An acetyl-coa carboxylase mutant resistant to herbicides and use thereof

Abstract

This invention provides a mutant acetyl-CoA carboxylase (ACC) protein, nucleic acid, and their applications, specifically relating to the mutant acetyl-CoA carboxylase (ACC) protein, nucleic acid, and their application in plant breeding. Specifically, this invention provides a mutant acetyl-CoA carboxylase (ACC) protein, wherein the mutant acetyl-CoA carboxylase (ACC) protein, compared to the parental acetyl-CoA carboxylase (ACC) protein, has a mutation at amino acid position 1878 and / or 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1, or at amino acid position 1791 and / or 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3; plants with acetyl-CoA carboxylase (ACC) mutation exhibit high herbicide resistance and have very broad application prospects in the breeding of herbicide-resistant plants.

Description

[0001] This application claims priority to Chinese patent application CN202311718231.1, filed on December 14, 2023. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of biotechnology and crop genetics and breeding, and specifically relates to a mutant protein of acetyl-CoA carboxylase, a nucleic acid, and its method and application in improving plant resistance to herbicides. Background Technology

[0003] Rice (Oryza sativa) is consumed by two-thirds of the world's population and is a primary energy source in the diets of at least half of them. Rice is a low-cost food that is easy and quick to prepare and can be paired with a variety of dishes.

[0004] Herbicides are widely used to control weeds and other plants in crops. As a crucial component of modern agricultural production systems, herbicides are the most reliable and economical means of weed control in farmland. Since the introduction of 2,4-D in the 1940s, the herbicide industry has a history of over 60 years, and a large number of selective herbicides have been successfully developed. Research on ACC enzyme inhibitors began in the 1970s. ACC herbicides are classified into three types: cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs), and phenoxypyrazolines (DENs). ACC herbicides inhibit fatty acid synthesis in gramineous plants, exhibit high selectivity, are translocated within the plant, and can control annual or perennial gramineous weeds post-emergence. They possess advantages such as high efficiency, low toxicity, long application period, and safety for subsequent crops, thus holding an important position in the herbicide market.

[0005] Acetyl-CoA carboxylase (ACCase) is an important target of chemical herbicides and a biotinylate discovered in 1958. It catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in vivo, providing substrates for the synthesis of fatty acids and many secondary metabolites. It is a key or rate-limiting enzyme in fatty acid biosynthesis. This carboxylase involves a two-step reversible reaction: ATP-dependent carboxylation of the biotin group on the substrate domain via biotin-carboxylase activity, followed by carboxyltransferase transfer of the carboxyl group from biotin to the acetyl-CoA substrate. Acetyl-CoA carboxylase is a crucial enzyme in plant fatty acid biosynthesis, a process that occurs in chloroplasts and mitochondria. ACC also plays a role in the formation of long-chain fatty acids and flavonoids, as well as malonylation in the cytoplasm. Summary of the Invention

[0006] The purpose of this invention is to provide a mutant acetyl-CoA carboxylase (ACC) protein or polynucleotide that can confer herbicide resistance to plants and its application.

[0007] In this article, ACCase or ACC refers to acetyl-CoA carboxylase. Mutant acetyl-CoA carboxylase (ACC)

[0008] On one hand, the present invention provides a mutant acetyl-CoA carboxylase (ACC), wherein the mutant acetyl-CoA carboxylase (ACC) is mutated at amino acid position 1878 corresponding to the amino acid sequence shown in SEQ ID No. 1, or at amino acid position 1791 corresponding to the amino acid sequence shown in SEQ ID No. 3, compared with the amino acid sequence of the parent acetyl-CoA carboxylase (ACC).

[0009] In one embodiment, the mutated acetyl-CoA carboxylase (ACC) has a mutation at amino acid position 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1, or a mutation at amino acid position 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3, compared to the amino acid sequence of the parental acetyl-CoA carboxylase (ACC).

[0010] In one embodiment, the mutated acetyl-CoA carboxylase (ACC) is mutated at amino acids 1878 and 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1, compared to the parental acetyl-CoA carboxylase (ACC).

[0011] In one embodiment, the mutated acetyl-CoA carboxylase (ACC) is mutated at amino acids 1791 and 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3, compared to the parental acetyl-CoA carboxylase (ACC).

[0012] In one embodiment, the amino acid at position 1878 is mutated to a non-N amino acid, such as A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably S, G, or D.

[0013] In one embodiment, the amino acid at position 1791 is mutated to a non-N amino acid, such as A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably S, G, or D.

[0014] In one embodiment, the amino acid at position 1879 is mutated to a non-I amino acid, such as A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0015] In one embodiment, the amino acid at position 1792 is mutated to a non-I amino acid, such as A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0016] In a preferred embodiment, the 1878th amino acid is mutated to S, G, or D; and the 1879th amino acid is mutated to V.

[0017] In a preferred embodiment, the 1791st amino acid is mutated to S, G, or D; and the 1792nd amino acid is mutated to V.

[0018] In one embodiment, the mutated acetyl-CoA carboxylase (ACC) further includes other mutation sites, particularly herbicide-resistant mutation sites.

[0019] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from any plant.

[0020] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from monocotyledonous or dicotyledonous plants.

[0021] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from a monocotyledonous plant.

[0022] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of: grasses, legumes, chenopodiaceae, and cruciferous plants.

[0023] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of: Arabidopsis thaliana, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0024] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of Arabidopsis thaliana, rice, corn, wheat, and soybean.

[0025] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of rice, corn, and wheat.

[0026] In a preferred embodiment, the parental acetyl-CoA carboxylase (ACC) of the present invention is derived from the genus Oryza, particularly rice.

[0027] In a preferred embodiment, the parental acetyl-CoA carboxylase (ACC) of the present invention is derived from rice.

[0028] In a preferred embodiment, the parental acetyl-CoA carboxylase (ACC) of the present invention is derived from japonica or indica rice.

[0029] In one embodiment, the parental acetyl-CoA carboxylase (ACC) has ACC activity, and the amino acid sequence of the parental ACC has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequences shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13.

[0030] In a preferred embodiment, the amino acid sequence of the parent ACC has the sequence shown in SEQ ID No. 1.

[0031] In a preferred embodiment, the amino acid sequence of the parent ACC is shown in SEQ ID No. 1.

[0032] In a preferred embodiment, the amino acid sequence of the parent ACC has the sequence shown in SEQ ID No. 3.

[0033] In a preferred embodiment, the amino acid sequence of the parent ACC is shown in SEQ ID No. 3.

[0034] In a preferred embodiment, the amino acid sequence of the parent ACC has the sequence shown in SEQ ID No. 1 or SEQ ID No. 3.

[0035] In a preferred embodiment, the amino acid sequence of the parent ACC is shown in SEQ ID No. 1 or SEQ ID No. 3.

[0036] In a preferred embodiment, the amino acid sequence of the parent ACC is shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

[0037] In a preferred embodiment, the amino acid sequence of the parent ACC is shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 10 or SEQ ID No. 11.

[0038] In one embodiment, the amino acid sequence of the parent ACC is as shown in SEQ ID No. 1, and the mutant acetyl-CoA carboxylase (ACC) is mutated at amino acid position 1878 corresponding to the amino acid sequence shown in SEQ ID No. 1, or at amino acids positions 1878 and 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1.

[0039] In one embodiment, the amino acid sequence of the parent ACC is as shown in SEQ ID No. 3, and the mutant acetyl-CoA carboxylase (ACC) is mutated at amino acid positions 1791 and / or 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3, or at amino acid positions 1791 and 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3.

[0040] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from wheat, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 10. Preferably, the mutated acetyl-CoA carboxylase (ACC) has a mutation at amino acid positions 1768 and / or 1769 corresponding to the amino acid sequence shown in SEQ ID No. 10, compared to the amino acid sequence of the parental acetyl-CoA carboxylase (ACC). Preferably, the mutated amino acid at position 1768 is a non-N amino acid, for example, A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably, S, G, or D. The mutated amino acid at position 1769 is a non-I amino acid, for example, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0041] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from maize, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 11. Preferably, the mutant acetyl-CoA carboxylase (ACC) has a mutation at amino acid positions 1783 and / or 1784 corresponding to the amino acid sequence shown in SEQ ID No. 11, compared to the amino acid sequence of the parental acetyl-CoA carboxylase (ACC). Preferably, the mutated amino acid at position 1783 is a non-N amino acid, for example, A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably, S, G, or D. The mutated amino acid at position 1784 is a non-I amino acid, for example, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0042] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from soybean, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 12. Preferably, the mutated acetyl-CoA carboxylase (ACC) has a mutation at amino acid positions 1718 and / or 1719 corresponding to the amino acid sequence shown in SEQ ID No. 12, compared to the amino acid sequence of the parental acetyl-CoA carboxylase (ACC). Preferably, the mutated amino acid at position 1718 is a non-N amino acid, for example, A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably, S, G, or D. The mutated amino acid at position 1719 is a non-I or non-L amino acid, for example, A, V, G, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0043] In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from Arabidopsis thaliana, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 13. Preferably, the mutant acetyl-CoA carboxylase (ACC) has a mutation at amino acid positions 1713 and / or 1714 corresponding to the amino acid sequence shown in SEQ ID No. 13, compared to the amino acid sequence of the parental acetyl-CoA carboxylase (ACC). Preferably, the mutated amino acid at position 1713 is a non-N amino acid, for example, A, V, G, L, Q, F, W, Y, D, R, E, K, M, S, T, C, P, H, I; preferably, S, G, or D. The mutated amino acid at position 1714 is a non-I or non-L amino acid, for example, A, V, G, Q, F, W, Y, D, N, E, K, M, S, T, C, P, H, R; preferably, V.

