ALS inhibitor broad-spectrum resistance mutant protein and application

Abstract

The invention relates to the technical field of agricultural biology, in particular to ALS inhibitor broad-spectrum resistance mutant protein and application. According to the invention, a gene editing technology is utilized to edit rice ALS genes, then resistance screening is carried out to obtain two new rice ALS mutant proteins, and compared with rice wild-type ALS proteins, the ALS mutant proteins have the advantages that the rice ALS mutant proteins have higher rice ALS mutant proteins; the mutation comprises the mutation that the 171st proline is mutated into threonine, the 172nd arginine is mutated into glycine and the 174th methionine is mutated into leucine, or the mutation comprises the mutation that the 169th glutamine is mutated into asparagine and the 170th valine is mutated into glutamine. The two mutant proteins are reported for the first time, so that rice has relatively high resistance to various ALS inhibitor herbicides; meanwhile, the mutant gene can be used as a plant endogenous transgenic selection marker and added with other functional elements for plant gene transformation, and has great value in commercial application of transgenic plants.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to ALS inhibitor broad-spectrum resistance mutant proteins and applications. Background Art

[0002] Acetolactate synthase (ALS) is a key enzyme in the synthesis of branched-chain amino acids (isoleucine, leucine and valine) in plants. If the function of ALS is blocked, the biosynthesis of branched-chain amino acids is inhibited, thus achieving the purpose of eliminating plant weeds. ALS inhibitors include a variety of herbicides (such as sulfonylureas, imidazolinones, pyrimidine salicylic acid, sulfonamide carbonyl triazolinones and sulfonamide herbicides, etc.) (Duggleby, RG, McCourt, JA and Guddat, LW (2008) Structure and mechanism of inhibition of plantacetohydroxyacid synthase. Plant Physiology and Biochemistry 46, 309-324). ALS is a plant-specific enzyme that does not exist in animals, so it is relatively safe for humans and animals. Changes in the amino acid sequence of ALS will cause changes in the protein structure, which may lead to the inability of ALS inhibitors to bind to ALS, thereby reducing the sensitivity of ALS to herbicides and making plants resistant to herbicides. The ALS mutations reported so far have low levels of herbicide resistance and a narrow resistance spectrum, so it is particularly necessary to create multi-resistant and highly resistant ALS mutations. Summary of the invention

[0003] The first object of the present invention is to provide a rice ALS mutant protein and a gene encoding the same.

[0004] The second object of the present invention is to provide a plant transgenic screening expression cassette and screening vector using plant endogenous genes as screening markers.

[0005] The third object of the present invention is to provide a method for preparing a transgenic plant.

[0006] Specifically, the present invention provides the following technical solutions: In the first aspect, the present invention provides rice ALS mutant proteins including ALS mutant protein I and ALS mutant protein II. Compared with the wild-type rice ALS protein, the ALS mutant protein I contains mutations in which proline at position 171 is mutated to threonine, arginine at position 172 is mutated to glycine, and methionine at position 174 is mutated to leucine; compared with the wild-type rice ALS protein, the ALS mutant protein II contains mutations in which glutamine at position 169 is mutated to asparagine, and valine at position 170 is mutated to glutamine.

[0007] The present invention finds that two mutations including ALS mutant protein I and ALS mutant protein II can significantly improve the resistance of ALS protein to ALS inhibitor herbicides, while increasing the types of herbicides resistant, thereby giving plants high resistance to multiple ALS inhibitor herbicides.

[0008] In some embodiments of the present invention, the amino acid sequence of the ALS mutant protein I is shown as SEQ ID NO.1.

[0009] Preferably, the rice ALS mutant protein provided by the present invention has high resistance to pyrimidine salicylic acid (such as bispyribac-sodium) and imidazolinone (such as imazethapyr, imipenem, and methoxazole) herbicides.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the rice ALS mutant protein described above, wherein the nucleic acid molecule comprises DNA or RNA.

[0011] Based on the amino acid sequence of rice ALS mutant protein and codon rules, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the rice ALS mutant protein. Based on the degeneracy of codons, the nucleotide sequence of the above nucleic acid molecule is not unique, but all nucleic acid molecules that can encode the rice ALS mutant protein are within the protection scope of the present invention.

