Method for improving sorghum herbicide tolerance by precisely editing SbALS gene

Abstract

The invention discloses a method for improving sorghum herbicide tolerance by accurately editing a SbALS gene. The method is realized by partially or completely mutating the SbALS gene with a nucleotide sequence shown as SEQ ID No.2 in a sorghum genome into a SbALSW545L gene with a nucleotide sequence shown as SEQ ID No.4. The SbALS gene has a nucleotide sequence shown as SEQ ID No.3. The invention also discloses a method for improving the sorghum herbicide tolerance by accurately editing the SbALS gene. According to the invention, the coding gene of the protein SbALS in the sorghum genome is subjected to directed mutation, so that the herbicide-resistant sorghum material is successfully obtained, and the herbicide-resistant germplasm resources of sorghum are enriched. The method has an important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a method for improving the herbicide tolerance of sorghum by precise editing SbALS of a gene. BACKGROUND

[0002] Field weeds, as one of biological stresses, compete with crops for nutrients and growing space, and seriously affect the growth and yield of crops. In addition, manual weeding and mechanical weeding increase the production cost of crops. Spraying herbicides is the most effective means for controlling weeds, which can promote the development of crop production towards high efficiency, low cost and simplification. In China, the types of sorghum field weeds are various, the quantity is large, and the harm is serious. Manual weeding increases the production cost, reduces the planting benefit and enthusiasm of farmers, and also restricts the mechanization and large-scale planting of sorghum. Therefore, cultivating new sorghum varieties with herbicide resistance is an important measure to increase the yield of sorghum, improve the enthusiasm of farmers and realize the large-scale planting of sorghum.

[0003] Acetolactate synthase (ALS) or acetyl hydroxy acid synthase (AHAS) is a key enzyme for the biosynthesis of three branched-chain amino acids (valine, leucine and isoleucine) in plants. ALS herbicides or ALS inhibitors are developed by targeting ALS. Spraying this kind of herbicides can reduce the activity of ALS in plants, block the synthesis of three branched-chain amino acids, affect the synthesis of proteins, further inhibit cell division, cause plant tissues to lose green and turn yellow, and finally lead to the death of plants. ALS herbicides have the advantages of strong selectivity, wide weed spectrum, low dosage, small toxicity to mammals, etc., and are widely used in production. SUMMARY

[0004] The purpose of the present application is to improve the herbicide tolerance of sorghum.

[0005] The present application first protects the protein SbALS W545L , which can be C1) or C2): C1) the protein obtained by replacing the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS with a leucine residue; The protein SbALS can be a1), a2) or a3): a1) the protein with the amino acid sequence shown in SEQ ID No. 1; a2) a fusion protein obtained by connecting a tag to the N terminus or / and C terminus of the protein shown in SEQ ID No. 1; a3) a protein related to the herbicide tolerance of sorghum obtained by substitution, deletion and / or addition of one or more amino acid residues in the protein shown in a1) or a2); C2) a fusion protein obtained by connecting a tag at the N terminus and / or C terminus of the protein shown in C1).

[0006] wherein SEQ ID No. 1 consists of 641 amino acid residues.

[0007] In order to facilitate the purification of the protein in C1), a tag shown in Table 1 can be connected at the amino terminus or carboxyl terminus of the protein shown in C1).

[0008]

[0009] Any of the above-mentioned proteins SbALS W545L may be S1) or S2) or S3): S1) a protein with the amino acid sequence shown in SEQ ID No. 3; S2) a fusion protein obtained by connecting a tag at the N terminus and / or C terminus of the protein shown in SEQ ID No. 3; S3) a protein related to the herbicide tolerance of sorghum obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the protein shown in S1) or S2).

[0010] In the protein in a3) or S3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0011] The protein in a3) or S3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0012] The encoding gene of the protein in a3) above can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutation of one or several base pairs, and / or connecting the encoding sequence of the tag shown in Table 1 at the 5' end and / or 3' end thereof.

