A recombinant acetyl-CoA carboxylase encoding gene and its application

Abstract

This invention provides a recombinant acetyl-CoA carboxylase encoding gene and its applications. Mutations at positions 5374, 5229, 5335, 5336, and 5493 in the recombinant acetyl-CoA carboxylase encoding gene provided by this invention can induce more significant resistance in rice expressing this recombinant acetyl-CoA carboxylase. In particular, it shows a significant increase in resistance to seven ACCase inhibitors from three major chemically distinct classes: arbutin (APP), quizalofop-P-ethyl, quizalofop-P-ethyl, and flupyrflupyr; cyclohexenone (CHD), clethodim and clodinafop-P-ethyl; and neophenylpyrazoline (DEN), clodinafop-P-ethyl.

Description

Technical Field

[0001] This invention relates to the fields of gene editing and molecular biology, and in particular to a recombinant acetyl-CoA carboxylase encoding gene and its applications. Background Technology

[0002] Rice is one of my country's important food crops, and the distribution of weeds in rice paddies varies depending on the cultivation method. The main weeds in dry-directed rice fields include barnyard grass, weedy rice, *Echinochloa crus-galli*, *Digitaria sanguinalis*, *Setaria viridis*, and *Cyperus difformis*. In water-directed rice fields, the main weeds include barnyard grass, *Echinochloa crus-galli*, *Gnaphalium affine*, *Eclipta prostrata*, *Monochoria vaginalis*, and *Cyperus difformis*. In transplanted and machine-transplanted rice fields, the main weeds include barnyard grass, *Gnaphalium affine*, *Sagittaria sagittifolia*, *Monochoria vaginalis*, *Gnaphalium affine*, *Echinochloa vaginalis*, *Cyperus difformis*, *Alternanthera philoxeroides*, *Eclipta prostrata*, and *Digitaria sanguinalis*. Weeds are a significant factor affecting rice quality and yield, causing an average yield loss of 15%, which can reach over 50% in severe cases. Weed control is an important agricultural activity, and the use of highly effective chemical herbicides can effectively reduce agricultural losses caused by weeds, save labor costs, and improve agricultural production efficiency.

[0003] Acetyl-CoA carboxylase (ACCase) is the rate-limiting enzyme in the de novo synthesis of fatty acids in plants. ACCase inhibitor herbicides inhibit ACCase, thereby hindering the biosynthesis of acyl esters in plants, altering the permeability of plant cell membranes, causing leakage of metabolic products, and ultimately leading to plant death.

[0004] ACCase inhibitor herbicides mainly include three categories: aryloxyphenoxycarboxylic acid esters (APP), cyclohexenones (CHD), and neophenylpyrazoline herbicides (DEN). APP includes quizalofop-e-ethyl, quizalofop-tefuryl, fenoxaprop-P-ethyl, diclofop-methyl, haloxyfop-P-methyl, clodinafop-propargyl, metamifop, and cyhalofop-butyl, etc. CHD includes clethodim, sethoxydim, alloxydim, and cycloxdim, etc. DEN includes pinoxaden.

[0005] Currently, chemical control of rice paddies in my country mainly relies on soil sealing combined with foliar application. The top ten registered herbicides for rice paddies in my country (single-agent) are, in order: cyhalofop-butyl, penflusulfuron, butachlor, bispyribac-sodium, bensulfuron-methyl, quinclorac, pyrimisulfuron-methyl, pretilachlor, oxadiazon, bentazon, and ethoxyfluazon. These top ten herbicides account for 75.6% of the total registered herbicides for rice (single-agent). Among selective herbicides for rice paddies, cyhalofop-butyl is the only ACCase inhibitor herbicide that is highly safe for rice. It is widely used to control grassy weeds, especially barnyardgrass. However, due to its long-term irrational use, grassy weeds have developed varying degrees of resistance to this herbicide, significantly threatening the product lifecycle of cyhalofop-butyl.