[0044] In this invention, the sequences shown in SEQ ID No. 1 and SEQ ID No. 3 are both derived from acetyl-CoA carboxylase (ACC) of rice; wherein, the 1878th amino acid site of the rice ACC shown in SEQ ID No. 1 and the 1791st amino acid site of the rice ACC shown in SEQ ID No. 3 are conserved amino acid sites, and the 1879th amino acid site of the rice ACC shown in SEQ ID No. 1 and the 1792nd amino acid site of the rice ACC shown in SEQ ID No. 3 are conserved amino acid sites.

[0045] The present invention has found that mutations in the conserved sites of acetyl-CoA carboxylase shown in SEQ ID No. 1 or acetyl-CoA carboxylase shown in SEQ ID No. 3 in rice can improve the resistance of rice to ACCase inhibitor herbicides.

[0046] In this invention, ACC from different plant sources, such as ACC naturally occurring in different plants or ACC from different plant sources that have been artificially modified, can all be used as parent ACC. Those skilled in the art can obtain, through conventional technical knowledge, amino acid sites that are homologous or conserved to amino acids 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1 or amino acids 1791 and 1792 of the amino acid sequence shown in SEQ ID No. 3 from ACC from different sources.

[0047] For example, in one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from wheat, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 10; amino acids 1768 and 1769 of the amino acid sequence shown in SEQ ID No. 10 are conserved amino acid sites with amino acids 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1. In other embodiments, the parental acetyl-CoA carboxylase (ACC) is derived from maize, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 11; amino acids 1783 and 1784 of the amino acid sequence shown in SEQ ID No. 11 are conserved amino acid sites with amino acids 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1. In other embodiments, the parental acetyl-CoA carboxylase (ACC) is derived from soybean, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 12; amino acids 1718 and 1719 of the amino acid sequence shown in SEQ ID No. 12 are conserved amino acid sites with amino acids 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1. In other embodiments, the parental acetyl-CoA carboxylase (ACC) is derived from Arabidopsis thaliana, and the amino acid sequence of the parental ACC is shown in SEQ ID No. 13; amino acids 1713 and 1714 of the amino acid sequence shown in SEQ ID No. 13 are conserved amino acid sites with amino acids 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1.

[0048] On the other hand, the present invention provides a mutant acetyl-CoA carboxylase (ACC), wherein the mutant acetyl-CoA carboxylase (ACC) is selected from any group I-IV below:

[0049] I. A mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at amino acid position 1878; or a mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 3 at amino acid position 1791; or a mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 10 at amino acid position 1768; or a mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 11 at amino acid position 1783; or a mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 12 at amino acid position 1718; or a mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 13 at amino acid position 1713.

[0050] II. A mutant ACC obtained by mutation at amino acid positions 1878 and 1879 of the amino acid sequence shown in SEQ ID No. 1; or a mutant ACC obtained by mutation at amino acid positions 1791 and 1792 of the amino acid sequence shown in SEQ ID No. 3; or a mutant ACC obtained by mutation at amino acid positions 1768 and 1769 of the amino acid sequence shown in SEQ ID No. 10; or a mutant ACC obtained by mutation at amino acid positions 1783 and 1784 of the amino acid sequence shown in SEQ ID No. 11; or a mutant ACC obtained by mutation at amino acid positions 1718 and 1719 of the amino acid sequence shown in SEQ ID No. 12; or a mutant ACC obtained by mutation at amino acid positions 1713 and 1714 of the amino acid sequence shown in SEQ ID No. 13.

[0051] III. Compared with the mutant ACC described in I or II, it has the mutation site described in I or II; and compared with the mutant ACC described in I or II, it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity, and retains herbicide resistance activity;

[0052] IV. Compared with the mutant ACC described in I or II, it has the mutation site described in I or II; and compared with the mutant ACC described in I or II, it has a sequence with one or more amino acid substitutions, deletions, or additions, and retains herbicide resistance activity; the one or more amino acids include substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0053] In other embodiments, the mutant acetyl-CoA carboxylase of the present invention also includes other herbicide-resistant mutation sites.

[0054] Those skilled in the art will understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., replacing another nonpolar amino acid residue with a nonpolar amino acid residue, replacing another polar uncharged amino acid residue with a polar uncharged amino acid residue, replacing another basic amino acid residue with a basic amino acid residue, and replacing another acidic amino acid residue with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where an amino acid is replaced by another amino acid belonging to the same group, fall within the scope of this invention, provided that the substitution does not lead to the inactivation of the protein's biological activity. Therefore, the proteins of this invention can contain one or more conserved substitutions in their amino acid sequence, preferably generated by substitutions according to Table 1. Furthermore, this invention also covers proteins that also contain one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the proteins of this invention.

[0055] Table 1

[0056] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0057] Conserved amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing their biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in non-conserved regions of the ACC. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved alterations in conserved regions.

[0058] As is well known in the art, one or more amino acid residues can be altered (replaced, deleted, truncated, or inserted) from the N and / or C ends of a protein while retaining its functional activity. Therefore, proteins with altered N and / or C ends of the ACC protein while retaining their desired functional activity are also within the scope of this invention. These alterations can include those introduced by modern molecular methods such as PCR, which includes PCR amplification that alters or lengthens the protein-coding sequence by means of oligonucleotides containing amino acid-coding sequences used in the PCR amplification.

[0059] It should be recognized that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such operations are generally known in the art. For example, amino acid sequence variants of the ACC protein can be prepared by mutating the DNA. This can also be accomplished through other forms of mutagenesis and / or directed evolution, for example, using known mutagenesis, recombination, and / or shuffling methods, combined with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0060] Those skilled in the art will understand that these minor amino acid changes in the ACC protein of this invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, a smaller impact can be expected.

[0061] Those skilled in the art can identify the essential amino acids of the ACC protein using methods known in the art, such as localized mutagenesis, protein evolution, or bioinformatics analysis. The protein's catalytic domains, active sites, or other functional domains can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key site amino acids.

[0062] Fusion protein

[0063] On the other hand, the present invention provides a fusion protein comprising the mutant ACC protein of the present invention; further, the fusion protein further comprises: a tag peptide, a histidine tag, 6×His, or a plasmid guiding peptide, such as a peptide guided into the chloroplast, or a regulatory element, such as a promoter sequence, a terminator sequence, a leader sequence, a polyadenylated sequence, a marker gene, etc.

[0064] Polynucleotides

[0065] On the other hand, the present invention provides a polynucleotide that encodes the mutated ACC protein or an active fragment thereof.

[0066] In one embodiment, the polynucleotide is selected from the group consisting of genomic sequences, cDNA sequences, RNA sequences, or combinations thereof.

[0067] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0068] In one embodiment, the polynucleotide further comprises, flanking the ORF of the mutant protein, an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), nuclear localization signals (NLS), or combinations thereof.

[0069] In one embodiment, the polynucleotide also includes a promoter operatively linked to the ORF sequence of the mutant polypeptide.

[0070] In one embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue-specific promoters, inducible promoters, or strong promoters.

[0071] Nucleic acid constructs

[0072] On the other hand, the present invention provides a nucleic acid construct containing the aforementioned polynucleotide and a regulatory element operatively linked thereto.

[0073] In one embodiment, the regulatory element is selected from one or more of the following: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, and marker genes.

[0074] carrier

[0075] The present invention also provides a vector comprising a nucleic acid sequence encoding a mutant ACCase or fusion protein of the present invention. Preferably, the vector further comprises an expression regulatory element operatively linked to the aforementioned nucleic acid sequence.

[0076] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0077] In one implementation, the vector may be a vector for gene editing of the endogenous ACC gene in the host cell.

[0078] In one embodiment, the expression vector further contains at least one replication origin to enable self-replication.

[0079] In one embodiment, the vector may be a vector that is integrated into the genome when introduced into a host cell and replicates along with the chromosome into which it is integrated.

[0080] The vector can be of the following types: plasmid, virus, granule, bacteriophage, etc., which are well known to those skilled in the art.

[0081] Preferably, the carrier in this invention is a plasmid.

[0082] Gene editing vector system

[0083] On the other hand, the present invention provides a gene editing vector system comprising one or more vectors, wherein the one or more vectors contain at least a guide sequence targeting the parental ACC.

[0084] The guide sequence contains a portion of the parental ACC nucleotide sequence, preferably at least 15 bp of ACC nucleotide sequence, and more preferably at least 20 bp of ACC nucleotide sequence. In one embodiment, the editing vector further includes a gene-editing enzyme. The gene-editing enzyme includes nucleases from CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease) editing tools.

[0085] In one embodiment, the gene editing vector system of the present invention can generate the above-mentioned mutated ACC in plants through gene editing.

[0086] Preferably, the gene editing enzyme is a Cas protein, also known as a CRISPR enzyme or Cas effector protein, and its types include, but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein.

[0087] Preferably, the Cas protein is operatively connected to the first regulatory element.

[0088] In one embodiment, the gene-editing enzyme is the Cas9 protein, and the vector further includes a scaffold sequence that specifically binds to the Cas9 protein. The scaffold sequence and the guide sequence are operatively linked to form a guide RNA (gRNA). Preferably, the gRNA is operatively linked to a second regulatory element.

[0089] In other embodiments, the gene-editing enzyme is a Cas12 protein, such as Cas12a, Cas12b, or Cas12i, and the vector further includes a direct repeat sequence that specifically binds to the Cas12 protein. The direct repeat sequence and the guide sequence are operatively linked to form a guide RNA (gRNA). Preferably, the direct repeat sequence is as shown in SEQ ID No. 8, and the guide sequence is as shown in SEQ ID No. 9; preferably, the gRNA is operatively linked to a second regulatory element.

