Rice ALS mutant protein ALS-ANTSL and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a rice ALS mutant protein ALS-ANTSL and application of the rice ALS mutant protein ALS-ANTSL. Compared with a wild type rice ALS protein, the rice ALS mutant protein disclosed by the invention contains mutation that valine at the 170th position is mutated into alanine, proline at the 171st position is mutated into asparagine, arginine at the 172nd position is mutated into threonine, arginine at the 173rd position is mutated into serine, and methionine at the 174th position is mutated into leucine. According to the rice ALS mutant protein disclosed by the invention, various plants can have relatively high resistance to various ALS inhibitor herbicides, and the rice ALS mutant protein can be used for cultivating herbicide-resistant plant varieties and has relatively high application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a rice ALS mutant protein, ALS-ANTSL, and its applications. Background Technology

[0002] Acetolactate synthase (ALS; EC2.2.1.6) is a key enzyme catalyzing the initiation of branched-chain amino acid (isoleucine, leucine, and valine) synthesis in plants (Herrera Estrella L, Block MD, Messens E, et al. Chimeric genes as dominant selectable markers in plant cells. The EMBO Journal, 1983, 2(6): 987-995.). It is a plant-specific enzyme, absent in animals. Therefore, it is possible to control weeds by inhibiting ALS to block the biosynthesis of branched-chain amino acids without harming humans or animals. Currently, various herbicides (such as sulfonylureas, imidazolinones, pyrimidine salicylic acid, and sulfonamides) have been developed and applied targeting this enzyme. Alterations in the amino acid sequence of ALS can lead to changes in its protein structure, preventing ALS inhibitors from binding to ALS and reducing its sensitivity to herbicides, thus leading to resistance to certain herbicides. Currently reported ALS mutations suffer from low levels of herbicide resistance and an insufficiently broad resistance spectrum, making the creation of highly resistant and multi-resistant ALS mutations particularly necessary. Summary of the Invention

[0003] One of the objectives of this invention is to provide a mutant protein that enables various plants to exhibit high resistance to a variety of ALS inhibitor herbicides.

[0004] This invention provides a rice ALS mutant protein, which, compared with wild-type rice ALS protein, contains mutations in the following positions: valine (V) at position 170 is mutated to alanine (A), proline (P) at position 171 is mutated to asparagine (N), arginine (R) at position 172 is mutated to threonine (T), arginine (R) at position 173 is mutated to serine (S), and methionine (M) at position 174 is mutated to leucine (L).

[0005] The gene encoding wild-type rice ALS protein in this invention is shown in SEQ ID NO.4.

[0006] The amino acid sequence of the rice ALS mutant protein of the present invention is shown in SEQ ID NO.1.

[0007] This invention utilizes CRISPR / Cas9 gene editing technology to edit the rice ALS gene, resulting in a novel rice ALS mutant protein. Containing this mutant protein significantly enhances plant resistance to ALS inhibitor herbicides, while also increasing the variety of herbicides resistant, thereby conferring high resistance to multiple ALS inhibitor herbicides. Specifically, it can enable rice and corn to exhibit high resistance to two or more ALS inhibitor herbicides, demonstrating significant market value and social benefits.

[0008] The rice ALS mutant protein provided by this invention enables plants to have high resistance to pyrimidine salicylic acid herbicides (such as bispyribac-sodium), imidazolinone herbicides (such as imidazolinone acetonide), and sulfonamide carbonyl triazolinone herbicides (such as flusulfuron-methyl).

[0009] The present invention also provides a nucleic acid molecule that encodes the above-mentioned rice ALS mutant protein.

[0010] The nucleotide sequence of the nucleic acid molecule of the present invention is shown in SEQ ID NO.2.

[0011] The nucleic acid molecules of this invention include DNA or RNA.

[0012] Based on the amino acid sequence and codon rules of the rice ALS mutant protein, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the rice ALS mutant protein. Due to the degeneracy of the codon, the nucleotide sequence of the above nucleic acid molecule is not unique, but all nucleic acid molecules that can encode the above rice ALS mutant protein are within the protection scope of this invention.

[0013] The present invention also provides a biomaterial comprising the above-mentioned nucleic acid molecules, wherein the biomaterial is an expression cassette, a recombinant vector, or a cell.

[0014] Furthermore, the host cell may be a microbial cell or an animal cell, but does not include plant cells capable of developing into a complete plant.

[0015] Preferably, the host cell is Escherichia coli or Agrobacterium, but the type of host cell is not limited to these and can be any microbial cell or animal cell that can be used for protein expression.

[0016] The present invention also provides the application of the above-mentioned rice ALS mutant protein or nucleic acid molecule or biological material in making plants resistant to ALS inhibitor herbicides or improving plant resistance to ALS inhibitor herbicides or reducing the phytotoxicity of ALS inhibitor herbicides to plants.

[0017] In the above applications, plants can be made resistant to ALS inhibitor herbicides, or have their resistance to ALS inhibitor herbicides increased, or their phytotoxicity reduced, by expressing the above-mentioned rice ALS mutant protein or introducing the above-mentioned nucleic acid molecules or expression cassettes or vectors containing the above-mentioned nucleic acid molecules.