[0012] In a preferred embodiment, the nucleic acid molecule I is used to encode ALS mutant protein I, and the nucleotide sequence of the nucleic acid molecule I is shown in SEQ ID NO.3; the nucleic acid molecule II is used to encode ALS mutant protein II, and the nucleotide sequence of the nucleic acid molecule II is shown in SEQ ID NO.4. In a third aspect, the present invention provides biological materials comprising the nucleic acid molecule or expressing the rice ALS mutant protein.

[0013] Preferably, the biological material is an expression cassette, a vector or a host cell.

[0014] Furthermore, the host cell may be a microbial cell or an animal cell, and does not include a plant cell that can develop into a complete plant.

[0015] Preferably, the expression cassette is obtained by operably linking a promoter and the nucleic acid molecule.

[0016] Preferably, the expression cassette is obtained by operably linking a promoter, the nucleic acid molecule and a terminator.

[0017] Preferably, the vector is a plasmid vector, including replicable vectors and non-replicable vectors.

[0018] Preferably, the host cell is Escherichia coli or Agrobacterium tumefaciens, but the type of host cell is not limited thereto and can be any microbial cell or animal cell that can be used for protein expression.

[0019] In a fourth aspect, the present invention provides a plant transgenic screening expression cassette, the expression cassette comprising the above-mentioned nucleic acid molecule and a promoter and a terminator for initiating and terminating the transcription of the nucleic acid molecule.

[0020] Preferably, the plant transgenic screening expression cassette comprises a gene encoding a rice ALS mutant protein with an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The present invention finds that the rice ALS mutant protein is more suitable as a plant transgenic screening marker, and can more efficiently play the function of a screening marker in the process of plant gene transformation, thereby achieving a higher screening efficiency.

[0021] Preferably, the plant transgenic screening expression cassette comprises a gene encoding a rice ALS mutant protein whose sequence is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0022] In the above expression cassette, the promoter may be a plant constitutive promoter or a plant tissue-specific promoter, and the terminator is a DNA sequence that can terminate gene transcription in plants.

[0023] Preferably, the promoter is a rice ALS gene promoter, a rice Ubi promoter, a corn Ubi promoter, a Rubisco small subunit promoter or a Cab promoter; and the terminator is a rice ALS gene terminator, a rice Ubi terminator or a NOS terminator.

[0024] In some embodiments of the present invention, the promoter is a rice ALS gene promoter, and the terminator is a rice Ubi terminator. The present invention finds that the use of a rice ALS gene promoter and a rice Ubi terminator to cooperate in regulating the transcription of the rice ALS mutant protein encoding gene can better control the efficient, stable, and moderate expression of the gene, so that the rice ALS mutant protein can better play the role of a screening marker.

[0025] Preferably, the nucleotide sequence of the plant transgenic screening expression cassette is shown as SEQ ID NO.5 or SEQ ID NO.6.

[0026] In a fifth aspect, the present invention further provides a plant genetic transformation screening vector, which comprises the plant transgenic screening expression cassette described above.

[0027] Preferably, the plant genetic transformation screening vector is genetically transformed by cloning other expression cassettes into the vector, wherein other expression cassettes refer to expression cassettes other than the above-mentioned plant transgenic screening expression cassettes, including but not limited to fluorescent protein expression cassettes, GUS reporter gene expression cassettes, insect resistance expression cassettes, herbicide resistance expression cassettes, etc.

[0028] Preferably, the plant genetic transformation screening vector is a plant binary expression vector.

[0029] Preferably, the plant genetic transformation screening vector also comprises a series of multiple cloning sites such as EcoRI, SacI, KpnI, MssI, HindIII, etc., to facilitate the subsequent cloning of target genes. In some embodiments of the present invention, the nucleotide sequence of the plant genetic transformation screening vector is shown as SEQ ID NO. 7 or SEQ ID NO.8.