[0090] The aforementioned regulatory elements include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and polyU sequences).

[0091] Preferably, the editing vector system further includes a base editing element selected from adenine deaminase and / or cytosine deaminase.

[0092] Preferably, the base editing element is adenine deaminase (also known as adenosine deaminase); more preferably, the base editing element is adenine deaminase TadA8e; even more preferably, the base editing element is adenine deaminase TadA8e, the amino acid sequence of which is shown in SEQ ID No. 6.

[0093] In one embodiment, the editing vector further includes resistance genes for screening purposes, said resistance genes including hyg, bar, kana, rif, spec, and amp, which are well known to those skilled in the art.

[0094] Preferably, the Cas protein is selected from dCas12i3 or other Cas12 proteins with inactivated nuclease activity. Here, "d" indicates a Cas protein with inactivated nuclease activity.

[0095] Preferably, the Cas protein is dCas12i3, which is an E844A mutated Cas12i3 protein; the amino acid sequence of the wild-type Cas12i3 is shown in SEQ ID No. 7, and the amino acid sequence of the dCas12i3 is mutated from amino acid E to A at position 844 relative to the amino acid sequence shown in SEQ ID No. 7.

[0096] host cells

[0097] On the other hand, the present invention provides a host cell containing one or more of the following: the mutated acetyl-CoA carboxylase (ACC), the gene encoding the mutated acetyl-CoA carboxylase (ACC), the fusion protein, the vector, and the nucleic acid construct; or, the host cell genome integrates the aforementioned polynucleotide.

[0098] In one embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0099] In one embodiment, the host cell is a plant cell, and the plant includes angiosperms and gymnosperms.

[0100] In one embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0101] In one embodiment, the plants include herbaceous plants and woody plants.

[0102] In one embodiment, the plants include Arabidopsis thaliana, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0103] Herbicide-resistant plants

[0104] On the other hand, the present invention provides a herbicide-resistant plant containing one or more of the following: the mutated acetyl-CoA carboxylase (ACC), the gene encoding the mutated acetyl-CoA carboxylase (ACC), the fusion protein, the polynucleotide, the vector, the nucleic acid construct, or the host cell; or the polynucleotide is integrated into the plant genome.

[0105] The herbicide resistance is preferably ACCase inhibitor, ACCase inhibitor herbicide, or ACCase-inhibiting herbicide.

[0106] Methods for preparing mutant peptides

[0107] On the other hand, the present invention provides a method for preparing the mutated ACC peptide or its active fragment, the method comprising the steps of:

[0108] (a) Under suitable expression conditions, host cells containing the mutated ACC peptide are cultured to express the mutated ACC peptide.

[0109] Preferably, the method further includes (b) the step of isolating the mutant ACC peptide.

[0110] Methods for obtaining herbicide-resistant plants

[0111] On the other hand, the present invention provides a herbicide-resistant plant cell, plant seed, plant tissue, plant part, or plant, wherein the plant cell, plant tissue, plant seed, plant part, or plant contains the mutated ACC polypeptide or its polynucleotide sequence.

[0112] On the other hand, the present invention provides a method for obtaining or preparing plant cells, plant seeds, plant tissues, plant parts or plants with herbicide resistance, the method comprising introducing the above-mentioned mutated ACC polypeptide or its polynucleotide sequence into the plant cells, plant seeds, plant tissues, plant parts or plants.

[0113] Furthermore, the method also includes the step of developing or regenerating the aforementioned plant cells, plant seeds, plant tissues, or plant parts into plants.

[0114] In one embodiment, the introduction of the ACC mutant polypeptide according to the present invention includes the step of expressing the ACC mutant polypeptide in plant cells, plant seeds, plant tissues, plant parts or plants, for example, by expressing the mutant polypeptide through an expression vector, or by integrating the mutant polypeptide into the plant genome for expression.

[0115] In another preferred embodiment, the above method includes the following steps:

[0116] (1) Provide an Agrobacterium carrying an expression vector, wherein the expression vector contains the DNA coding sequence of the mutated ACC polypeptide or its active fragment;

[0117] (2) Contacting plant cells, plant tissues, or plant parts with Agrobacterium in step (1) thereby transferring the DNA coding sequence of the mutated ACC polypeptide or its active fragment into the plant cells and integrating it into the chromosomes of the plant cells; and

[0118] (3) Select plant cells that have been transformed into the DNA coding sequence of the mutated ACC polypeptide or its active fragment.

[0119] In one embodiment, the introduction of the ACC mutant polypeptide includes the step of mutating the endogenous ACC of the plant to introduce the mutant polypeptide; for example, the mutant polypeptide is introduced by means of mutagenesis, fragment substitution, homologous recombination or gene editing; preferably, the mutant polypeptide can be introduced by gene editing.

[0120] In one embodiment, the gene encoding the endogenous ACC of a plant can be mutated to produce the aforementioned mutation at amino acid positions 1878 and / or 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1.

[0121] In one embodiment, the gene encoding the endogenous ACC of a plant can be mutated to produce the aforementioned mutation at amino acid positions 1791 and / or 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3.

[0122] In one embodiment, the gene encoding the plant's endogenous ACC can be mutated to produce the aforementioned mutation at amino acid positions 1768 and / or 1769 corresponding to the amino acid sequence shown in SEQ ID No. 10.

[0123] In one embodiment, the gene encoding the endogenous ACC of a plant can be mutated to produce the aforementioned mutation at amino acid positions 1783 and / or 1784 corresponding to the amino acid sequence shown in SEQ ID No. 11.

[0124] In one embodiment, the gene encoding the endogenous ACC of a plant can be mutated to produce the aforementioned mutation at amino acid positions 1718 and / or 1719 corresponding to the amino acid sequence shown in SEQ ID No. 12.

[0125] In one embodiment, the gene encoding the endogenous ACC of a plant can be mutated to produce the aforementioned mutation at amino acid positions 1713 and / or 1714 corresponding to the amino acid sequence shown in SEQ ID No. 13.

[0126] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1878 and / or 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1.

[0127] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1791 and / or 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3.

[0128] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1768 and / or 1769 corresponding to the amino acid sequence shown in SEQ ID No. 10.

[0129] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1783 and / or 1784 corresponding to the amino acid sequence shown in SEQ ID No. 11.

[0130] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1718 and / or 1719 corresponding to the amino acid sequence shown in SEQ ID No. 12.

[0131] In another preferred embodiment, the method includes the step of mutating the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues, or plant parts at amino acid positions 1713 and / or 1714 corresponding to the amino acid sequence shown in SEQ ID No. 13.

[0132] In another preferred embodiment, the method includes the following steps:

[0133] (1) Introduce the aforementioned gene editing vector system into plant cells, plant seeds, plant tissues, and plant parts;

[0134] (2) The step of applying a gene editing tool to its endogenous ACC and causing a mutation at amino acid position 1878 and / or 1879 corresponding to the amino acid sequence shown in SEQ ID No. 1.

[0135] In another preferred embodiment, the method includes the following steps:

[0136] (1) Introduce the aforementioned gene editing vector system into plant cells, plant seeds, plant tissues, and plant parts;

[0137] (2) The step of applying a gene editing tool to its endogenous ACC and causing a mutation at amino acid position 1791 and / or 1792 corresponding to the amino acid sequence shown in SEQ ID No. 3.

[0138] Furthermore, the above method also includes the step of screening for mutated plant cells, plant tissues, plant parts, and optionally, isolating the gene-editing tool.

[0139] In another preferred embodiment, the gene editing tools include CRISPR, TALEN, and ZFN.

[0140] In another preferred embodiment, the plants include angiosperms and gymnosperms.

[0141] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0142] In another preferred embodiment, the plants include herbaceous plants and woody plants.

[0143] In another preferred embodiment, the plants include Arabidopsis thaliana, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0144] The herbicide resistance is preferably ACCase inhibitor, ACCase inhibitor herbicide, or ACCase-inhibiting herbicide.

[0145] On the other hand, the present invention also provides a method for preparing hybrid plants, the method comprising the step of hybridizing the herbicide-resistant plants obtained by the above-described method with other plants.

[0146] On the other hand, the present invention also provides hybrid plants obtained using the above-described method for preparing hybrid plants.

[0147] Methods of controlling weeds

[0148] On the other hand, the present invention also provides a method for controlling the growth of weeds near plants, comprising:

[0149] a) Provide the above-mentioned herbicide-resistant plants or the above-mentioned hybrid plants;

[0150] b) Apply an effective amount of herbicide to the plant and the weeds nearby to control the weeds near the plant.

[0151] The preferred plant is rice.

[0152] In one embodiment, the herbicide is an ACCase inhibitor or an ACCase inhibitor class herbicide, wherein the herbicide is one or more of cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs), or phenylpyrazoline (DEN) herbicides.

[0153] In one embodiment, the herbicide includes cyclohexanediones (DIMs) such as clethodim and ketadiazon, aryloxyphenoxypropionates (FOPs) such as haloxyfop-methyl and quizalofop-p-ethyl, and phenoxypyrazoline (DEN) such as clodinafop-propargyl.

[0154] Preferably, the herbicide comprises one or more of the following: quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, butylbenzyl, clethodim, cyclohexyl, thiamethoxam, pyranoben, styraclostrobin, benzoyl propargite, quizalofop-P-ethyl, clodinafop-P-ethyl, chlorpyrifos, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, isofen, cyclohexyl, haloxyfop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, trifluorophenoxypropionic acid, clodinafop-P-ethyl, quizalofop-P-ethyl, butyryl clethodim, or their salts or esters.