[0006] While herbicides have brought convenience to agricultural production, the long-term, singular use of a single herbicide or herbicide with the same mechanism has led to herbicide resistance in weeds. The types of resistant weeds are gradually increasing, and the mechanisms of resistance formation are becoming more complex, making weed control in farmland more difficult. By improving the broad-spectrum and high-resistance properties of rice to herbicides with different mechanisms, and by combining broad-spectrum and high-resistance rice with corresponding herbicides, we can effectively solve the problems of weeds (especially weedy rice and resistant weeds) and their resistance in rice production. This will expand the range of herbicides that can be safely used in rice paddies, improve the control effect on gramineous weeds, reduce the base number of weeds resistant to existing herbicides, and is of great significance for enriching weed control programs in rice paddies and protecting the life cycle of older herbicide varieties. Summary of the Invention

[0007] This invention provides a gene encoding recombinant acetyl-CoA carboxylase, wherein the gene, based on the wild-type rice ACC2 gene with the nucleotide sequence SEQ ID NO:1, includes a mutation at position 5374 where A is changed to C, and the codon corresponding to this site is changed from ATA to CTA, and the isoleucine at position 1792 of the corresponding encoded protein is changed to leucine; it also includes a mutation at position 5229 where G is changed to C, at position 5335 where T is changed to A, at position 5336 where C is changed to G, and at position 5493 where C is changed to T.

[0008] Furthermore, the nucleotide sequence of the encoding gene is SEQ ID NO:2.

[0009] The present invention also provides a gene editing combinatorial vector, the combinatorial vector comprising a first vector and a second vector, the first vector comprising a Cas9 gene fused with a Gal4BD sequence (Cas9-Gal4BD), two target sequences derived from the ACC2 gene (ACC2-g1 and ACC2-g2) and corresponding sgRNA sequences, the target sequences being shown in SEQ ID NO:3 (target 1, ACC2-g1) and SEQ ID NO:4 (target 2, ACC2-g2);

[0010] The second vector contains a UAS sequence and an ACC2Donor sequence; wherein the ACC2Donor sequence includes a first homologous arm (HA1) sequence, a substitution sequence (HDR) and a second homologous arm (HA2) sequence; wherein the ACC2Donor sequence is SEQ ID NO:5; and the substitution sequence contains the aforementioned recombinant coding gene.

[0011] Furthermore, the first homologous arm sequence (HA1) is SEQ ID NO:6, the second homologous arm sequence is (HA2) SEQ ID NO:7, and the replacement sequence (HDR) is SEQ ID NO:11.

[0012] The present invention also provides the application of the aforementioned encoding gene or combination vector in constructing rice resistant to ACCase inhibitor herbicides; further, the rice variety is Nanjing 9108.

[0013] The present invention also provides a method for preparing herbicide-resistant rice, which enables the rice to acquire tolerance to ACCase inhibitor herbicides by containing the aforementioned recombinant acetyl-CoA carboxylase encoding gene in the rice genome and being able to express it. Further, the rice variety is Nanjing 9108.

[0014] Specifically, including:

[0015] The aforementioned combined vector was transformed into rice using Agrobacterium-mediated transformation, resulting in gene-edited rice transformants.

[0016] Rice plants tolerant to herbicides were screened by spraying with ACCase inhibitor herbicides or mixtures containing ACCase inhibitor herbicides.

[0017] The present invention also provides a method for controlling grassy weeds in paddy fields, comprising sowing rice varieties or their derivative lines prepared using the aforementioned method for preparing herbicide-tolerant rice in the field;

[0018] During the rice seedling to flowering stage, spray with ACCase inhibitor herbicides or mixtures containing ACCase inhibitor herbicides to remove weeds that are sensitive to ACCase inhibitor herbicides.

[0019] The present invention also provides a method for detecting the presence of DNA in rice samples containing the aforementioned herbicide-resistant rice, comprising:

[0020] (1) Use primer pairs ACC2F1-F1 and ACC2F1-R1 to perform nucleic acid amplification reaction on sample DNA, wherein the nucleotide sequence of primer ACC2F1-F1 is SEQ ID NO:8 and the nucleotide sequence of ACC2F1-R1 is SEQ ID NO:9;

[0021] (2) Sequencing analysis was performed using sequencing primers ACC2F1-F2, the nucleotide sequence of which is SEQ ID NO:10;

[0022] The amplification product includes the aforementioned nucleotide mutation site in the recombinant acetyl-CoA carboxylase encoding gene, indicating that the test sample contains the aforementioned herbicide-resistant rice DNA.

