one or more of the following: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine thiobenzoic acids, pyrimidine salicylic acids, or sulfonamide carbonyl triazolinones; and / or, The plant is a grass or a legume. The grasses mentioned in this invention include plants from the genera *Oryza*, *Triticum*, *Zea*, *Barley*, *Oat*, *Rye*, *Sorghum*, *Paspalum*, *Sorghum*, *Pennisetum*, *Eragrostis*, *Saccharum*, *Citronella*, *Phyllostachys*, *Phyllostachys*, *Lyrfalfa*, *Festuca*, *Tetracentron*, or *Orchardgrass*.

[0022] The legumes mentioned in this invention include plants from the genera *Glycine*, *Peanut*, *Phaseolus*, *Cowpea*, *Vicia*, *Lentinula*, *Chickpea*, *Alfalfa*, *Trifolium*, *Astragalus*, *Lupinus*, *Tamarind*, *Carob*, *Acacia*, or *Leucaena*.

[0023] In some embodiments of the present invention, the grasses include: rice, corn, wheat, soybean, sorghum or millet; In some embodiments of the present invention, the legume includes soybean, peanut, pea, or green bean.

[0024] Fifthly, the present invention provides a method for improving plant resistance to ALS inhibitor herbicides, comprising: expressing the aforementioned mutant or expressing the aforementioned nucleic acid molecule in the plant.

[0025] Furthermore, the mutant is expressed in the plant by any one or more of the following methods: (1) Gene editing of the gene encoding wild-type ALS protein in plants using CRISPR technology; (2) The mutant was expressed exogenously in plants using a plasmid vector; (3) The mutant was expressed exogenously in plants using a plant virus vector; (4) Use plants that stably express the mutant to carry out hybridization breeding with wild-type plants.

[0026] The ALS inhibitor herbicides include one or more of the following: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine thiobenzoic acids, pyrimidine salicylic acids, or sulfonamide carbonyl triazolinones; and / or, The plant is a grass or a legume. In some embodiments of the present invention, the grasses include: rice, corn, wheat, soybean, sorghum or millet; In some embodiments of the present invention, the legume includes soybean, peanut, pea, or green bean.

[0027] In some embodiments of the present invention, the imidazolinone herbicide is metronidazole nicotinic acid.

[0028] In some embodiments of the present invention, the pyrimidine salicylic acid herbicide is bispyribac-sodium.

[0029] The present invention has the following beneficial effects: This invention obtained a mutant of rice ALS protein through resistance stress screening. This mutant enables various plants, including rice, corn, and soybean, to exhibit high resistance to multiple ALS-inhibiting herbicides. Transgenic plants incorporating this mutant protein gene demonstrate high resistance to herbicides such as pyrimidine salicylates and imidazolinones. The mutant protein provided by this invention can significantly improve the resistance level of plants to ALS-inhibiting herbicides and significantly increase the types of ALS-inhibiting herbicides that plants are resistant to. It exhibits good effects in various plant species and can be used for the breeding of herbicide-resistant plant varieties, thus possessing high application value. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of resistant plants obtained under the screening pressure of ALS inhibitor herbicides provided in Example 1 of the present invention; wherein the concentration of nicotinic acid is 360 mg / L; the red arrows point to the corresponding gene-edited mutant materials, and the white arrows point to wild-type materials.

[0032] Figure 2 This is an analysis diagram of the ALS gene sequencing results of the resistant strain that survived after herbicide application, provided in Example 2 of this invention; where A is a schematic diagram of the target site and a comparison diagram of the ALS gene sequence of the resistant strain and the ALS gene sequence of WT, B is the corresponding sequencing peak diagram, and C is the corresponding amino acid sequence comparison result.

[0033] Figure 3 The images show the phenotypic results of the transgenic rice and wild-type material ZH11 provided in Example 3 of this invention after being sprayed with 1200 mg / L bispyribac-sodium and 720 mg / L mefenoxam for 28 days, respectively. WT represents wild-type ZH11.