[0155] Preferably, the herbicide is one or any combination of quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, haloxyfop-R-methyl, and clodinafop-P-ethyl.

[0156] Preferably, the herbicide is chlorpyrifos.

[0157] In one embodiment, the herbicide is an ACCase inhibitor or an ACCase inhibitor class of herbicides, comprising one or more of the following: aryloxyphenoxypropanoates (APP), oxime ethers (CHD), aryloxyphenylcyclohexanediones (APCHD), and triketones (CTR).

[0158] use

[0159] On the other hand, the present invention provides the use of the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the polynucleotide, the vector, the nucleic acid construct, or the host cell in reagents or kits for preparing herbicide-resistant plants.

[0160] On the other hand, the present invention provides the use of the gene editing vector system in reagents or kits for preparing herbicide-resistant plants.

[0161] On the other hand, the present invention provides the use of the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the polynucleotide, the vector, the nucleic acid construct, or the host cell in weed control.

[0162] On the other hand, the present invention provides the use of the gene editing vector system in weed control.

[0163] On the other hand, the present invention provides the use of the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the polynucleotide, the vector, the nucleic acid construct, or the host cell in the preparation of herbicide-resistant plants.

[0164] On the other hand, the present invention provides the use of the gene editing vector system in the preparation of herbicide-resistant plants.

[0165] herbicide

[0166] In one embodiment, the herbicide of the present invention is an ACCase-inhibiting herbicide, which includes, but is not limited to, one or more of cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs), and phenoxypyrazoline (DEN) herbicides. The cyclohexanediones (DIMs) class of herbicides include clethodim, haloxyfop-methyl, butylbenzyl, clethodim, thiamethoxam, pyranoben, and cyclobenzyl; the aryloxyphenoxypropionates (FOPs) class of herbicides include quizalofop-p-ethyl, haloxyfop-methyl, quizalofop-p-ethyl, quizalofop-p-ethyl, cyhalofop-butyl, clodinafop-propargyl, quizalofop-p-ethyl, quizalofop-p-ethyl, oxadiazon, and oxadiazon; the phenoxypyrazoline (DEN) class of herbicides includes clodinafop-propargyl.

[0167] In one embodiment, the herbicide of the present invention is an ACCase-inhibiting herbicide, which includes, but is not limited to, one or more of the following: aryloxyphenoxypropanoates (APP), oxime ethers (CHD), aryloxyphenylcyclohexanediones (APCHD), and triketones (CTR).

[0168] Preferably, the herbicides of the present invention include, but are not limited to, clethodim, haloxyfop-R-methyl, quizalofop-P-ethyl, clethodim, cyclohexyl ketone, thiamethoxam, pyranofop-P-ethyl, styraclostrobin, benzoyl ketone, quizalofop-P-ethyl, quizalofop-P-ethyl, chlorpyrifos, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, isofen, cyclohexyl ketone, haloxyfop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, quizalofop-P-ethyl, trifluorophenoxypropionic acid, clodinafop-P-ethyl, quizalofop-P-ethyl, butyryl clethodim, or one or more of their salts or esters.

[0169] In one embodiment, the herbicide includes cyclohexanediones (DIMs) such as clethodim and ketadiazon, aryloxyphenoxypropionates (FOPs) such as haloxyfop-methyl and quizalofop-p-ethyl, and phenoxypyrazoline (DEN) such as clodinafop-propargyl.

[0170] Preferably, the herbicide is one or more of the following: quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, haloxyfop-R-methyl, and clodinafop-P-ethyl.

[0171] Preferably, the herbicide is haloxyfop-R-methyl.

[0172] General definition

[0173] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0174] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0175] The term "homology" or "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Therefore, the compositions and methods of the present invention also comprise homologs of the nucleotide and polypeptide sequences of the present invention. Homology can be calculated using known methods including, but not limited to, the following: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.), Stockton. Stockton Press, New York (1991).

[0176] The specific amino acid positions (numbers) within the protein described in this invention are determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID No. 1 or SEQ ID No. 3. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, with the sequence considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4. The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10.0, gap extension penalty: 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID No. 1 or SEQ ID No. 3.

[0177] The term "encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes that have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological characteristics they produce. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein.

[0178] The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

[0179] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0180] The term "mutant protein" or "mutant protein" refers to a protein that has one or more amino acid residues substituted, inserted, deleted, and / or added compared to the amino acid sequence of the parent protein.

[0181] In this invention, amino acid residues can be represented by a single letter or a three-letter symbol, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0182] The term "AxxB" indicates that amino acid A at position xx is replaced by amino acid B. For example, N1878S indicates that N at position 1878 is replaced by S. For double or multiple mutations, the mutations are separated by " / ". For example, N1878S / I1879V indicates that, relative to the amino acid sequence of SEQ ID No. 1, N at position 1878 is replaced by S and I at position 1879 is replaced by V.

[0183] The term "regulatory element," as used herein, is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), for detailed description in Goeddel, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of that nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a time-dependent manner (e.g., cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0184] As used herein, the term "promoter" has the meaning known to those skilled in the art, referring to a non-coding nucleotide sequence located upstream of a gene that initiates the expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to that promoter.

[0185] A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein to allow it to be transported to the cell nucleus via nuclear transport; that is, a protein with an NLS is transported to the cell nucleus. Typically, an NLS contains positively charged Lys or Arg residues exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from the following: SV40 large T antigen, EGL-13, c-Myc, and TUS protein.

[0186] As used herein, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0187] The term "vector" refers to an element that allows the vector to integrate into the host cell's genome or to replicate autonomously within the cell independently of the genome. The vector may contain any element that guarantees self-replication. It typically carries a gene that is not part of the cell's central metabolism and is usually in the form of double-stranded DNA. The choice of vector generally depends on its compatibility with the host cell to which it is to be introduced. If a vector is used, the choice of vector depends on methods well-known to those skilled in the art for transforming host cells. For example, plasmid vectors may be used.

[0188] The term "ACC inhibitor herbicide" refers to a class of herbicides that inhibit fatty acid synthesis in grasses, exhibit high selectivity, are translocated within the plant, and can control annual or perennial grass weeds post-emergence. They possess advantages such as high efficiency, low toxicity, long application period, and safety for subsequent crops, thus occupying an important position among herbicides.

[0189] ACC inhibitor herbicides can be divided into three categories: cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs), and phenoxypyrazolines (DENs). Cyclohexanediones (DIMs) include clethodim, haloxyfop-methyl, butyrazanol, clethodim, thiamethoxam, pyrazosulfuron, and cyclohexane. Aryloxyphenoxypropionates (FOPs) include quizalofop-p-ethyl, haloxyfop-methyl, quizalofop-p-ethyl, cyhalofop-butyl, clodinafop-propargyl, quizalofop-p-ethyl, quizalofop-p-ethyl, oxadiazon, and oxychlorpyrifos. Phenylpyrazoline (DENs) include clodinafop-propargyl.

[0190] In other classification methods, ACC inhibitor herbicides can be further divided into four types: aryloxyphenoxypropanoates (APP), oxime ethers (Cyclohexanedioneoximes, CHD), aryloxyphenylcyclohexanediones (APCHD), and triketones (Cyclict riketones, CTR), as shown below.

[0191]

[0192] The herbicides include, but are not limited to: clethodim, haloxyfop-R-methyl, quizalofop-P-ethyl, butaniloxane, clethodim, cyclohexyl ketone, thiamethoxam, clethodim, pyranolol, styraclostrobin, benzoyl haloxyfop-R-methyl, clodinafop-R-methyl, chlorpyrifos, quizalofop-P-methyl, quizalofop-P-methyl, pyranolol-P-methyl, quizalofop-P-methyl, isofen, cyclohexyl ketone, haloxyfop-R-methyl, quizalofop-P-methyl, quizalofop-P-methyl, quizalofop-P-methyl, quizalofop-P-methyl, quizalofop-P-methyl, trifluorophenoxypropionic acid, clodinafop-P-methyl, quizalofop-P-methyl, butaniloxane.

[0193] "Herbicide resistance" or "herbicide tolerance" refers to the genetic ability of a plant to survive and reproduce after exposure to a dose of a herbicide typically lethal to the wild type. In plants, resistance can be naturally occurring or induced by techniques such as genetic engineering or selection of variants produced by tissue culture or mutagenesis. Unless otherwise stated, herbicide "resistance" is heritable and allows a plant to grow and reproduce in the presence of a herbicide treatment typically herbicide-effective against a given plant, as suggested in the current version of the herbicide manual at the time of filing of this invention. As will be appreciated by those skilled in the art, a plant may still be considered "resistant" even if a degree of plant damage due to herbicide exposure is evident. As used herein, the terms "tolerance" or "resistance" include "resistant" or "tolerant" plants as defined herein, and the improved ability of a particular plant to tolerate various degrees of herbicide-induced damage, typically ethyl, in wild-type plants of the same genotype, at the same herbicide dose.

[0194] In one embodiment, the mutant peptide, compared with the parent peptide, has a tolerance concentration to the maximum ACC-inhibiting herbicide that is increased by at least 1 time, for example, by at least 1.5 times, preferably by at least 2 times, preferably by at least 3 times, preferably by at least 4 times, preferably by at least 5 times, preferably by at least 6 times, and preferably by at least 10 times.

[0195] In one embodiment, the plant containing the mutant peptide can tolerate at least 1, 2, 3, 4, 5, 6, or 10 times the recommended concentration of ACC inhibitory herbicide.