[0030] The plant genetic transformation screening vector described above can be prepared by the following method: (1) Construction of a plant transgenic screening expression cassette: The ALS mutant protein encoding gene with a sequence as shown in SEQ ID NO. 3 or 4 is expressed under the drive of the gene's own promoter ALSpro, and the expression is terminated downstream with a rice Ubi terminator to obtain a plant transgenic screening expression cassette with a sequence as shown in SEQ ID NO. 5 or 6; (2) Connecting the plant transgenic screening expression cassette obtained in step (1) into the plant binary expression vector pC1300 to obtain a plant genetic transformation screening vector.

[0031] In a sixth aspect, the present invention provides any one of the following uses of the rice ALS mutant protein, the nucleic acid molecule, the biological material, or the plant transgenic screening expression cassette: (1) Application in making plants resistant to ALS inhibitor herbicides; (2) Application in improving plant resistance to ALS inhibitor herbicides; (3) Application in reducing the phytotoxicity of ALS inhibitor herbicides to plants; (4) Application in weed control; (5) Application in plant genetic breeding or seed production.

[0032] The applications described in (1), (2) and (3) can make plants resistant to ALS inhibitor herbicides, increase plant resistance to ALS inhibitor herbicides or reduce the phytotoxicity of ALS inhibitor herbicides to plants by causing the plants to express the rice ALS mutant protein or introduce the nucleic acid molecule or an expression cassette or vector containing the nucleic acid molecule.

[0033] The use described in (4) comprises: making the plant contain the nucleic acid molecule or express the ALS mutant protein, and applying an effective dose of ALS inhibitor herbicide during the plant planting process.

[0034] In a seventh aspect, the present invention provides a method for preparing a plant resistant to ALS inhibitor herbicides, the method comprising: allowing the plant to contain the nucleic acid molecule or express the rice ALS mutant protein.

[0035] In the present invention, the ALS inhibitor herbicide includes but is not limited to imidazolinone, pyrimidine salicylic acid, sulfonylurea, sulfonamide carbonyl triazolinone or sulfonamide herbicide. Among them, imidazolinone herbicides include imazapyr, imipenem and methazaquin; pyrimidine salicylic acid herbicides include bispyribac-sodium.

[0036] In the present invention, the plant includes but is not limited to rice, corn, wheat, soybean, sorghum, peanut, sesame, cotton, linseed, indica, oat, rapeseed, barley, rye, millet, tobacco, highland barley, Arabidopsis, etc. Preferably, the plant is rice, corn, wheat, soybean, sorghum or millet.

[0037] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The present invention provides a rice ALS mutant protein, which can make rice, corn, soybean and other plants have high resistance to various ALS inhibitor herbicides, and the transgenic plants introduced with the mutant protein gene have high resistance to pyrimidine salicylic acid, imidazolinone and other herbicides. The mutant protein can significantly improve the resistance level of plants to ALS inhibitor herbicides, and at the same time significantly increase the types of ALS inhibitor herbicides to which plants are resistant. It can play a good effect in various plants, can be used for the cultivation of herbicide-resistant plant varieties, and can also be used for the development of plant genetic transformation screening systems using herbicide resistance genes as screening markers.