[0034] Figure 4 The plant height phenotype of the transgenic rice material provided in Example 3 of this invention is shown. WT is wild-type ZH11 (without herbicide spraying), IMP is the line sprayed with methyl methacrylate, and BS is the line sprayed with bispyribac-sodium. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0037] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0038] Example 1: Gene Editing to Create Herbicide-Resistant Mutants in Rice This invention utilizes CRISPR / Cas9 gene editing technology, designs a target sequence (5´-CGATCATGCGGCGGGGGACCTGG-3´), with the underlined PAM sequence, constructs a gene editing vector to edit the ALS gene in rice plants, adds an ALS inhibitor herbicide (bispyribac-sodium) throughout the transformation process to increase selection pressure, and sprays the seedlings with an ALS inhibitor herbicide (360 mg / L methomyl nicotinic acid). Resistant plants were obtained in the T0 generation. The process is as follows: Figure 1 As shown.

[0039] Example 2: Identification of ALS gene sequence in herbicide-resistant mutants Based on the resistant plants obtained in Example 1, this invention amplified the rice ALS gene by genomic PCR and then sequenced it, obtaining a rice ALS protein mutant with high resistance to bispyribac-sodium and metribuzin (the amino acid sequence of this rice ALS protein mutant is shown in SEQ ID NO.1, and the nucleotide sequence of its corresponding mutant gene is shown in SEQ ID NO.2). Compared with the wild-type rice ALS protein (the amino acid sequence of the wild-type rice ALS protein is shown in SEQ ID NO.3), this rice ALS protein mutant has a serine mutation at position 627 to histidine, a glycine mutation at position 628 to histidine, and a glycine deletion at position 629. The mutation sequences are shown in [the original text is missing]. Figure 2 This mutant protein can confer greater and broader resistance to imidazolinone and pyrimidine salicylic acid herbicides in rice.

[0040] Example 3: Identification of herbicide resistance in rice In this invention, the mutant gene obtained in Example 2 was placed in the pC1300-ALSpro-MCS-OsubiT vector (modified by the inventors based on the pC1300 vector backbone, containing the constitutive promoter ALSpro and the terminator OsubiT), and expression was driven by ALSpro. The constructed plasmid was provided for electroporation transformation of Agrobacterium tumefaciens EHA105, and the rice variety ZH11 was infected by Agrobacterium-mediated genetic transformation, resulting in 50 transgenic lines. The T0 generation transgenic plants were sprayed with 1200 mg / L bispyribac-sodium (25 lines) and 720 mg / L methomyl nicotinic acid (25 lines) herbicides at the 3-leaf stage for verification. After 4 weeks of observation, the wild-type control had died, while 23 and 21 transgenic lines survived, respectively. Most of the surviving lines were able to grow normally. Figure 3 As shown. Plant height measurements of more than 10 lines after 4 weeks showed no significant difference between the bispyribac- and mefenoxam treatments and the untreated wild-type. (See figure) Figure 4 The results of the transgenic experiments further demonstrated that the ALS mutant gene can confer high resistance to bispyribac-sodium and mefenoxam. Therefore, the ALS mutant gene in this invention can be used to create highly herbicide-resistant plants and to create new germplasm resources resistant to ALS inhibitor herbicides.

[0041] Example 4: Identification of herbicide resistance in transferred rice materials For the rice with the ALS protein mutant obtained in Example 2, this invention further introduces it into other rice varieties through hybridization and breeding methods. The resulting offspring are then self-crossed or backcrossed. Each generation is sprayed with 1200 mg / L bispyribac-sodium or 360 mg / L methomyl-sodium herbicide to obtain the breeding material containing the above-mentioned mutation site. After multiple generations of self-crossing or backcrossing, stable germplasm resources with high resistance to bispyribac-sodium or methomyl-sodium can be obtained.