[0196] The terms "parental ACC polypeptide" and "parental ACC polypeptide" refer to the polypeptide from which the ACC mutant polypeptide originates. In a preferred embodiment, the parental ACC polypeptide is a nucleic acid molecule or protein (polypeptide) that can be found in nature. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and its amino acid sequence can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type ACC polypeptide is, for example, shown in SEQ ID No. 1 or SEQ ID No. 3. In some embodiments, the parental ACC polypeptide can be a polypeptide that has undergone one or more amino acid residue modifications to the wild-type ACC polypeptide without affecting its enzymatic activity.

[0197] The terms “mutated ACC protein”, “mutated ACC protein”, “mutated ACC”, “mutated ACC enzyme”, “mutated protein”, “mutated polypeptide”, “polypeptide of the present invention”, and “protein of the present invention” are used interchangeably.

[0198] The term “host organism” should be understood as any single-celled or multicellular organism into which a mutated ACC protein-encoded nucleic acid can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0199] The term "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, particularly monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, longleaf lettuce), bok choy, taro, cucurbits (e.g., melons, watermelons, crenshaw, cantaloupes, Roman melons), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, headless cabbage, Chinese cabbage, baby bok choy), artichokes, carrots, napa cabbage, okra, onions, celery, parsley, chickpeas, parsnip, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, baby zucchini, squash, pumpkin), radishes, and dried heads. Onions, rutabagas, purple eggplant (also known as eggplant), ginseng, lettuce, scallions, chicory, garlic, spinach, green onions, squash, leafy greens, beets (sugar beets and fodder beets), sweet potatoes, romaine lettuce, wasabi, tomatoes, turnips, and spices; fruits and / or vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus fruits, blueberries, boysenberries. Raspberries, cranberries, currants, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pears, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silvergrass, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas) Beans, soybeans), oil plants (rapeseed, mustard, olive, sunflower, coconut, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugarcane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0200] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant masses, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0201] The term “plant cell” should be understood as any cell that is derived from or found in a plant and is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or seeds.

[0202] The term "gene editing" technology includes CRISPR, TALEN, and ZFN technologies. CRISPR technology refers to clustered, regularly interspaced short palindromic repeats derived from the microbial immune system. Gene editing tools include guide RNA and Cas proteins (such as Cas9, Cpf1, and Cas12i). The gene editing tools referred to in TALEN technology are restriction enzymes capable of cleaving specific DNA sequences, comprising a TAL effector DNA-binding domain and a DNA-cleaving domain. The gene editing tools referred to in ZFN technology are also restriction enzymes capable of cleaving specific DNA sequences, comprising a zinc finger DNA-binding domain and a DNA-cleaving domain. Those skilled in the art know that by constructing nucleotides and other regulatory elements encoding gene editing tools into suitable vectors and then transforming them into cells, intracellular genome editing can be achieved, including gene knockout, insertion, and base editing.

[0203] As used herein, the term "gene editing enzyme" refers to a nuclease suitable for editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease). Preferably, the gene editing enzyme is a CRISPR enzyme, also known as a Cas protein, which includes, but is not limited to, Cas9, Cas12, Cas13, Cas14, Csm1, and FDK1 proteins. The Cas protein refers to a protein family that can have different structures depending on its origin, such as SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. It can also be classified according to structural features (such as domains), such as the Cas12 family, which includes Cas12a (also known as Cpf1), Cas12b, Cas12c, and Cas12i. The Cas protein can be double-stranded, single-stranded, or have no cleavage activity. The Cas protein of this invention can be wild-type or a mutant thereof. The mutation types of the mutant include amino acid substitution, substitution, or deletion. The mutant may or may not alter the cleavage activity of the Cas protein. Preferably, the Cas protein of this invention has only single-stranded cleavage activity or no cleavage activity, and is a mutant of the wild-type Cas protein. Preferably, the Cas protein of this invention is Cas9, Cas12, Cas13, or Cas14 with single-stranded cleavage activity. In a preferred embodiment, the Cas12 protein of the present invention includes Cas12i3 and dCas12i3, wherein "d" indicates a Cas protein with inactivated nuclease activity. As is known to those skilled in the art, various Cas proteins with nucleic acid cleavage activity have been reported in the prior art, and these known proteins or their modified variants can achieve the functions of the present invention; they are included in the scope of protection herein by reference.

[0204] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments (e.g., amino acid fragments containing the mutant sites of the present invention) from the N and / or C-terminus of a mutant ACC protein that have deleted one or more amino acid residues while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant ACC protein of the present invention that retains the biological activity of the mutant ACC protein of the present invention. For example, a bioactive fragment of a mutant ACC protein may be a portion of the protein whose N and / or C-terminus has been deleted one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues, but which still retains the biological activity of the full-length protein.

[0205] Furthermore, the mutant proteins of this invention can be modified. Modifications (generally without altering the primary structure) include chemically derived forms of the mutant protein, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during or after the synthesis and processing of the mutant protein, or in further processing steps. This modification can be accomplished by exposing the mutant protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Mutant proteins modified to improve their resistance to proteolysis or optimize their solubility are also included.

[0206] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0207] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed nucleotide sequences, especially the open reading frame sequences, according to this invention, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. The obtained nucleotide sequences can be cloned into vectors, transformed into cells, and then isolated in large quantities from the proliferated host cells using conventional methods. The mutation sites of this invention can also be introduced artificially.

[0208] One or more copies of the polynucleotide of the present invention can be inserted into host cells to increase the yield of gene products. The number of polynucleotide copies can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide. In the latter case, cells containing an amplified copy of the selectable marker gene and the resulting additional copy of the polynucleotide can be selected by culturing the cells artificially in the presence of a suitable selectable agent.

[0209] Methods well known to those skilled in the art can be used to construct vectors containing an ACC mutant polypeptide encoding a DNA sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the vector to guide mRNA synthesis. The vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0210] The vectors applicable in this invention include plasmids that are available from commercial sources, such as, but not limited to: pBR322 (ATCC37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (PromegaBiotec, Madison, WI, USA), pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript IIKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), etc.

[0211] This invention also provides host cells comprising the nucleic acid sequence encoding the ACC mutant polypeptide of this invention, a nucleic acid construct, or an expression vector. Introducing a vector encoding this invention into a host cell allows the vector to exist as part of a chromosomal integrase or as a self-replicating extrachromosomal vector as previously described, or the vector can perform gene editing on the endogenous ACC gene within the host cell. The host cell can be any host cell familiar to those skilled in the art, including prokaryotic and eukaryotic cells.

[0212] The nucleic acid sequences, nucleic acid constructs, or expression vectors of the present invention can be introduced into host cells through a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid-mediated gene delivery, as well as calcium phosphate transfection, DEAE-glucan-mediated transfection, lipid transfection, or electroporation.

[0213] In the production method of the present invention, the cells are cultured on a nutrient medium suitable for the production of the polypeptide using methods well known in the art. If the polypeptide is secreted into the nutrient medium, it can be directly recovered from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

[0214] As used herein, the terms “guide RNA,” “mature crRNA,” “guide sequence,” and “gRNA” are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, a guide RNA may comprise a direct repeat (DR) sequence and a guide sequence, or consist essentially of or comprise of a direct repeat sequence and a guide sequence (also referred to as a spacer in the context of an endogenous CRISPR system).

[0215] In some cases, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, the complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimal alignment is achieved. Determining the optimal alignment is within the capabilities of a person skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan. The main advantages of this invention are:

[0216] 1. This invention screened out a group of mutant ACC proteins.

[0217] 2. Plants containing the mutant ACC protein of this invention have significantly enhanced herbicide resistance compared to their parent plants. Attached Figure Description

[0218] Figure 1 .iABE base editor.

[0219] Figure 2 Part A is the target sequence of the iABE base editor, and Part B is the codons for the 1878th and 1879th amino acids of the first amino acid sequence encoded by the japonica rice ACC protein, as well as the replacement codons after base editing.

[0220] Figure 3 A comparison of the amino acid sequences encoded by the ACCase gene in japonica rice and indica rice.

[0221] Figure 4 Wild-type japonica rice plants (WT) and edited plants (ACC) N1878D ACC N1878S ACC N1878G ACC I1879V ACC N1878S / I1879V Herbicide resistance.

[0222] Figure 5 Wild-type japonica rice plants (WT) and edited plants (ACC) I1879V ACC N1878D / I1879V ACC N1878S / I1879V ACC N1878G / I1879V Herbicide resistance.

[0223] Figure 6 Wild-type japonica rice plants (WT) and edited plants (ACC) N1878S / I1879V Comparison of effective panicle number, panicle length, single panicle grain filling rate, and thousand-grain weight.

[0224] Figure 7 Wild-type indica rice plants (Indica-WT) and edited plants (ACC) N1791S / I1792V Resistance to herbicides quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, clethodim, and clodinafop-P-ethyl.

[0225] Figure 8 A comparative diagram of the amino acid sequences encoded by the ACCase gene in rice, wheat, corn, soybean, and Arabidopsis thaliana.

[0226] Sequence information

[0227] Detailed Implementation

[0228] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0229] Example 1: Construction of gene editing vectors and screening of edited plants

[0230] 1. Constructing an iABE base editor targeting the endogenous ACC gene in rice.

[0231] The ABE base editor can achieve A / T->G / C base conversion within a certain sequence window. This invention uses the iABE(dCas12i3-ABE) base editor as a vector to design gRNA in the rice endogenous ACCase gene, clone it into the iABE vector, and form a base editor that targets the rice endogenous ACCase gene.