[0038] 2. The present invention also provides a plant transgenic screening expression cassette and a plant genetic transformation screening vector using a rice ALS mutant gene as a screening marker. The plant genetic transformation screening vector of the present invention can be used as a transgenic screening vector with other functional elements added for plant gene transformation, which can achieve a high positive screening efficiency. The screening marker contained in the plant genetic transformation screening vector is an endogenous gene of the plant, and no exogenous screening marker genes such as bacterial sources are introduced during the transgenic process. This not only enriches the plant transgenic screening method, but also effectively reduces the potential safety risks of transgenic plants caused by exogenous genes, which is beneficial to the commercial application of transgenic plants and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The results of spraying herbicides on the gene-edited strains of Example 1 of the present invention; wherein the concentration of imazethapyr is 150 mg / L and the concentration of bispyribac-sodium is 1200 mg / L; Figure 2 The following is an analysis diagram of the sequencing results of the ALS gene of the resistant strain that survived the spraying of the herbicide in Example 1 of the present invention; wherein A is a schematic diagram of the target point and a sequence diagram of the ALS gene of the resistant strain and the ALS gene of the WT, B is a corresponding sequencing peak diagram, and C is a corresponding amino acid sequence change result; Figure 3 This is the vector map in Example 2 of the present invention; A is pCALSTM, B is pCALSDM; Figure 4 The electrophoresis diagram of the pCALSTM recombinant plasmid in Example 1 of the present invention after single digestion by HindⅢ and double digestion by kpnⅠ+MssⅠ is verified; wherein M is a marker, CK + It is pCALSTM or pCALSDM recombinant plasmid, 1-3 is the pCALSTM recombinant plasmid digested with enzymes, 4-5 is the pCALSDM recombinant plasmid digested with enzymes, single enzyme digestion can cut out a fragment of about 1.1kb, double enzyme digestion can cut out a fragment of about 2.5kb, and the enzyme digestion bands are correct; Figure 5 : is the PCR electrophoresis result of the transformed Agrobacterium in Example 2 of the present invention; wherein, M is a marker, ck+ is a positive control of the pCALSTM recombinant plasmid, 1-5 are the PCR results of the Agrobacterium monoclonal colony transformed with the pCALSTM recombinant plasmid, and 6-10 are the PCR results of the Agrobacterium monoclonal colony transformed with the pCALSDM recombinant plasmid; Figure 6 This is a diagram showing the spraying results of certain concentrations of bispyribac-sodium, imazapyr and imipenem at the T1 rice seedling stage of the pCALSTM transgenic strain in Example 5 of the present invention; wherein, bispyribac-sodium 50× represents 1500 mg / L, imazapyr 20× represents 1500 mg / L, imipenem 15× represents 10800 mg / L, and the arrow represents the negative control ZH11. DETAILED DESCRIPTION

[0040] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1 Gene editing to create rice ALS mutant protein like Figure 1As shown, the present invention uses gene editing technology to design the target sequence (5'- CCA GGTCCCCCGCCGCATGATCG-3´), the underline is the PAM sequence, and a gene editing vector was constructed to edit the rice ALS gene. ALS inhibitor herbicides such as bispyribac-sodium and methoxyfen-sodium were added during the entire transformation period to increase the selection pressure, and ALS inhibitor herbicides were sprayed at the seedling stage to obtain resistant plants in the T0 generation. After genome PCR amplification of rice ALS gene, two rice ALS mutant proteins with high resistance to bispyribac, imazapic acid, methazapic acid and methoxazole were obtained by sequencing. Compared with the wild-type rice ALS protein, the ALS mutant proteins contain a mutation of proline at position 171 to threonine, arginine at position 172 to glycine, and methionine at position 174 to leucine (the mutant gene is referred to as ALS-TM, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3); or a mutation of glutamine at position 169 to asparagine, and a mutation of valine at position 170 to glutamine (the mutant gene is referred to as ALS-DM, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4). The mutant sequences are shown in Figure 2 The mutant protein confers rice with higher and broader resistance to imidazolinone, pyrimidine salicylic acid, sulfonylurea, sulfonamide carbonyl triazolinone, and sulfonamide herbicides.

[0044] Example 2 Construction of a Plant Genetic Transformation Screening Vector Containing Rice ALS Mutant Protein The construction methods of the plant transgenic screening expression cassettes OsALSP-ALS-TM-OsubiT and OsALSP-ALS-DM-OsubiT (sequences are shown in SEQ ID NOs. 5 and 6, respectively) and the plant genetic transformation screening vectors pCALSTM and pCALSDM (sequences are shown in SEQ ID NOs. 7 and 8) of the present invention are briefly described as follows: The OsALSP, OsubiT, ALS-TM and ALS-DM sequences were amplified from the genome of the above mutant rice, respectively, and the OsALSP-ALS-TM-OsubiT expression cassette and the OsALSP-ALS-DM-OsubiT expression cassette (sequences are shown in SEQ ID NOs. 5 and 6, respectively) were obtained by overlap PCR, and the above expression cassettes were connected with the plant binary expression vector pCAMBIA1300 after being digested with SacI and HindIII by the Gibson assemble method.