[0013] The encoding gene of the protein in S3) above can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID No. 4, and / or performing missense mutation of one or several base pairs, and / or connecting the encoding sequence of the tag shown in Table 1 at the 5' end and / or 3' end thereof.

[0014] The nucleic acid molecule encoding any of the above-mentioned proteins SbALS W545L also belongs to the protection scope of the present application.

[0015] The expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules also belongs to the protection scope of the present application.

[0016] The expression cassette can comprise a promoter, a nucleic acid molecule encoding the protein SbALS W545L , and a terminator.

[0017] The recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any of the above-mentioned protein SbALS W545L into an expression vector.

[0018] The recombinant microorganism can be obtained by introducing any of the above-mentioned recombinant vector into a starting microorganism.

[0019] The starting microorganism can be a yeast, a bacterium, an alga, or a fungus. The bacterium can be a gram-positive bacterium or a gram-negative bacterium. The gram-negative bacterium can be Agrobacterium tumefaciens. The Agrobacterium tumefaciens can be Agrobacterium tumefaciens EHA105.

[0020] The present application also protects a method for breeding herbicide-tolerant sorghum, which can comprise the following steps: partially or completely mutating any of the above-mentioned protein SbALS W545L in a recipient sorghum into any of the above-mentioned protein SbALS to obtain herbicide-tolerant sorghum; and the herbicide tolerance of the herbicide-tolerant sorghum is improved compared with the recipient sorghum.

[0021] In the above method, the protein SbALS W545L is a protein obtained by replacing the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS with a leucine residue.

[0022] In the above method, the partial or complete mutation of the protein SbALS into the protein SbALS W545L can be specifically achieved by partially or completely mutating TGG at positions 1633-1635 in the gene encoding any of the above-mentioned protein SbALS into CTC. The nucleotide sequence of the gene encoding any of the above-mentioned protein SbALS (i.e., the SbALS gene) can be specifically as shown in SEQ ID No. 2. SbALS

[0023] In the above method, the partial or complete mutation is achieved by gene editing. The target sequence of the gene editing can be as shown in SEQ ID No. 5.

[0024] The present application also protects the use of a substance that partially or completely mutates any of the above-mentioned protein SbALS into any of the above-mentioned protein SbALS W545L in improving the herbicide tolerance of sorghum.​

[0025] In the above use, the protein SbALS W545L is a protein obtained by substituting the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS with a leucine residue.

[0026] In the above use, the "substance that partially or completely mutates any of the above-mentioned proteins SbALS W545L to any of the above-mentioned proteins SbALS SbALS may be a substance that partially or completely mutates the gene shown in SEQ ID No. 2 to the gene shown in SEQ ID No. 4. SbALS W545L may be a substance that partially or completely mutates the gene shown in SEQ ID No. 2 to the gene shown in SEQ ID No. 4.

[0027] In the above use, the "substance that partially or completely mutates any of the above-mentioned proteins SbALS W545L to any of the above-mentioned proteins SbALS may also include a gene editing system. The target sequence of the gene editing can be as shown in SEQ ID No. 5.

[0028] The present application also protects the use of any of the above-mentioned proteins SbALS W545L , any of the above-mentioned nucleic acid molecules or expression cassettes, recombinant vectors or recombinant microorganisms containing any of the above-mentioned nucleic acid molecules, which can be A1) or A2): A1) improving herbicide tolerance; A2) breeding herbicide-tolerant sorghum.

[0029] Any of the above-mentioned nucleic acid molecules (i.e. nucleic acid molecules encoding any of the above-mentioned proteins SbALS W545L may be a DNA molecule as shown in b1), b2), b3) or b4): b1) a DNA molecule encoding the sequence shown in SEQ ID No. 4; b2) a DNA molecule having the nucleotide sequence shown in SEQ ID No. 4; b3) a DNA molecule having 90% or more homology with the nucleotide sequence defined in b1) or b2), derived from sorghum and encoding any of the above-mentioned proteins SbALS W545L ; and b4) a DNA molecule derived from sorghum and encoding any of the above-mentioned proteins SbALS W545L hybridizing to the nucleotide sequence defined in b1) or b2) under stringent conditions.