[0042] Example 5: Identification of herbicide resistance in maize Using a genetic transformation method similar to that in Example 3, the same ALS mutant gene was transferred into maize to obtain transgenic maize plants. The T0 generation transgenic maize plants were sprayed with 1200 mg / L bispyribac-sodium or 360 mg / L methomyl nicotinic acid herbicides for verification, and highly resistant plants were obtained. Based on this method, new herbicide-resistant maize germplasm resources can be created.

[0043] Example 6: Identification of herbicide resistance in soybeans Using a genetic transformation method similar to that in Example 3, this invention transfers the same ALS mutant gene into soybeans to obtain transgenic plants. The T0 generation transgenic plants were then sprayed with 1200 mg / L bispyribac-sodium herbicide for verification, and highly resistant plants were obtained. Based on this method, new herbicide-resistant soybean germplasm resources can be created.

[0044] The above results indicate that transgenic rice lines obtained through breeding or transformation are highly resistant to bispyribac-sodium and also resistant to nicotinic acid; transgenic maize lines obtained through transformation are highly resistant to bispyribac-sodium and also resistant to nicotinic acid; and transgenic soybean lines obtained through transformation are highly resistant to bispyribac-sodium.

[0045] The two herbicides mentioned above are only representative of the ALS inhibitor herbicide category. The transgenic lines obtained not only have high resistance to these three herbicides, but may also have high resistance to other sulfonylurea, imidazolinone, and pyrimidine salicylic acid herbicides.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of rice ALS protein, characterized in that, The mutant is obtained by the following mutations in the amino acid sequence of wild-type rice ALS protein: serine at position 627 is mutated to threonine, glycine at position 628 is mutated to histidine, and amino acid at position 629 is deleted.

2. The mutant according to claim 1, characterized in that, The mutants include the amino acid sequence shown in SED ID NO.

1.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule is used to encode the mutant as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO.

2.

5. A biomaterial, characterized in that, The biomaterial includes the mutant as described in claim 1 or 2, or the nucleic acid molecule as described in claim 3 or 4; the biomaterial is an expression cassette, vector, or transgenic cell.

6. The use of a mutant of the rice ALS protein according to claim 1 or 2, or the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5, in any of the following: (1) To make plants resistant to ALS inhibitor herbicides; (2) Improve plant resistance to ALS inhibitor herbicides; (3) Reduce the phytotoxicity of ALS inhibitor herbicides to plants; (4) Weed control; (5) Plant genetic breeding or seed production; (6) As a marker for plant transgenic selection.

7. The application according to claim 6, characterized in that, The ALS inhibitor herbicides include one or more of the following: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine thiobenzoic acids, pyrimidine salicylic acids, or sulfonamide carbonyl triazolinones; and / or, The plant is a grass or a legume. Preferably, the grasses include: rice, corn, wheat, soybean, sorghum, or millet; Preferably, the legume includes soybeans, peanuts, peas, or green beans.

8. A method for improving plant resistance to ALS inhibitor herbicides, characterized in that, include: In plants, the mutant of claim 1 or 2 is expressed, or the nucleic acid molecule of claim 3 or 4 is expressed.

9. The method according to claim 8, characterized in that, The mutant is expressed in plants by any one or more of the following methods: (1) Gene editing of the gene encoding wild-type ALS protein in plants using CRISPR technology; (2) The mutant was expressed exogenously in plants using a plasmid vector; (3) The mutant was expressed exogenously in plants using a plant virus vector; (4) Use plants that stably express the mutant to carry out hybridization breeding with wild-type plants.

10. The method according to claim 8 or 9, characterized in that, The ALS inhibitor herbicides include one or more of the following: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine thiobenzoic acids, pyrimidine salicylic acids, or sulfonamide carbonyl triazolinones; and / or, The plant is a grass or a legume. Preferably, the grasses include: rice, corn, wheat, soybean, sorghum, or millet; Preferably, the legume includes soybeans, peanuts, peas, or green beans.

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