[0232] Different rice varieties may have different amino acid sequences encoded by the ACCase gene due to variations in gene annotations across different gene databases. Specifically:

[0233] There are two amino acid sequences encoded by the ACCase gene in japonica rice (due to different annotations in different gene databases): In the Rice Genome Annotation Project database, the Locusidentifier of the ACCase gene in japonica rice is LOC_Os05g22940, and its encoded amino acid sequence is shown in SEQ ID No. 1, while its nucleotide sequence (CDS sequence) is shown in SEQ ID No. 2; In the Genebank database, the ACCase gene in japonica rice is defined as LOC4338322, and its encoded amino acid sequence is shown in SEQ ID No. 3, while its nucleotide sequence (CDS sequence) is shown in SEQ ID No. 4.

[0234] The Genebank number for the indica rice ACCase gene is EAY97401.1, and its encoded amino acid sequence is exactly the same as the amino acid sequence of the japonica rice ACCase gene in the Genebank database, as shown in SEQ ID No. 3. The nucleotide sequence (genomic sequence) is shown in SEQ ID No. 5.

[0235] iABE base editor, such as Figure 1As shown, pU6 is the U6 promoter, pUBI is the UBI promoter, tNOS is the terminator, and NLS is the nuclear localization signal; ABE is adenosine deaminase, and its amino acid sequence is shown in SEQ ID No. 6; dCas12i3 is a Cas12i3 mutant protein, Cas12i3 is Cas12f.4 in CN111757889B, and its amino acid sequence is shown in SEQ ID No. 7; dCas12i3 is the E844A mutant Cas12i3, which is inactivated by nuclease activity; gRNA includes a direct repeat sequence (DR) and a guide sequence, the direct repeat sequence is agagaaugugugcauagucacac (SEQ ID No. 8), the guide sequence is gtgtggagaatatacatgga (SEQ ID No. 9), and PAM is TTG.

[0236] In other embodiments, those skilled in the art may also choose other types of base editors, such as combining adenosine deaminases from different sources with different Cas proteins to achieve the effect of base editing.

[0237] The first amino acid sequence encoded by the ACCase gene in japonica rice is as follows (SEQ ID No. 1):

[0238] mpmrpwefiilgrtapisppplgisafsrplrlrlrlrlaavvdaariqtpprvafsaffffffffseglsregtaarlpaliyrvpfipcllqlqira

[0239] phadprpagfscahhflalaqglsssllsgtishiwgfifslygtthlrnraillfglagiidlpndaasevdishgsedprgptvpgsyqmng

[0240] iinethngrhasvskvvefctalggktpihsvlvanngmaaakfmrsvrtwandtfgsekaiqliamatpedlrinaehiriadqfvevpg

[0241] gtnnnnyanvqliveiaertgvsavwpgwghasenpelpdaltakgivflgppassmhalgdkvgsaliaqaagvptlawsgshvev

[0242] pleccldsipdemyrkacvttteeavascqvvgypamikaswggggkgirkvhnddevrtlfkqvqgevpgspifimrlaaqsrhlev

[0243] qllcdqygnvaalhsrdcsvqrrhqkiieegpvtvapretvkeleqaarrlakavgyvgaatveylysmetgeyyflelnprlqvehpvte

[0244] wiaevnlpaaqvavgmgiplwqipeirrfygmnhgggydlwrktaalatpfnfdevdskwpkghcvavritsedpddgfkptggkv

[0245] keisfkskpnvwayfsvksgggihefadsqfghvfaygttrsaaittmalalkevqirgeihsnvdytvdllnasdfrenkihtgwldtria

[0246] mrvqaerppwyisvvggalyktvtantatvsdyvgyltkgqippkhislvyttvalnidgkkytidtvrsghgsyrlrmngstvdanvqi

[0247] lcdggllmqldgnshviyaeeeasgtrllidgktcmlqndhdpskllaetpckllrflvadgahvdadvpyaevevmkmcmpllspas

[0248] gvihvvmsegqamqagdliarldlddpsavkraepfedtfpqmglpiaasgqvhklcaaslnacrmilagyehdidkvvpelvycldt

[0249] pelpflqweelmsvlatrlprnlkselegkyeeykvkfdsgiindfpanmlrviieenlacgsekekatnerlveplmsllksyeggresh

[0250] ahfvvkslfeeylyveelfsdgiqsdvierlrlqhskdlqkvvdivlshqsvrnktklilklmeslvypnpaayrdqlirfsslnhkayykla

[0251] lkaselleqtklselrariarslselemfteeskglsmhkreiaikesmedlvtaplpvedalislfdcsdttvqqrvietyiarlyqphlvkdsi

[0252] kmkwiesgvialwefpeghfdarnggavlgdkrwgamvivksleslsmairfalketshytssegnmmhiallgadnkkmhiiqesg

[0253] dadriaklplilkdnvtdlhasgvktisfivqrdearmtmrrtflusdeklsyeeepilrhvepplsalleldklkvkgynemkytpsrdr

[0254] qwhiytlrntenpkmlhrvffrtlvrqpsvsnkfssgqigdmevgsaeeplsftstsilrslmtaielelhairtghshmylhvlkeqklldl

[0255] vpvsgntvldvgqdeataysllkemamkihelvgarmhhlsvcqwevklkldcdgpasgtwrivttnvtshtctvdiyremedkesrk

[0256] lvyhpatpaagplhgvalnnpyqplsvidlkrcsarnnrttycydfplafetavrkswssstsgaskgvenaqcyvkatelvfadkhgsw

[0257] gtplvqmdrpaglndigmvawtlkmstpefpsgreiivvanditfragsfgpredaffeavtnlacekklpliylaansgarigiadevks

[0258] cfrvgwsddgspergfqyiylseedyarigtsviahkmqldsgeirwvidsvvgkedglgvenihgsaaiasaysrayketftltfvtgrtv

[0259] gigaylarlgirciqrldqpiiltgysalnkllgrevysshmqlggpkimatngvvhltvsddlegvsnilrwlsyvpayiggplpvttpldp

[0260] pdrpvayipenscdpraairgvddsqgkwlggmfdkdsfvetfegwaktvvtgraklggipvgviavetqtmmqtipadpgqldsre

[0261] qsvpragqvwfpdsatktaqalldfnreglplfilanwrgfsggqrdlfegilqagstivenlrtynqpafvyipmaaelrggawvvvdsk

[0262] inpdriecyaertakgnvlepqglieikfrseelqdcmsrldptlidlkaklevankngsadtkslqenieartkqlmplytqiairfaelhdt

[0263] slrmaakgvikkvvdweesrsffykrlrrrisedvlakeiravageqfshqpaielikkwysashaaewddddafvawmdnpenykdyiqylkaqrvsqslsslsdsssdlqalpqglsmlldkmdpsrraqlveeirkvlg;

[0264] The first nucleotide sequence of the japonica rice ACCase gene is as follows (SEQ ID No.2):

[0265] atgccgatgcggccgtgggaatttattattttaggccgcacagcccccatctctcccccacccctcgggatatccgccttctcccgcccgctcc

[0266] gcctccgcctccgcctccgcctcgccgccgttgtcgacgccgcaaggatccaaacgccgccccgcgtcgccttctccgccttcttcttcttctt

[0267] cttcttcttctccgagggtctctctcgggaaggtacagctgcccgcctccctgctcttatttatcgcgtgccgttcattccgtgccttctccagctcc

[0268] agatccgcgcgccgcacgccgatccgcgcccggccggttttagctgcgctcatcatttcctcgcgttggctcagggtttaagctcctctttgtta

[0269] agtgggacgatttcacacatctggggatttatcttctctttgtatggcactacacatttgagaaaccgtgcaattctactgtttggtcttgctggcatc

[0270] attgacctcccaaatgacgcagcttcagaagttgatatttcacatggttccgaagatcccagggggcctacggtcccaggttcctaccaaatga

[0271] atgggattatcaatgaaacacataatgggaggcatgcttcagtctccaaggttgttgagttttgtacggcacttggtggcaaaacaccaattcac

[0272] agtgtattagtggccaacaatggaatggcagcagctaagttcatgcggagtgtccgaacatgggctaatgatacttttggatcagagaaggca

[0273] attcagctgatagctatggcaactccggaggatctgaggataaatgcagagcacatcagaattgccgatcaatttgtagaggtacctggtgga

[0274] acaaacaacaacaactatgcaaatgtccaactcatagtggagatagcagagagaacaggtgtttctgctgtttggcctggttggggtcatgcat

[0275] ctgagaatcctgaacttccagatgcgctgactgcaaaaggaattgtttttcttgggccaccagcatcatcaatgcatgcattaggagacaaggtt

[0276] ggctcagctctcattgctcaagcagctggagttccaacacttgcttggagtggatcacatgtggaagttcctctggagtgttgcttggactcaata

[0277] cctgatgagatgtatagaaaagcttgtgttactaccacagaggaagcagttgcaagttgtcaggtggttggttatcctgccatgattaaggcatct

[0278] tggggtggtggtggtaaaggaataaggaaggttcataatgatgatgaggttaggacattatttaagcaagttcaaggcgaagtacctggttccc

[0279] caatatttatcatgaggctagctgctcagagtcgacatcttgaagttcagttgctttgtgatcaatatggcaacgtagcagcacttcacagtcgag

[0280] attgcagtgtacaacggcgacaccaaaagataatcgaggaaggaccagttactgttgctcctcgtgagactgtgaaagagcttgagcaggca

[0281] gcacggaggcttgctaaagctgtgggttatgttggtgctgctactgttgaatacctttacagcatggaaactggtgaatattattttctggaactta