[0030] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0031] Of these, SEQ ID No. 4 consists of 1926 nucleotides, and the nucleotides shown in SEQ ID No. 4 encode the amino acid sequence shown in SEQ ID No. 3.

[0032] Those skilled in the art can readily employ known methods, such as directed evolution and point mutation, to modify the protein SbALS encoding the present invention. W545L The nucleotide sequence was mutated. Those artificially modified proteins, having the same characteristics as the SbALS protein isolated in this invention, were also mutated. W545L Nucleotides with 90% or higher nucleotide sequence identity, as long as encoding the protein SbALS. W545L All of these are nucleotide sequences derived from and equivalent to those of the present invention.

[0033] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes the protein SbALS, which encodes the amino acid sequence shown in SEQ ID No. 4 of this invention. W545L The nucleotide sequences have 90% or higher, or 95% or higher, nucleotide sequences with the same identity. Identity can be evaluated visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0034] Any of the herbicides mentioned above may be acetolactate synthase inhibitors.

[0035] The acetolactate synthase inhibitory herbicide mentioned above may be at least one of sulfonylurea herbicides, imidazolinone herbicides, triazole pyrimidine herbicides, pyrimidine oxy(sulfur)benzoic acid herbicides, and sulfonamide hydroxytriazolinone herbicides.

[0036] The herbicide mentioned above may specifically be imidazoline.

[0037] Experiments have shown that using a gene editing system to edit the genome of sorghum variety P898012 on two homologous chromosomes, as shown in SEQ ID No. 2, can effectively remove the virus. SbALS The gene mutation is as shown in SEQ ID No. 4. SbALS W545L Gene editing was used to obtain gene-edited sorghum with improved herbicide tolerance. This demonstrates that mutating the protein SbALS to the protein SbALS in the sorghum genome... W545LThis invention can significantly improve herbicide tolerance. The method provided by this invention can directionally improve sorghum traits, and is faster, more efficient, and more precise than chemical mutagenesis. Furthermore, this invention is the first to target the sorghum genome... SbALS By performing targeted gene mutations, herbicide-resistant sorghum materials were successfully obtained, enriching the germplasm resources of herbicide-resistant sorghum. This invention has significant application value. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of the gene editing vector PEplus-SbALS.

[0039] Figure 2 For step two of Example 2, 4. Detection of mutant strains #1-#10 SbALS Types of gene mutations.

[0040] Figure 3 In step three of Example 2, the negative homozygous mutant strain obtained by the removal of the editing tool was detected using a bar test strip.

[0041] Figure 4 To identify the imidazole nicotinic acid tolerance of mutant strain Z1. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0045] In the following examples, the amino acids are denoted by the commonly accepted IUPAC letter abbreviations, wherein each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0046] In the following examples, regarding the mutation of an amino acid, it is denoted as “original amino acid, position, substituted amino acid”. For example, the mutation of tryptophan W at position 545 to leucine L is denoted as W545L.

[0047] In the following examples, wt% means weight percent, unless otherwise specified.

[0048] Sorghum variety P898012 is a common market variety and is described in the following document: PT Do, H Lee, K Nelson-Vasilchik, A Kausch, ZJ Zhang. Rapid and Efficient Genetic Transformation of Sorghum via Agrobacterium-Mediated Method. Current Protocols in Plant Biology. 2018. Its name in the document is P898012.