[0023] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0024] This invention achieves point mutations in the acetyl-CoA carboxylase gene through gene editing. Unexpectedly, it was discovered that compared to a single mutation at position 1792 of the ACCase protein, simultaneous mutations at positions 5374, 5229, 5335, 5336, and 5493 can significantly increase rice's resistance to various ACCase inhibitor herbicides. In particular, it shows a significant increase in resistance to seven ACCase inhibitors with different chemical structures from three major classes: acetophenoxycarboxylic acid esters (APP) such as quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, and flupyrflupyr; cyclohexenones (CHD) such as clethodim and clodinafop-P-ethyl; and neophenylpyrazoline (DEN) such as clodinafop-P-ethyl. All of them can achieve a tolerance level of at least 100% survival at 4 times the application rate. Using rice varieties expressing the recombinant acetyl-CoA carboxylase provided in this invention can not only expand the range of herbicides available for paddy fields, but also kill grassy weeds, resistant weeds, weedy rice, and free-growing rice seedlings that do not have tolerance to ACCase herbicides, while preventing weeds from developing new herbicide resistance. Furthermore, by increasing the concentration or dosage of herbicides, weeds can be eliminated more effectively while ensuring that rice plants do not suffer from herbicide damage. Attached Figure Description

[0025] Figure 1A comparison diagram of sequencing results of the recombinant acetyl-CoA carboxylase encoding genes of KJR0801 and Nanjing 9108;

[0026] Figure 2 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of clodinafop-propargyl (5% EC) for 4 weeks. In the image, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 75 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 150g ai / hm 2 (Second dose, twice the medium dose), treatment 3 concentration was 300g ai / hm 2 (4 times the medium dose);

[0027] Figure 3 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of haloxyfop-R-methyl (12.5% ​​emulsifiable concentrate) for 4 weeks. In the image, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 169 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 338g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 675g ai / hm 2 (4 times the medium dose);

[0028] Figure 4 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of clethodim (240 g / L emulsifiable concentrate) for 4 weeks. In the image, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 108 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 216g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 432g ai / hm 2 (4 times the medium dose);

[0029] Figure 5 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of quizalofop-P-ethyl (8% EC) for 4 weeks. In the image, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 36 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 72g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 144 g ai / hm 2 (4 times the medium dose)

[0030] Figure 6Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of quizalofop-P-ethyl (5% EC) for 4 weeks. A represents Nanjing 9108, B represents KJR0801, and treatment 1 had a concentration of 45 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 90g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 180g ai / hm 2 (4 times the medium dose)

[0031] Figure 7 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of quizalofop-p-ethyl (7.5% water-in-oil emulsion) for 4 weeks. In the treatment, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 90 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 180g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 360g ai / hm 2 (4 times the medium dose)

[0032] Figure 8 Photographs comparing the resistance of KJR0801 and Nanjing 9108 after spraying with different concentrations of high-efficiency flupyradifurone (108 g / L emulsifiable concentrate) for 4 weeks. In the photo, A represents Nanjing 9108, B represents KJR0801, and treatment 1 has a concentration of 49 g ai / hm. 2 (1x the medium dose), treatment 2 concentration was 98g ai / hm 2 (2 times the medium dose), treatment 3 concentration was 195g ai / hm 2 (4 times the medium dose);

[0033] Figure 9 This is a schematic diagram of the T-DNA region structure of the first vector pK194;

[0034] Figure 10 This is a schematic diagram of the T-DNA region structure of the second vector pK195. Detailed Implementation

[0035] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0036] Example 1: Obtaining KJR0801 material

[0037] 1. DNA sequence characteristics of the KJR0801 editing vector

[0038] Gene-edited rice transformants were obtained by co-transformation of CRISPR-Cas9-Gal4BD vectors pk194 and pk195, which carry the CRISPR-Cas9-Gal4BD system vectors carrying the ACC2 gene encoding acetyl-CoA carboxylase as the editing site, into the rice variety Nanjing 9108 using Agrobacterium-mediated transformation. The pk194 vector was obtained through artificial modification based on the pCAMBIA1300 vector backbone. Figure 9 As shown, the vector pK194 contains a Cas9 gene fused with Gal4BD (Cas9-Gal4BD), two target sequences derived from the ACC2 gene (ACC2-g1 and ACC2-g2), and corresponding sgRNA guide sequences (sgRNA). The target sequences are SEQ ID NO:3 (target 1, ACC2-g1) and SEQ ID NO:4 (target 2, ACC2-g2). The pK194 vector is also obtained through artificial modification based on the pCAMBIA1300 vector backbone. Figure 10 As shown, the vector pK195 contains a UAS sequence and an ACC2 Donor sequence, wherein the ACC2 Donor sequence consists of a first homologous arm (HA1) sequence, a substitution sequence (HDR) and a second homologous arm (HA2) sequence; the ACC2 Donor sequence is SEQ ID NO:5, the first homologous arm (HA1) sequence is SEQ ID NO:6, the second homologous arm (HA2) sequence is SEQ ID NO:7, and the substitution sequence (HDR) is SEQ ID NO:11.