[0018] The present invention also provides the application of the above-mentioned rice ALS mutant protein or nucleic acid molecule or biological material in plant genetic breeding or weed control.

[0019] Applications for weed control include: ensuring that plants contain the aforementioned nucleic acid molecules or express the aforementioned ALS mutant proteins, and applying effective doses of ALS inhibitor herbicides during plant cultivation.

[0020] In the application of this invention, the plant is rice, corn, wheat, soybean, sorghum, peanut, sesame, cotton, flaxseed, bulrush, oats, rapeseed, barley, rye, millet, tobacco, highland barley, or Arabidopsis thaliana. Preferably, the plant is rice, corn, wheat, soybean, sorghum, or millet.

[0021] In the application of this invention, the ALS inhibitor herbicides are imidazolinones, pyrimidine salicylic acids, sulfonylureas, and / or sulfonamide carbonyl triazolinones.

[0022] Among them, imidazolinone herbicides include imidazoline, methyl imidazoline, and / or methoxyfenozide; pyrimidine salicylic acid herbicides include bispyribac-sodium; and sulfonamide carbonyl triazolinone herbicides include flusulfuron.

[0023] The present invention also provides a method for preparing plants containing anti-ALS inhibitor herbicides, comprising the step of making the plant contain the above-mentioned nucleic acid molecules or expressing the above-mentioned rice ALS mutant protein.

[0024] The beneficial effects of this invention are at least as follows: This invention provides a novel rice ALS mutant protein that enables various plants, including rice, corn, and soybeans, 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, imidazolinones, and sulfonamide carbonyl triazolinones. This mutant protein significantly improves plant resistance to ALS-inhibiting herbicides and also significantly increases the variety of ALS-inhibiting herbicides that plants are resistant to. It exhibits good efficacy in various plant species and can be used for the breeding of herbicide-resistant plant varieties, demonstrating high application value. Attached Figure Description

[0025] Figure 1The results of spraying herbicide on the gene-edited strains of Example 1 of the present invention are shown; the concentration of flusulfuron is 120 mg / L; the red arrow points to the gene-edited mutant material created in this invention, the white arrow points to the wild-type material, and the remaining surviving materials are other mutants.

[0026] Figure 2 This is a diagram showing the sequencing results of the ALS gene of the resistant strain that survived after herbicide application in Example 1 of the present invention; where A is a schematic diagram of the target site and the ALS gene sequence diagram of the resistant strain and WT, B is the corresponding sequencing peak diagram, and C is the corresponding amino acid sequence change result.

[0027] Figure 3 Phenotypic images of transgenic rice and wild-type material ZH11 after 28 days of spraying with 1200 mg / L bispyribac-sodium, 750 mg / L imidacloprid, and 120 mg / L flusulfuron-methyl, respectively. WT represents wild-type ZH11.

[0028] Figure 4 The plant heights of the transgenic rice lines and the wild-type material ZH11 are shown. WT represents the wild-type ZH11 (without herbicide spraying), IMT represents the line sprayed with imidacloprid, BS represents the line sprayed with bispyribac-sodium, and FLZ represents the line sprayed with flusulfuron-methyl. This means p < 0.01. Detailed Implementation

[0029] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0031] Example 1: Gene Editing to Create Herbicide-Resistant Mutants in Rice This invention utilizes gene editing technology to design the target sequence: 5'-CGATCATGCGGCGGGGGACC TGG -3' (SEQ ID NO.3, italicized portion is PAM sequence) was used to construct a gene editing vector to edit the rice ALS gene. ALS inhibitor herbicides such as bispyribac-sodium and methoxyfenozide were added throughout the transformation process to increase selection pressure. Seedlings were then sprayed with 120 mg / L fluazinam herbicide. Resistant plants were obtained in the T0 generation. Phenotypic observation results are as follows: Figure 1 As shown.

[0032] Example 2: Identification of ALS gene sequence in herbicide-resistant mutants After amplifying the ALS gene of rice resistant to ALS inhibitor herbicides obtained in Example 1 by genomic PCR, sequencing yielded a rice ALS mutant protein exhibiting high resistance to bispyribac-sodium, imidacloprid, and fluoxetine. Compared to the wild-type rice ALS protein, this mutant protein contains mutations at positions 170 (valine to alanine), 171 (proline to asparagine), 172 (arginine to threonine), 173 (arginine to serine), and 174 (methionine to leucine). The mutation details are shown below. Figure 2 The amino acid sequence of the specific ALS mutant protein is shown in SEQ ID No. 1, and the nucleotide sequence encoding it is shown in SEQ ID No. 2. This mutant protein confers higher and broader resistance to imidazolinone and pyrimidine salicylic acid herbicides in rice.