[0049] Example 1, cloning of protein SbALS and its encoding gene The inventors of the present application cloned the gene SbALS from sorghum variety P898912. SbALS The nucleotide sequence of the gene SbALS in the genomic DNA of sorghum variety P898012 is shown in SEQ ID No. 2. SbALS The nucleotide sequence of the gene SbALS in the genomic DNA of sorghum variety P898012 is shown in SEQ ID No. 2. SbALS The gene SbALS encodes the protein SbALS, and the amino acid sequence of the protein SbALS is shown in SEQ ID No. 1.

[0050] Example 2, mutation of protein SbALS to protein SbALS W545L Increasing imidazolinone tolerance of sorghum I. Construction of gene editing vector PEplus-SbALS The gene editing vector PEplus-SbALS was constructed according to the method in the reference (P Ni, Y Zhao, X Zhou, Z Liu, Z Huang, Z Ni, Q Sun, Y Zong. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biology. 2023.). The specific steps are as follows: 1. The PEplus vector was digested with restriction endonuclease BsaI, and the vector backbone of about 1.8 kb was recovered.

[0051] The PEplus vector is described in the following reference: P Ni, Y Zhao, X Zhou, Z Liu, Z Huang, Z Ni, Q Sun, Y Zong. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biology. 2023. 2. Using SEQ ID No. 5 as a template, PCR was performed with primer SgRNA-F: 5'-ctgccgtataggcagGGGGATGGTGGTGCAGTGGG-3' and primer SgRNA-R: 5'-ctagatagaaccgcgGTGGTGCAGTtGGAaGACAG-3' to recover a DNA fragment of about 160 bp.

[0052] 3. The vector backbone recovered in step 1 and the DNA fragment recovered in step 2 were ligated to obtain the gene editing vector PEplus-SbALS.

[0053] The structure of the gene editing vector PEplus-SbALS is shown in Figure 1 .

[0054] The gene editing vector PEplus-SbALS was sequenced. The sequencing results showed that the gene editing vector PEplus-SbALS inserted the DNA sequence as shown in SEQ ID No. 5 into the recognition site of restriction endonuclease BsaI of the PEplus vector, and the recombinant plasmid was obtained.

[0055] II. Obtaining of positive regenerated plants of T0 generation and identification of mutation type Since sorghum is a diploid plant, when gene editing is performed, both alleles on the two homologous chromosomes in the same cell can be edited, resulting in the same type or different types of mutations, so the two alleles in a plant are considered as two gene editing events. Homozygous mutant refers to the same mutation of the two homologous chromosomes of the plant. SbALS Double-allele mutant refers to the mutation of the two homologous chromosomes of the plant but different mutation forms. SbALS Heterozygous mutant refers to the mutation of one of the two homologous chromosomes of the plant. SbALS Heterozygous mutant refers to the mutation of one of the two homologous chromosomes of the plant. SbALS Wild type refers to the non-mutation of the two homologous chromosomes of the plant. SbALS Wild type refers to the non-mutation of the two homologous chromosomes of the plant.

[0056] 1. Introducing the gene editing vector PEplus-SbALS into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105 / PEplus-SbALS.

[0057] 2. Transforming the recombinant Agrobacterium EHA105 / PEplus-SbALS to the sorghum variety P898012 to obtain regenerated plants. The specific steps are as follows: (1) Preparation of infection solution Filter the infection medium (MS liquid medium containing 0.5 g / L MES and 30 mg / L 2,4-D) with a bacterial filter, then add acetyl-syringone (AS) and mix well; then add the recombinant Agrobacterium EHA105 / SbALS and blow to a particle-free state, adjust the OD 600nm value to between 0.8-1.0. Then wrap with tin foil paper, shake culture at 22℃, 65rpm for 2h to obtain the infection solution.

[0058] (2) Preparation of callus Take 2-5cm long sorghum variety P898012 young spikes, remove the outer stems and leaves to leave only two layers, then soak in 75% (v / v) ethanol aqueous solution for 2min, then completely remove the young spikes and inoculate on the induction medium (MS solid medium containing 3mg / L 2,4-D and 0.5mg / L KT), 25℃ culture for 10 days; then inoculate on the pre-culture medium (MS solid medium containing 3mg / L 2,4-D, 0.5mg / L KT and 200µmol / L AS), 25℃ culture for 2 days to obtain callus.