[0282] atccacggctacaggttgagcatcctgtcactgagtggatagctgaagtaaatttgcctgcggctcaagttgctgttggaatgggtataccccttt

[0283] ggcagattccagagatcaggcgcttctacggaatgaaccatggaggaggctatgacctttggaggaaaacagcagctctagcgactccattt

[0284] aactttgatgaagtagattctaaatggccaaaaggccactgcgtagctgttagaataactagcgaggatccagatgatgggtttaagcctactg

[0285] gtggaaaagtaaaggagataagtttcaagagtaaaccaaatgtttgggcctatttctcagtaaagtctggtggaggcatccatgaattcgctgat

[0286] tctcagttcggacatgtttttgcgtatggaactactagatcggcagcaataactaccatggctcttgcactaaaagaggttcaaattcgtggagaa

[0287] attcattcaaacgtagactacacagttgacctattaaatgcctcagattttagagaaaataagattcatactggttggctggataccaggatagcc

[0288] atgcgtgttcaagctgagaggcctccatggtatatttcagtcgttggaggggctttatataaaacagtaactgccaacacggccactgtttctgat

[0289] tatgttggttatcttaccaagggccagattccaccaaagcatatatcccttgtctatacgactgttgctttgaatatagatgggaaaaaatatacaat

[0290] cgatactgtgaggagtggacatggtagctacagattgcgaatgaatggatcaacggttgacgcaaatgtacaaatattatgtgatggtgggctt

[0291] ttaatgcagctggatggaaacagccatgtaatttatgctgaagaagaggccagtggtacacgacttcttattgatggaaagacatgcatgttaca

[0292] gaatgaccatgacccatcaaagttattagctgagacaccatgcaaacttcttcgtttcttggttgctgatggtgctcatgttgatgctgatgtaccat

[0293] atgcggaagttgaggttatgaagatgtgcatgcccctcttatcacccgcttctggtgtcatacatgttgtaatgtctgagggccaagcaatgcag

[0294] gctggtgatcttatagctaggctggatcttgatgacccttctgctgttaagagagctgagccgttcgaagatacttttccacaaatgggtctcccta

[0295] ttgctgcttctggccaagttcacaaattatgtgctgcaagtctgaatgcttgtcgaatgatccttgcggggtatgagcatgatattgacaaggttgt

[0296] gccagagttggtatactgcctagacactccggagcttcctttcctgcagtgggaggagcttatgtctgttttagcaactagacttccaagaaatct

[0297] taaaagtgagttggagggcaaatatgaggaatacaaagtaaaatttgactctgggataatcaatgatttccctgccaatatgctacgagtgataa

[0298] ttgaggaaaatcttgcatgtggttctgagaaggagaaggctacaaatgagaggcttgttgagcctcttatgagcctactgaagtcatatgaggg

[0299] tgggagagaaagtcatgctcactttgttgtcaagtccctttttgaggagtatctctatgttgaagaattgttcagtgatggaattcagtctgatgtgat

[0300] tgagcgtctgcgccttcaacatagtaaagacctacagaaggtcgtagacattgtgttgtcccaccagagtgttagaaataaaactaagctgata

[0301] ctaaaactcatggagagtctggtctatccaaatcctgctgcctacagggatcaattgattcgcttttcttcccttaatcacaaagcgtattacaagtt

[0302] ggcacttaaagctagtgaacttcttgaacaaacaaaacttagtgagctccgtgcaagaatagcaaggagcctttcagagctggagatgtttact

[0303] gaggaaagcaagggtctctccatgcataagcgagaaattgccattaaggagagcatggaagatttagtcactgctccactgccagttgaagat

[0304] gcgctcatttctttatttgattgtagtgatacaactgttcaacagagagtgattgagacttatatagctcgattataccagcctcatcttgtaaaggac

[0305] agtatcaaaatgaaatggatagaatcgggtgttattgctttatgggaatttcctgaagggcattttgatgcaagaaatggaggagcggttcttggt

[0306] gacaaaagatggggtgccatggtcattgtcaagtctcttgaatcactttcaatggccattagatttgcactaaaggagacatcacactacactag

[0307] ctctgagggcaatatgatgcatattgctttgttgggtgctgataataagatgcatataattcaagaaagtggtgatgatgctgacagaatagccaa

[0308] acttcccttgatactaaaggataatgtaaccgatctgcatgcctctggtgtgaaaacaataagtttcattgttcaaagagatgaagcacggatgac

[0309] aatgcgtcgtaccttcctttggtctgatgaaaagctttcttatgaggaagagccaattctccggcatgtggaacctcctctttctgcacttcttgagt

[0310] tggacaagttgaaagtgaaaggatacaatgaaatgaagtataccccatcacgggatcgtcaatggcatatctacacacttagaaatactgaaa

[0311] accccaaaatgttgcaccgggtatttttccgaacccttgtcaggcaacccagtgtgtatccaacaagttttcttcgggccagattggtgacatggaa

[0312] gttgggagtgctgaagaacctctgtcatttacatcaaccagcatattaagatctttgatgactgctatagaggaattggagcttcacgcaattaga

[0313] actggccattcacacatgtatttgcatgtattgaaagaacaaaagcttcttgatcttgttccagtttcagggaatacagttttggatgttggtcaaga

[0314] tgaagctactgcatattcacttttaaaagaaatggctatgaagatacatgaacttgttggtgcaagaatgcaccatctttctgtatgccaatgggaa

[0315] gtgaaacttaagttggactgcgatggtcctgccagtggtacctggaggattgtaacaaccaatgttactagtcacacttgcactgtggatatcta

[0316] ccgtgagatggaagataaagaatcacggaagttagtataccatcccgccactccggcggctggtcctctgcatggtgtggcactgaataatcc

[0317] atatcagcctttgagtgtcattgatctcaaacgctgttctgctaggaataatagaactacatactgctatgattttccactggcatttgaaactgcag

[0318] tgaggaagtcatggtcctctagtacctctggtgcttctaaaggtgttgaaaatgcccaatgttatgttaaagctacagagttggtatttgcggaca

[0319] aacatgggtcatggggcactcctttagttcaaatggaccggcctgctgggctcaatgacattggtatggtagcttggaccttgaagatgtccact

[0320] cctgaatttcctagtggtagggagattattgttgttgcaaatgatattacgttcagagctggatcatttggcccaagggaagatgcattttttgaag

[0321] ctgttaccaacctagcctgtgagaagaaacttcctcttatttatttggcagcaaattctggtgctcgaattggcatagcagatgaagtgaaatcttg

[0322] cttccgtgttgggtggtctgatgatggcagccctgaacgtgggtttcagtacatttatctaagcgaagaagactatgctcgtattggcacttctgtc

[0323] atagcacatagatgcagctagacagtggtgaaattaggtgggttattgattctgttgtgggcaaggaagatggacttggtgtggagaatatac

[0324] atggaagtgctgctattgccagtgcttattctagggcatataaggagacatttacacttacatttgtgactggaagaactgttggaataggagctt

[0325] atcttgctcgacttggcatccggtgcatacagcgtcttgaccagcctattattcttacaggctattctgcactgaacaagcttcttgggcgggaag

[0326] tgtacagctcccacatgcagttgggtggtcccaaaatcatggcaactaatggtgttgtccatcttactgtttcagatgaccttgaaggcgtttctaa

[0327] tatattgaggtggctcagttatgttcctgcctacattggtggaccacttccagtaacaacaccgttggacccaccggacagacctgttgcataca

[0328] ttcctgagaactcgtgtgatcctcgagcggctatccgtggtgttgatgacagccaagggaaatggtaggtggtatgtttgataaagacagcttt

[0329] gtggaaacatttgaaggttgggctaagacagtggttactggcagagcaaagcttggtggaattccagtgggtgtgatagctgtgggagactcag

[0330] accatgatgcaaactatccctgctgaccctggtcagcttgattcccgtgagcaatctgttcctcgtgctggacaagtgtggtttccagattctgca

[0331] accaagactgcgcaggcattgctggacttcaaccgtgaaggattacctctgttcatcctcgctaactggagaggcttctctggtggacaaaga

[0332] gatctttttgaaggaattcttcaggctggctcgactattgttgagaaccttaggacatacaatcagcctgcctttgtctacattcccatggctgcag

[0333] agctacgaggaggggcttgggttgtggttgatagcaagataaacccagaccgcattgagtgctatgctgagaggactgcaaaaggcaatgtt

[0334] ctggaaccgcaagggttaattgagatcaagttcaggtcagaggaactccaggattgcatgagtcggcttgacccaacattaattgatctgaaa

[0335] gcaaaactcgaagtagcaaataaaaatggaagtgctgacacaaaatcgcttcaagaaaatatagaagctcgaacaaaacagttgatgcctcta

[0336] tatactcagattgcgatacggtttgctgaattgcatgatacatccctcagaatggctgcgaaaggtgtgattaagaaagttgtggactgggaaga

[0337] atcacgatctttcttctataagagattacggaggaggatctctgaggatgttcttgcaaaagaaattagagctgtagcaggtgagcagttttccca

[0338] ccaaccagcaatcgagctgatcaagaaatggtattcagcttcacatgcagctgaatggggatgatgacgatgcttttgttgcttggatggataacc

[0339] ctgaaaactacaaggattatattcaatatcttaaggctcaaagagtatcccaatccctctcaagtctttcagattccagctcagatttgcaagccctgccacagggtctttccatgttactagataagatggatccctctagaagagctcaacttgttgaagaaatcaggaaggtccttggttga;