[0045] Take 1 μl of the above ligation product, add it to the competent E. coli cells, and mix it lightly; use an electroporator to transform the competent E. coli cells at 1.8KV; add 1ml of SOC medium, shake and culture at 37℃ and 220 rpm for 1h, centrifuge at 5000rpm for 30s, discard 800 μl of supernatant, mix the remaining bacteria with the culture medium, and spread it on the LB plate containing kanamycin. Culture at 37℃ for about 16 hours, pick a single colony, and use specific primers (pC-F2 and pC-R2, the sequence is shown in SEQ ID NO.9 and 10) for colony PCR verification. The amplified fragment is about 954bp. Select positive colonies, shake the bacteria at 37℃ and 220 rpm overnight, extract the plasmid, and the correct result of enzyme digestion detection is as follows: Figure 4 The bacteria were preserved and sent for sequencing. The verified plasmids were named pCALSTM and pCALSDM (sequences are shown in SEQ ID NO.7 and 8). The vector map is shown in Figure 3 shown.

[0046] The primer sequences are as follows: The specific primer pC-F2 has a sequence of SEQ ID NO.9; The specific primer pC-R2 has a sequence of SEQ ID NO.10.

[0047] Example 3 Agrobacterium transformation, identification and rice genetic transformation Take Agrobacterium EHA105 competent cells stored at -80℃, add 1μl of the correctly sequenced pCALSTM and pCALSDM plasmids constructed in Example 1, and transform them by electroporation at 2.5 KV. Spread on YEP culture plates containing kanamycin, rifampicin and streptomycin, culture at 28℃ for about 48h, pick a single colony and shake overnight, and perform PCR verification of the bacterial solution using specific primers (pC-F2 and pC-R2) ( Figure 5 ), amplify the target fragment of about 954bp, select the positive clone (engineered Agrobacterium), shake the bacteria for 36-48h, and save the bacterial solution for infection.

[0048] Taking rice as an example, the engineered Agrobacterium obtained above was used to infect rice callus tissue through Agrobacterium-mediated genetic transformation, and then screened, differentiated, and rooted under screening pressures such as bispyribac-sodium (BS) and imidazolinone. A total of 300 calli were screened, and 54 positive calli were obtained. After differentiation and rooting, 26 transformed strains were obtained, and the final transformation efficiency was 8.67%. Since one callus can differentiate into multiple seedlings, when rooting, one strain rooted 2-4 seedlings for subsequent identification.

[0049] Example 4 Identification of transgenic lines In order to identify whether the obtained strains are transgenic strains, this example performs PCR verification on some positive transgenic plants obtained through the screening culture, differentiation culture and rooting culture in the above examples.

[0050] First, extract the sample DNA. The DNA extraction steps are as follows: take a rice leaf of about 2 cm in length and place it in a 2 ml centrifuge tube; add 800 μl 1.5×CTAB in a mortar, grind the leaf into a homogenate and pour it back into the centrifuge tube; place in a 65℃ water bath for 20-30 min, and mix once every 5 min; centrifuge at 12000 rpm for 10 min; pipette 400 μl of supernatant into a new centrifuge tube, add 2 times the volume of ice-cold anhydrous ethanol, and place at -20℃ for 20 min; centrifuge at 12000 rpm for 10 min; discard the supernatant, add 500 μl 75% ethanol, rinse by inversion, and centrifuge at 8000 rpm for 5 min; discard the supernatant, place in a clean bench to blow dry or dry naturally, and add 100 μl ddH2O to dissolve the DNA.

[0051] Primers (pC-F2 and pC-R2) were used to perform PCR amplification on the genomic DNA samples of the transgenic lines. This primer pair was unable to amplify a fragment using the endogenous rice genome as a template, but the fragment size amplified using the transgenic expression cassette as a template was about 954 bp.

[0052] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 55-65°C for 45 s; extension at 72°C for 1.5 min; 30-35 cycles; extension at 72°C for another 10 min; and termination at 16°C.

[0053] The PCR reaction system is shown in Table 4.

[0054] Table 4 PCR reaction system The PCR products were subjected to agarose gel electrophoresis, and the results showed that most of the transgenic samples contained a 954 bp transgenic band.