[0059] (3) Infection The callus prepared in step 2 was placed in the infection solution prepared in step 1 and infected for 10 minutes.

[0060] (4) Co-cultivation After step (3) was completed, the infection solution on the surface of the callus was absorbed with filter paper, and then the callus was inoculated into co-cultivation medium (MS solid medium containing 2 mg / L 2,4-D, 0.5 mg / L KT and 200 μmol / L AS, pH 5.5) and cultured in dark at 25 °C for 2 days.

[0061] (5) Selection culture After step (4) was completed, the callus was inoculated into selection medium (MS solid medium containing 0.5 mg / L KT, 500 mg / L Cef and 50 mg / L Km) and cultured at 25 °C until regenerated seedlings were obtained.

[0062] (6) Rooting culture After step (5) was completed, the regenerated seedlings obtained were inoculated into rooting medium (MS solid medium containing 0.5 mg / L NAA) and cultured at 25 °C, and then transplanted into seedlings when the roots grew to about 1 cm. A total of 20 regenerated plants were obtained.

[0063] 3. Identification of regenerated plants (1) The genomic DNA of the leaves of the 20 regenerated plants was extracted by the CTAB method (Mou Z, He Y, Dai Y, et al. Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology. Plant Cell. 2000, 12, 405-418).

[0064] (2) After step (1) was completed, the genomic DNA of the leaves of the 20 regenerated plants was used as a template, and a primer pair composed of primer BarTest-F: 5'-ACGCTCTTGAAGCCCTGTG-3' and primer BarTest-R: 5'-TCTCGAGTCTACCATGAGCCC-3' was used for PCR amplification to obtain a PCR amplification product; the following judgment was made: if a regenerated plant could PCR-amplify a product containing a DNA fragment with a size of 380 bp, then the regenerated plant was a T0 generation positive regenerated plant.

[0065] Through identification, a total of 10 T0 generation positive regenerated plants were obtained from the 20 regenerated plants.

[0066] 4. Identification of mutation types of T0 generation positive regenerated plants (1) Genomic DNA was extracted from the leaves of 10 positive T0 generation regenerated plants and sorghum variety P898012 using the CTAB method.

[0067] (2) After completing step (1), genomic DNA from leaves of sorghum variety P898012 (as a control) and genomic DNA from leaves of 10 T0 generation positive regenerated plants were used as templates. PCR amplification was performed using primer pairs consisting of primer SbALS545-F: 5′-ATCATTGCCACAGGTGTTGGG-3′ and primer SbALS545-R: 5′-TGCATGTCAAAGAAAGGCAGGG-3′. The PCR amplification product with a size of 620bp was recovered.

[0068] (3) After completing step (2), perform Sanger sequencing on the PCR amplification products. The sequencing results are compared with... SbALS The gene editing target sequence (i.e., SEQ ID No. 2) is compared to count the mutation types.

[0069] Five heterozygous mutants were obtained, named mutant #1, mutant #2, mutant #4, mutant #8, and mutant #9, respectively; two homozygous mutants were obtained, named mutant #3 and mutant #7. They were identified by Sanger sequencing (see...). Figure 2 WT represents one homologous chromosome from one of the two homologous chromosomes of mutant strains #1, #2, #4, #8, and #9 (for sorghum variety P898012). SbALS No gene mutation occurred, but the other homologous chromosome... SbALS The same mutation occurred in both genes; the two homologous chromosomes of mutant strain #3 and mutant strain #7... SbALS The genes simultaneously undergo the same mutation, specifically as shown in SEQ ID No. 2. SbALS The TGG mutation at positions 1633-1635 of the gene is changed to CTC (i.e., shown in SEQ ID No. 2). SbALS The gene mutation is as shown in SEQ ID No. 4. SbALS W545L (Gene), which in turn leads to the mutation of the protein SbALS into the protein SbALS. W545L Protein SbALS W545L It is a protein obtained by replacing tryptophan at position 545 in the amino acid sequence of protein SbALS with leucine (amino acid sequence as shown in SEQ ID No. 3).