[0039] The specific design of the substitution sequence (HDR) is as follows: the full length is 275 bp (from base 5223 to base 5497 of the full-length ACC2 gene sequence, a total of 275 bp), containing the functional mutation site I1792L, corresponding to the codon ATA being mutated to CTA (i.e., base 5374 changing from A to C); in addition, to avoid secondary editing after recombination, GGG corresponding to the PAM sequence of target site 1 is mutated to GGC (i.e., base 5229 changing from G to C), and CCG corresponding to the PAM sequence of target site 2 is mutated to TCG (i.e., base 5493 changing from C to T);

[0040] In addition, the HinfI restriction site GATTC in the substitution sequence (HDR) is mutated to GATAG (i.e., the 5335th base changes from T to A and the 5336th base changes from C to G), which makes the restriction site disappear and facilitates the detection of recombination events after emergence.

[0041] When the T-DNA of the pK194 vector (the first vector) is transferred into the rice genome, the Cas9-Gal4BD protein guided by sgRNA cuts the corresponding sites of the two target sites (ACC2-g1 and ACC2-g2) of the ACC2 gene, deleting the sequence between the target sites. The T-DNA of the pK195 vector (the second vector) provides a sequence with homology to the target sites (first homologous arm HA1 SEQ ID NO: 6 and second homologous arm HA2 SEQ ID NO: 7), and carries a donor sequence containing 5 variant sites (replacement sequence HDR SEQ ID NO: 11). This allows plant cells to use their own homologous targeted repair mechanism to use the ACC2Donor sequence (SEQ ID NO: 5, including the first homologous arm HA1, the replacement sequence HDR, and the second homologous arm HA2) in the pK195 vector (the second vector) as a template for homologous recombination repair, thereby accurately replacing the artificially designed and synthesized sequence into the wild-type ACC2 gene, obtaining material KJR0801, and achieving precise gene editing.

[0042] 2. Detection methods for the ACC2 gene

[0043] Primers designed based on the DNA sequence of the ACC2 gene

[0044] ACC2F1-F1(5'-AGATGGAAGATAAAGAATCACGGAAG-3')(SEQ ID NO:8)

[0045] ACC2F1-R1(5'-GCGGTCTGGGTTTATCTTGCTATC-3')(SEQ ID NO:9)

[0046] PCR amplification was performed using genomic DNA from rice varieties KJR0801 and Nanjing 9108 as templates, respectively.

[0047] PCR reaction system:

[0048] Add 5 μL of 10× buffer, 1 μL of dNTPs (10M each of dATP, dCTP, dGTP, and dTTP), 1 μL each of primers ACC2F1-F1 and ACC2F1-R1, approximately 25-50 ng of DNA template, and 1 μL of KOD high-fidelity DNA polymerase. Add sterile redistilled water to bring the PCR reaction volume to 50 μL.

[0049] The reaction conditions are:

[0050] 32 cycles of 95℃ for 3 min, 94℃ for 1 min, 57℃ for 1 min, and 72℃ for 1 min, with an extension of 5 min at 72℃.

[0051] After amplifying the target fragment of the expected size (1769bp), DNA Sanger sequencing analysis was performed using the sequencing primers ACC2F1-F2(5'-GCTGTTACCAACCTAGCCTGTGAG-3') (SEQ ID NO:10).

[0052] Sequencing alignment results as follows Figure 1 As shown, the bases in the boxes represent the five nucleotide variation sites in the recombinant acetyl-CoA carboxylase encoding genes of KJR0801 and Nanjing 9108 rice.