[0055] Example 5 Identification of herbicide resistance phenotypes Some rooting materials of transgenic rice lines with rice ALS mutant genes (ALS-TM or ALS-DM) correctly identified in Example 4 were selected and tested for herbicide spraying concentration 7-14 days after transplanting (about 3-5 leaf stage). 1200mg / L (40x), 1500mg / L (50x) and 1800mg / L (60x) of bispyribac-sodium were sprayed. 3-7 days after spraying, the leaves of wild-type control ZH11 turned yellow. After 21 days, the wild-type control ZH11 had turned yellow and died. The transgenic lines grew normally under the spraying of 1200mg / L (40x) and 1500mg / L (50x), and a small number of leaves turned yellow. Most of the transgenic lines died under the spraying of 1800mg / L (60x) of bispyribac-sodium, and only a few plants had low resistance and survived but were slightly dwarfed.

[0056] Some rooting materials of transgenic rice lines with rice ALS mutant genes (ALS-TM or ALS-DM) identified correctly in Example 4 were selected and sprayed with 750mg / L (10x), 1500mg / L (20x) and 3000mg / L (40x) imidazole acetylcholine 7-14 days after transplanting (about 3-5 leaf stage). After 3-7 days of spraying, the leaves of wild-type control ZH11 turned yellow. After 21 days, the wild-type control ZH11 had turned yellow and died, and the transgenic lines could still grow healthily at 750mg / L (10x) and 1500mg / L (20x). However, most seedlings survived but were slightly dwarfed under the spraying of 3000mg / L imidazole acetylcholine acetylcholine.

[0057] Some rooting materials of transgenic rice lines with rice ALS mutant genes (ALS-TM or ALS-DM) identified correctly in Example 4 were selected and sprayed with 7200mg / L (10x), 10800mg / L (15x) and 14400mg / L (20x) of imipenem 7-14 days after transplanting (about 3-5 leaf stage). 3-7 days after spraying, the leaves of wild-type control ZH11 turned yellow. After 21 days, the wild-type control ZH11 had turned yellow and died. Most of the transgenic lines could grow normally under the spraying of 7200mg / L (10x) and 10800mg / L (15x); but they basically died under the spraying of 14400mg / L (20x), and there were no resistant seedlings.

[0058] The T0 generation transgenic seeds resistant to bispyribac-sodium, imazapic acid or imipenem as verified above were collected and first screened with rooting medium containing the corresponding herbicide before planting. The T1 generation transgenic plants were sprayed with 1500mg / L (50x) bispyribac-sodium, 1500mg / L (20x) imazapic acid or 10800mg / L (15x) imazapic acid 7-14 days after transplanting (around the 3-5 leaf stage). 3-7 days after spraying, the leaves of the wild-type control turned yellow. After 7 days, the wild-type control had turned yellow and died, while most plants of the transgenic lines grew normally, with a small number of leaves showing yellowing. After 21 days, the wild-type control and the sensitive transgenic lines had completely died, and the surviving lines were herbicide-resistant lines, such as Figure 6 shown.

[0059] Example 6 Identification of herbicide resistance in corn A similar method to the rice genetic transformation in Example 2 was used to obtain transgenic corn plants, and the T0 transgenic plants were sprayed with herbicides for verification, and plants with high resistance to bispyribac-sodium, imazapyr, and imipenem were obtained. T0 seeds of plants with high resistance to the three herbicides were collected and planted to obtain T1 transgenic plants and perform herbicide phenotype identification to obtain T1 generation strains with high resistance to bispyribac-sodium, imazapyr, and imipenem.

[0060] Example 7 Identification of herbicide resistance in soybean A similar method to the rice genetic transformation in Example 2 was used to obtain soybean transgenic plants, and the T0 transgenic plants were sprayed with bispyribac-sodium for verification, and highly resistant bispyribac-sodium plants were obtained. T0 seeds of highly resistant bispyribac-sodium plants were collected and planted to obtain T1 transgenic plants and perform bispyribac-sodium phenotype identification to obtain highly resistant bispyribac-sodium T1 generation strains.