[0033] Example 3: Identification of herbicide resistance in transgenic rice The mutant gene described above (as shown in SEQ ID No. 2) was placed in the pC1300-ALSpro-MCS-OsubiT vector (which is a modification 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 rice ZH11 was infected by Agrobacterium-mediated genetic transformation to obtain 80 transgenic lines with the mutant gene introduced above. The T0 generation transgenic plants were sprayed with herbicides at the 3-leaf stage with 1200 mg / L bispyribac-sodium (25 lines), 750 mg / L imidacloprid (30 lines), and 120 mg / L fluazinam (25 lines) for validation. Four weeks after spraying, all wild-type controls (WT) had died, while 18, 23, and 16 transgenic lines survived, respectively, for a total of 57 surviving lines. Representative phenotypic observation results are shown below. Figure 3 Most surviving lines grew normally, while a few weakly resistant lines grew more slowly, possibly related to the level of transgene expression. Four weeks later, plant height measurements of more than 10 lines showed no significant difference between the bispyribac- and imidacloprid-treated wild-type plants and those not treated with herbicides. Flufenoxuron-treated plants were slightly shorter than the untreated wild-type plants. (See [link to relevant documentation]). Figure 4 The results of the transgenic experiments further demonstrated that the ALS mutant gene can confer high resistance to bispyribac-sodium and imidacloprid, and relatively high resistance to flusulfuron-methyl. Therefore, the ALS mutant gene in this invention can be used to create highly herbicide-resistant plants and to create new herbicide-resistant germplasm resources.

[0034] Example 4: Identification of herbicide resistance in transferred rice materials The above-mentioned mutant genes were introduced into other rice varieties through hybridization and breeding methods to obtain self-cross or backcross progeny. Spraying 120 mg / L flusulfuron or 150 mg / L imidacloprid with each generation can quickly obtain breeding materials containing the above-mentioned mutant sites. After multiple generations of self-crossing or backcrossing, stable germplasm resources with high resistance to bispyribac-sodium, imidacloprid, and flusulfuron were obtained.

[0035] Example 5: Identification of herbicide resistance in maize The ALS resistance gene was transferred into maize to obtain transgenic plants through genetic transformation. The T0 generation transgenic plants were sprayed with herbicides of 1200 mg / L bispyribac-sodium, 750 mg / L imidacloprid, and 120 mg / L fluazinam. Two weeks after spraying, the wild-type control had died, while most of the transgenic lines could grow normally. Therefore, new herbicide-resistant maize germplasm resources can be created.

[0036] Example 6: Identification of herbicide resistance in soybeans The ALS resistance gene was transferred into soybeans using genetic transformation to obtain transgenic plants. The T0 generation transgenic plants were then sprayed with 1200 mg / L bispyribac-sodium and 120 mg / L fluazinam herbicides for validation, yielding highly resistant plants. This method can be used to create new herbicide-resistant soybean germplasm resources.

[0037] The above results indicate that transgenic lines obtained through breeding or transformation in rice are highly resistant to bispyribac-sodium, and also resistant to imidacloprid and flusulfuron-methyl; transgenic lines obtained through transformation in maize are highly resistant to bispyribac-sodium, and also resistant to imidacloprid and flusulfuron-methyl; and transgenic lines obtained through transformation in soybeans are highly resistant to bispyribac-sodium and flusulfuron-methyl.

[0038] The three herbicides mentioned above are only representative of their respective herbicide categories. The transgenic lines obtained not only possess high resistance to these three herbicides, but may also possess high resistance to other sulfonylurea herbicides.

[0039] The mutants obtained by gene editing-induced mutations in this invention possess resistance to ALS inhibitors. Furthermore, their introduction into target plants can induce resistance to ALS inhibitors, thus demonstrating high market application value.

[0040] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A rice ALS mutant protein, characterized in that, Compared with wild-type rice ALS protein, the rice ALS mutant protein contains mutations at position 170 (valine to alanine), position 171 (proline to asparagine), position 172 (arginine to threonine), position 173 (arginine to serine), and position 174 (methionine to leucine).

2. The rice ALS mutant protein according to claim 1, characterized in that, The amino acid sequence of the rice ALS mutant protein is shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the rice ALS mutant protein as described in claim 1 or 2.

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

2.

5. A biomaterial, characterized in that, It comprises the nucleic acid molecule of claim 3 or 4, wherein the biological material is an expression cassette, a recombinant vector, or a cell.

6. The application of the rice ALS mutant protein of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, or the biomaterial of claim 5 in equipping plants with resistance to ALS inhibitor herbicides, improving plant resistance to ALS inhibitor herbicides, or reducing phytotoxicity caused by ALS inhibitor herbicides.

7. The application of the rice ALS mutant protein of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, or the biomaterial of claim 5 in plant genetic breeding or weed control.

8. The application according to claim 6 or 7, characterized in that, The plants mentioned are rice, corn, wheat, soybean, sorghum, peanut, sesame, cotton, flaxseed, yam, oats, rapeseed, barley, rye, millet, tobacco, barley or Arabidopsis thaliana.

9. The application according to claim 6, characterized in that, The ALS inhibitor herbicides are imidazolinones, pyrimidine salicylic acids, sulfonylureas, and / or sulfonamide carbonyl triazolinones.

10. A method for preparing a plant resistant to ALS inhibitor herbicides, characterized in that, The steps include making the plant contain the nucleic acid molecule as described in claim 3 or 4 or expressing the rice ALS mutant protein as described in claim 1 or 2.

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