[0053] Example 2: Seedling stage herbicide spraying experiment of KJR0801

[0054] KJR0801 and Nanjing 9108 were sown and cultivated in a greenhouse, and at the 4-leaf stage, they were treated with 75, 150, and 300 g a.i. / hm, respectively. 2 The concentrations of clopyralid, 169, 338, and 675 g ai / hm 2 Gluconidine, 108, 216 and 432 g ai / hm 2 Clethodim, 36, 72 and 144 g ai / hm 2 quizalofop-P-ethyl, 45, 90 and 180 g ai / hm 2 Quizalofop-P-ethyl, 90, 180 and 360 g ai / hm 2 49, 98 and 195 g ai / hm of quizalofop-p-ethyl. 2 Highly effective fluroxypyr herbicide for rice treatment.

[0055] On the day of herbicide treatment, the plant height of KJR0801 and Nanjing 9108 rice under each treatment was investigated and recorded. Four weeks after treatment (referring to GB / T 17980.40-2000, this period is generally considered the period of best field efficacy), the number of surviving plants and plant height of KJR0801 and Nanjing 9108 under each treatment were investigated and recorded. The average survival rate of KJR0801 and Nanjing 9108 under each treatment was calculated. According to the requirements of NY / T 1155.8-2007, the safety of herbicide to rice was evaluated using the growth inhibition method (plant height), where the average inhibition rate (plant height) = (control plant height - treatment plant height) / control plant height × 100%). The results are shown in Table 1 and... Figure 2-8 As shown.

[0056] Table 1. Survival rate and plant height inhibition rate of KJR0801 and control Nanjing 9108 rice under different herbicide treatments.

[0057]

[0058]

[0059] like Figure 2 As shown, after spraying different concentrations of oxychlorpyrifos (5% EC) for 4 weeks, KJR0801 at 300g ai / hm 2 The survival rate was 100% at the given dose, and the average inhibition rate (plant height) was 14.2%; Nanjing 9108 at 75g ai / hm 2 All rice plants died after treatment with the specified dosage. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to clopyralid.

[0060] like Figure 3 As shown, after spraying different concentrations of tebufenozide (12.5% ​​emulsifiable concentrate) for 4 weeks, KJR0801 showed a reduction in saturation at 675 g ai / hm. 2 At the specified dosage, the survival rate was 100%, and plant height was not inhibited; Nanjing 9108 at 675 g ai / hm 2 The survival rate at the given dose was 20.4%, and the average inhibition rate (plant height) was 39%. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to oxadiazine.

[0061] like Figure 4 As shown, after spraying different concentrations of clethodim (240 g / L emulsifiable concentrate) for 4 weeks, KJR0801 showed a reduction in efficacy at 432 g ai / hm. 2 The survival rate at the given dose was 47.4%, and the average inhibition rate (plant height) was 45.3%; Nanjing 9108 at 108 g ai / hm 2 All rice plants died after treatment with the specified dosage. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to clethodim.

[0062] like Figure 5 As shown, after spraying different concentrations of quizalofop-P-ethyl (8% emulsifiable concentrate) for 4 weeks, KJR0801 showed a growth rate of 144 g ai / hm. 2 At the given dose, the survival rate was 100%, and the average inhibition rate (plant height) was 14.3%; Nanjing 9108 at 144 g ai / hm 2 The survival rate under the given dosage treatment was 20%, and the average inhibition rate (plant height) was 46.6%. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to quizalofop-P-ethyl.

[0063] like Figure 6 As shown, after spraying different concentrations of quizalofop-P-ethyl (5% EC) for 4 weeks, KJR0801 showed the effect at 180g ai / hm2 At the given dose, the survival rate was 100%, and the average inhibition rate (plant height) was 2.3%; Nanjing 9108 at 45g ai / hm 2 All rice plants died after treatment with the specified dosage. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to quizalofop-P-ethyl.

[0064] like Figure 7 As shown, after spraying different concentrations of quizalofop-p-ethyl (7.5% water-in-oil emulsion) for 4 weeks, KJR0801 showed a growth rate of 360 g a.i. / hm. 2 At the specified dosage, the survival rate was 100%, and plant height was not inhibited; Nanjing 9108 at 360 g ai / hm 2 The survival rate under the given dosage treatment was 86.7%, and the average inhibition rate (plant height) was 21.9%. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to S. oxadiazon.

[0065] like Figure 8 As shown, after spraying different concentrations of high-efficiency flupyradifurone (108 g / L emulsifiable concentrate) for 4 weeks, KJR0801 showed a growth rate of 195 g / L. 2 At the specified dosage, the survival rate was 100%, and plant height was not inhibited; Nanjing 9108 showed a survival rate of 100% at 195 g ai / hm. 2 The survival rate under the given dosage treatment was 76.7%, and the average inhibition rate (plant height) was 36.8%. This indicates that the rice material obtained by this invention, containing the recombinant acetyl-CoA carboxylase encoding gene as shown in SEQ ID NO:2, significantly improved its tolerance to highly effective flupyradifurone.