[0070] III. Elimination of Negative Homozygous Mutants from Editing Tools—Obtaining Mutant Z1 1. The mutant strain #3 obtained in step 2 was bagged and self-crossed to obtain T1 generation seeds of mutant strain #3. The T1 generation seeds of mutant strain #3 were germinated and then seedlings were raised to obtain 35 T1 generation plants of mutant strain #3, hereinafter referred to as T1 generation plants.

[0071] 2. Genomic DNA was extracted from the leaves of 35 T1 generation plants using the CTAB method.

[0072] 3. After completing step 2, genomic DNA from leaves of sorghum variety P898012 (as a control) and genomic DNA from leaves of 35 T1 generation plants were used as templates for PCR amplification. Primers SbALS545-F: 5′-ATCATTGCCACAGGTGTTGGG-3′ and SbALS545-R: 5′-TGCATGTCAAAGAAAGGCAGGG-3′ were used to recover the PCR amplification product of 620 bp.

[0073] 4. After completing step 3, perform Sanger sequencing on the PCR amplification products. The sequencing results are compared with... SbALS The gene editing target sequence (i.e., SEQ ID No. 2) is compared to count the mutation types.

[0074] The results showed that all the offspring of mutant strain #3 were homozygous mutants and the mutation types were the same as those of mutant strain #3.

[0075] 5. During the self-pollination process of sorghum, the editing tool Cas9 also undergoes homologous recombination, resulting in trait segregation of the editing tool Cas9 in the offspring plants. Therefore, 8 mutants were randomly selected from the 35 mutants obtained in step 4, and their vector residue was detected using bar test strips. The following judgments were made: if the bar test strip showed two bands, the mutants tested had vector residue and the editing tool Cas9 had not been removed; if the bar test strip showed one band (detection band), the mutants tested had no vector residue and the editing tool Cas9 had been removed, i.e., negative mutants with the editing tool removed.

[0076] Test results are shown Figure 3 The mutant strain shown in the red box is the negative homozygous mutant strain that was removed from the editing tools; it is named mutant strain Z1.

[0077] The mutant strain Z1 not only eliminated the editing tool Cas9, but also removed it from two homologous chromosomes. SbALS The same mutation occurred in all genes, specifically: SbALS The TGG mutation at positions 1633-1635 of the gene is changed to CTC (i.e., shown in SEQ ID No. 2). SbALS The gene mutation is as shown in SEQ ID No. 4. SbALSW545L gene), thereby encoding protein SbALS W545L .

[0078] IV. Identification of the tolerance of mutant strain Z1 to imidazolinone The experiment was repeated three times to take the average value, and 4 sorghum seedlings were observed and counted each time. The steps of each experiment were as follows: 4-5 leaf stage sorghum (mutant strain Z1 or sorghum variety P898012) plants were taken, and 0 mg / L, 200 mg / L, 400 mg / L or 800 mg / L imidazolinone was sprayed, and after 8 days or 16 days, the plant phenotype, especially the leaf phenotype, was observed.

[0079] Some detection results are shown in Figure 4 (the left side of each group of sorghum plants is sorghum variety P898012, and the right side is mutant strain Z1). The results show that after 8 days or 16 days of treatment with 200 mg / L, 400 mg / L or 800 mg / L imidazolinone, the mutant strain Z1 plant grows normally, while the leaves of the sorghum variety P898012 turn yellow and even wither. It can be seen that compared with the sorghum variety P898012, the imidazolinone tolerance of the mutant strain Z1 is significantly improved.