[0061] The above results show that the transgenic strains obtained through transformation in rice are highly resistant to bispyribac-sodium, and are also resistant to imazapic acid and imipyrac; the transgenic strains obtained through transformation in corn are highly resistant to bispyribac-sodium, and are also resistant to imazapic acid and imipyrac; the transgenic strains obtained through transformation in soybean are highly resistant to bispyribac-sodium.

[0062] The above three herbicides are only representatives of their respective herbicide categories. The transgenic strains obtained not only have high resistance to these three herbicides, but also have high resistance to other sulfonylurea, imidazolinone, and pyrimidine salicylic acid herbicides (such as benzylsulfuron-methyl, chlorsulfuron-methyl, pyrimidinesulfuron-butyl, pyrimidinesulfuron-butyl, etc.).

[0063] The present invention not only obtains ALSTM or ALSDM mutants induced by gene editing, but also constructs vectors pCALSTM or pCALSDM as plant binary expression vectors, which can make the target plants resistant to ALS inhibitors and have high market application value. Although the present invention has been described in detail above with general descriptions and specific implementation plans, it is obvious to those skilled in the art that some modifications or improvements can be made to it on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A rice ALS mutant protein I, compared with a rice wild-type ALS protein, characterized in that: The ALS mutant protein I contains mutations in which proline at position 171 is mutated to threonine, arginine at position 172 is mutated to glycine, and methionine at position 174 is mutated to leucine; the amino acid sequence of the ALS mutant protein I is shown in SEQ ID NO.

1.

2. A rice ALS mutant protein II, compared with the rice wild-type ALS protein, the ALS mutant protein II contains a mutation of glutamine at position 169 to asparagine and a mutation of valine at position 170 to glutamine. The amino acid sequence of the ALS mutant protein II is shown in SEQ ID NO.

2.

3. A nucleic acid molecule I, characterized in that The nucleic acid molecule I encodes the rice ALS mutant protein I according to claim 1, and the nucleotide sequence of the nucleic acid molecule I is shown in SEQ ID NO.

3.

4. A nucleic acid molecule II, characterized in that: The nucleic acid molecule encodes the rice ALS mutant protein II according to claim 3, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A biomaterial, characterized in that: It comprises any one or more of the ALS mutant protein I, ALS mutant protein II, and nucleic acid molecules capable of expressing ALS mutant protein I or ALS mutant protein II as described in claims 1-2.

6. A biomaterial according to claim 5, characterized in that The biological material is a plant transgenic screening expression box, which contains a nucleic acid molecule that can express ALS mutant protein I or ALS mutant protein II, and a promoter and terminator for starting and terminating the transcription of the nucleic acid molecule; the nucleotide sequence of the expression box is shown in SEQ ID NO.5 or SEQ ID NO.

6.

7. The plant transgenic screening expression cassette according to claim 6, characterized in that: The promoter is any one of rice ALS gene promoter, rice Ubi promoter, corn Ubi promoter, Actin promoter, Rubisco small subunit promoter and Cab promoter; the terminator is any one of rice ALS gene terminator and rice Ubi terminator.

8. A plant genetic transformation screening vector comprising the plant transgenic screening expression cassette according to any one of claims 6 to 7, characterized in that: The nucleotide sequence of the plant genetic transformation screening vector is shown in SEQ ID NO. 7 or SEQ ID NO.

8.

9. Any one of the following uses of the rice ALS mutant protein according to claims 1-2, the nucleic acid molecule according to claims 3-4, the biological material according to claim 5, or the plant transgenic screening expression cassette according to claims 6-8: (1) Application in making plants resistant to ALS inhibitor herbicides; (2) Application in improving plant resistance to ALS inhibitor herbicides; (3) Application in reducing the phytotoxicity of ALS inhibitor herbicides to plants; (4) Application in weed control; (5) Application in plant genetic breeding or seed production; (6) Application in plant transgenic screening markers.

10. A method for preparing a plant resistant to ALS inhibitor herbicides, characterized in that: The method comprises: making a plant contain the nucleic acid molecule according to claim 3 or 4 or express the rice ALS mutant protein according to claim 1 or 2.

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