[0066] In summary, rice varieties expressing the recombinant acetyl-CoA carboxylase provided by this invention exhibit more significant resistance to various ACCase inhibitor herbicides. In particular, they show significantly improved resistance to seven ACCase inhibitors with different chemical structures from three major classes: aroxyphenoxycarboxylic acid esters (APP) such as quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, and flupyrflupyr; cyclohexenones (CHD) such as clethodim and clodinafop-P-ethyl; and neophenylpyrazoline (DEN) such as clodinafop-P-ethyl. All of these inhibitors can achieve a tolerance level of at least 100% survival at four times the application rate.

Claims

1. A recombinant acetyl-CoA carboxylase encoding gene, characterized in that, The encoding gene, based on the wild-type rice ACC2 gene with SEQ ID NO:1, includes a mutation at position 5374 where A is changed to C, and the codon corresponding to this site changes from ATA to CTA, resulting in a change from isoleucine to leucine at position 1792 of the corresponding encoded protein; it also includes a mutation at position 5229 where G is changed to C, position 5335 where T is changed to A, position 5336 where C is changed to G, and position 5493 where C is changed to T.

2. The recombinant acetyl-CoA carboxylase encoding gene as described in claim 1, characterized in that, The nucleotide sequence of the recombinant acetyl-CoA carboxylase encoding gene is SEQ ID NO:

2.

3. A gene-editing combinatorial vector, characterized in that, The combined vector includes a first vector and a second vector. The first vector contains a Cas9 gene fused with a Gal4BD sequence, two target sequences derived from the ACC2 gene, and corresponding sgRNA sequences. The target sequences are SEQ ID NO:3 and SEQ ID NO:

4. The second vector contains a UAS sequence and an ACC2 Donor sequence; wherein the ACC2 Donor sequence includes a first homologous arm sequence, a substitution sequence, and a second homologous arm sequence; the ACC2 Donor sequence is SEQ ID NO:5, and the substitution sequence contains the mutation site of the encoding gene of claim 1.

4. The combined carrier as described in claim 3, characterized in that, The first homologous arm sequence is SEQ ID NO:6, the second homologous arm sequence is SEQ ID NO:7, and the substitution sequence is SEQ ID NO:

11.

5. The use of the encoding gene of claim 1 or 2, or the combined vector of claim 3 or 4, in the construction of rice resistant to ACCase inhibitor herbicides.

6. A method for preparing herbicide-resistant rice, characterized in that, By incorporating and expressing the recombinant acetyl-CoA carboxylase encoding gene as described in claim 1 or 2 into the rice genome, rice can acquire tolerance to ACCase inhibitor herbicides.

7. The method for preparing herbicide-resistant rice as described in claim 6, characterized in that, Includes the following steps: The combined vector as described in claim 3 or 4 was transformed into rice using Agrobacterium-mediated transformation. Rice plants tolerant to herbicides were screened by spraying with ACCase inhibitor herbicides or mixtures containing ACCase inhibitor herbicides.

8. A method for controlling grassy weeds in paddy fields, characterized in that, This includes sowing rice varieties or their derivatives prepared using the preparation method described in claim 6 or 7 in the field; During the rice seedling to flowering stage, spray with ACCase inhibitor herbicides or mixtures containing ACCase inhibitor herbicides to remove weeds that are sensitive to ACCase inhibitor herbicides.

9. A method for detecting the presence of herbicide-resistant rice DNA as described in claim 6 in a sample, characterized in that, include: (1) Use primer pairs ACC2F1-F1 and ACC2F1-R1 to perform nucleic acid amplification reaction on the DNA of the detection sample, wherein the nucleotide sequence of primer ACC2F1-F1 is SEQ ID NO:8 and the nucleotide sequence of ACC2F1-R1 is SEQ ID NO:9; (2) Sequencing analysis was performed using sequencing primers ACC2F1-F2, the nucleotide sequence of which is SEQ ID NO:10; The amplification product includes the nucleotide mutation site described in claim 1, indicating that the test sample contains the DNA of herbicide-resistant rice as described in claim 6.

Images