[0080] It can be seen that the mutation of protein SbALS to protein SbALS W545L can change the imidazolinone tolerance, specifically to improve the imidazolinone tolerance.

[0081] Sorghum is a diploid plant. SbALS The gene can cause SbALS gene mutation, specifically the mutation of the gene shown as SEQ ID No. 2 to the gene shown as SEQ ID No. 4. SbALS The gene mutation, specifically the mutation of the gene shown as SEQ ID No. 2 to the gene shown as SEQ ID No. 4. SbALS SbALS W545L The gene can cause the mutation of protein SbALS to protein SbALS W545L , thereby improving the imidazolinone tolerance.

[0082] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.​

Claims

1. Protein SbALS W545L is either C1) or C2): C1) a protein obtained by substituting the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS with a leucine residue; Said protein SbALS is a1) or a2) or a3): a1) a protein with the amino acid sequence shown in SEQ ID No. 1; a2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein shown in SEQ ID No. 1; a3) a protein related to herbicide tolerance of sorghum obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the protein shown in a1) or a2); C2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein shown in C1).

2. A nucleic acid molecule encoding the protein SbALS of claim 1. W545L 2. A nucleic acid molecule encoding the protein SbALS of claim 1.

3. An expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule of claim 2.

4. A method for breeding herbicide-tolerant sorghum, comprising the steps of: partially or completely mutating a protein SbALS in a recipient sorghum into a protein SbALS W545L , to obtain herbicide-tolerant sorghum; the herbicide tolerance of the herbicide-tolerant sorghum is improved compared with the recipient sorghum.​ Said protein SbALS is a1) or a2) or a3): a1) a protein with the amino acid sequence shown in SEQ ID No. 1; a2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein shown in SEQ ID No. 1; a3) a protein related to herbicide tolerance of sorghum obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the protein shown in a1) or a2); The protein SbALS W545L is a protein obtained by substituting the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS with a leucine residue.

5. The method of claim 4, wherein: Said partial or total mutation of the protein SbALS to the protein SbALS W545L Specifically, by partially or totally mutating the TGG at positions 1633-1635 of the gene shown in SEQ ID No. 2 to CTC. SbALS Said partial or total mutation of the protein SbALS to the protein SbALS 6. The method according to claim 4 or 5, characterized in that: Said partial mutation or total mutation is achieved by gene editing; Said target sequence of the gene editing is shown in SEQ ID No.

5.

7. partially or totally mutating the protein SbALS into the protein SbALS W545L for use in increasing the herbicide tolerance of sorghum. Said protein SbALS is a1) or a2) or a3): a1) a protein with the amino acid sequence shown in SEQ ID No. 1; a2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein shown in SEQ ID No. 1; a3) a protein related to herbicide tolerance of sorghum obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the protein shown in a1) or a2); The protein SbALS W545L is a protein in which the tryptophan residue at position 545 in the amino acid sequence of the protein SbALS is substituted with a leucine residue.

8. Use according to claim 7, characterized in that: Said "mutating partially or wholly the protein SbALS into a substance of the protein SbALS W545L Said "mutating partially or wholly the protein SbALS into a substance of the protein SbALS SbALS Said "mutating partially or wholly the protein SbALS into a substance of the protein SbALS SbALS W545L Said "mutating partially or wholly the protein SbALS into a substance of the protein SbALS 9. Use according to claim 7 or 8, characterized in that: The phrase "mutating protein SbALS partially or completely into protein SbALS" is used. W545L "Substances" include gene editing systems; Said target sequence of the gene editing is shown in SEQ ID No.

5.

10. The protein SbALS of claim 1 W545L the use of the nucleic acid molecule of claim 2 or of an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule of claim 2 for A1 ) or A2): A1) improving herbicide tolerance; A2) breeding sorghum with improved herbicide tolerance.

Images