[0340] The second amino acid sequence encoded by the ACCase gene in japonica rice (which is also the amino acid sequence encoded by the ACCase gene in indica rice) is as follows (SEQ ID No. 3):

[0341] mtsthvatlgvgaqapprhqkksagtafvssgssrpsyrkngqrtrslreesnggvsdskklnhsirqglagiidlpndaasevdishgse

[0342] dprgptvpgsyqmngiinethngrhasvskvvefctalggktpihsvlvanngmaaakfmrsvrtwandtfgsekaiqliamatpedlr

[0343] inaehiriadqfvevpggtnnnnyanvqliveiaertgvsavwpgwghasenpelpdaltakgivflgppassmhalgdkvgsaliaqa

[0344] agvptlawsgshvevpleccldsipdemyrkacvttteeavascqvvgypamikaswggggkgirkvhnddevrtlfkqvqgevpg

[0345] spifimrlaaqsrhlevqllcdqygnvaalhsrdcsvqrrhqkiieegpvtvapretvkeleqaarrlakavgyvgaatveylysmetgey

[0346] yflelnprlqvehpvtewiaevnlpaaqvavgmgiplwqipeirrfygmnhgggydlwrktaalatpfnfdevdskwpkghcvavrit

[0347] sedpddgfkptggkvkeisfkskpnvwayfsvksgggihefadsqfghvfaygttrsaaittmalalkevqirgeihsnvdytvdllnas

[0348] dfrenkihtgwldtriamrvqaerppwyisvvggalyktvtantatvsdyvgyltkgqippkhislvyttvalnidgkkytidtvrsghgs

[0349] yrlrmngstvdanvqilcdggllmqldgnshviyaeeeasgtrllidgktcmlqndhdpskllaetpckllrflvadgahvdadvpyaev

[0350] evmkmcmpllspasgvihvvmsegqamqagdliarldldpsavkraepfedtfpqmglpiaasgqvhklcaaslnacrmilagye

[0351] hdidkvvpelvycldtpelpflqweelmsvlatrlprnlkselegkyeeykvkfdsgiindfpanmlrviieenlacgsekekatnerlve

[0352] plmsllksyeggreshahfvvkslfeeylyveelfsdgiqsdvierlrlqhskdlqkvvdivlshqsvrnktklilklmeslvypnpaayrd

[0353] qlirfsslnhkayykalkaselleqtklselrariarslselemfteeskglsmhkreiaikesmedlvtaplpvedalislfdcsdttvqqrvi

[0354] etyiarlyqphlvkdsikmkwiesgvialwefpeghfdarnggavlgdkrwgamvivksleslsmairfalketshytssegnmmhia

[0355] llgadnkmhiiqesgddadriaklplilkdnvtdlhasgvktisfivqrdearmtmrrtflwsdeklsyeeepilrhvepplsalleldklkv

[0356] kgynemkytpsrdrqwhiytlrntenpkmlhrvffrtlvrqpsvsnkfssgqigdmevgsaeeplsftstsilrslmtaieelelhairtghs

[0357] hmylhvlkeqklldlvpvsgntvldvgqdeataysllkemamkihelvgarmhhlsvcqwevklkldcdgpasgtwrivttnvtshtc

[0358] tvdiyremedkesrklvyhpatpaagplhgvalnnpyqplsvidlkrcsarnnrttycydfplafetavrkswssstsgaskgvenaqcy

[0359] vkatelvfadkhgswgtplvqmdrpaglndigmvawtlkmstpefpsgreiivvanditfragsfgpredaffeavtnlacekklpliyl

[0360] aansgarigiadevkscfrvgwsddgspergfqyiylseedyarigtsviahkmqldsgeirwvidsvvgkedglgvenihgsaaiasa

[0361] ysrayketftltfvtgrtvgigaylarlgirciqrldqpiiltgysalnkllgrevysshmqlggpkimatngvvhltvsddlegvsnilrwlsy

[0362] vpayiggplpvttpldppdrpvayipenscdpraairgvddsqgkwlggmfdkdsfvetfegwaktvvtgraklggipvgviavetqt

[0363] mmqtipadpgqldsreqsvpragqvwfpdsatktaqalldfnreglplfilanwrgfsggqrdlfegilqagstivenlrtynqpafvyip

[0364] maaelrggawvvvdskinpdriecyaertakgnvlepqglieikfrseelqdcmsrldptlidlkaklevankngsadtkslqenieartk

[0365] qlmplytqiairfaelhdtslrmaakgvikkvvdweesrsffykrlrrrisedvlakeiravageqfshqpaielikkwysashaaewddddafvawmdnpenykdyiqylkaqrvsqslsslsdsssdlqalpqglsmlldkmdpsrraqlveeirkvlg;

[0366] The second nucleotide sequence of the japonica rice ACCase gene is as follows (SEQ ID No.4):

[0367] atgacatccacacatgtggcgacattgggagttggtgcccaggcacctcctcgtcaccagaaaaagtcagctggcactgcatttgtatcatctg

[0368] ggtcatcaagaccctcataccgaaagaatggtcagcgtactcggtcacttagggaagaaagcaatggaggagtgtctgattccaaaaagctt

[0369] aaccactctattcgccaaggtcttgctggcatcattgacctcccaaatgacgcagcttcagaagttgatatttcacatggttccgaagatcccag

[0370] ggggcctacggtcccaggttcctaccaaatgaatgggattatcaatgaaacacataatgggaggcatgcttcagtctccaaggttgttgagtttt

[0371] gtacggcacttggtggcaaaacaccaattcacagtgtattagtggccaacaatggaatggcagcagctaagttcatgcggagtgtccgaacat

[0372] gggctaatgatacttttggatcagagaaggcaattcagctgatagctatggcaactccggaggatctgaggataaatgcagagcacatcagaa

[0373] ttgccgatcaatttgtagaggtacctggtggaacaaacaacaacaactatgcaaatgtccaactcatagtggagatagcagagagaacaggtg

[0374] tttctgctgtttggcctggttggggtcatgcatctgagaatcctgaacttccagatgcgctgactgcaaaaggaattgtttttcttgggccaccagc

[0375] atcatcaatgcatgcattaggagacaaggttggctcagctctcattgctcaagcagctggagttccaacacttgcttggagtggatcacatgtgg

[0376] aagttcctctggagtgttgcttggactcaatacctgatgagatgtatagaaaagcttgtgttactaccacagaggaagcagttgcaagttgtcag

[0377] gtggttggttatcctgccatgattaaggcatcttggggtggtggtggtaaaggaataaggaaggttcataatgatgatgaggttaggacattattt

[0378] aagcaagttcaaggcgaagtacctggttccccaatatttatcatgaggctagctgctcagagtcgacatcttgaagttcagttgctttgtgatcaa

[0379] tatggcaacgtagcagcacttcacagtcgagattgcagtgtacaacggcgacaccaaaagataatcgaggaaggaccagttactgttgctcct

[0380] cgtgagactgtgaaagagcttgagcaggcagcacggaggcttgctaaagctgtgggttatgttggtgctgctactgttgaatacctttacagca

[0381] tggaaactggtgaatattattttctggaacttaatccacggctacaggttgagcatcctgtcactgagtggatagctgaagtaaatttgcctgcgg

[0382] ctcaagttgctgttggaatgggtatacccctttggcagattccagagatcaggcgcttctacggaatgaaccatggaggaggctatgacctttg

[0383] gaggaaaacagcagctctagcgactccatttaactttgatgaagtagattctaaatggccaaaaggccactgcgtagctgttagaataactagc

[0384] gaggatccagatgatgggtttaagcctactggtggaaaagtaaaggagataagtttcaagagtaaaccaaatgtttgggcctatttctcagtaaa

[0385] gtctggtggaggcatccatgaattcgctgattctcagttcggacatgtttttgcgtatggaactactagatcggcagcaataactaccatggctctt

[0386] gcactaaaagaggttcaaattcgtggagaaattcattcaaacgtagactacacagttgacctattaaatgcctcagattttagagaaaataagatt

[0387] catactggttggctggataccaggatagccatgcgtgttcaagctgagaggcctccatggtatatttcagtcgttggaggggctttatataaaac

[0388] agtaactgccaacacggccactgtttctgattatgttggttatcttaccaagggccagattccaccaaagcatatatcccttgtctatacgactgtt

[0389] gctttgaatatagatgggaaaaaatatacaatcgatactgtgaggagtggacatggtagctacagattgcgaatgaatggatcaacggttgacg

[0390] caaatgtacaaatattatgtgatggtgggcttttaatgcagctggatggaaacagccatgtaatttatgctgaagaagaggccagtggtacacg

[0391] acttcttattgatggaaagacatgcatgttacagaatgaccatgacccatcaaagttattagctgagacaccatgcaaacttcttcgtttcttggttg

[0392] ctgatggtgctcatgttgatgctgatgtaccatatgcggaagttgaggttatgaagatgtgcatgcccctcttatcacccgcttctggtgtcataca

[0393] tgttgtaatgtctgagggccaagcaatgcaggctggtgatcttatagctaggctggatcttgatgacccttctgctgttaagagagctgagccgtt

[0394] cgaagatacttttccacaaatgggtctccctattgctgcttctggccaagttcacaaattatgtgctgcaagtctgaatgcttgtcgaatgatccttg

[0395] cggggtatgagcatgatattgacaaggttgtgccagagttggtatactgcctagacactccggagcttcctttcctgcagtgggaggagcttat

[0396] gtctgttttagcaactagacttccaagaaatcttaaaagtgagttggagggcaaatatgaggaatacaaagtaaaaatttgactctgggataatca

[0397] atgatttccctgccaatatgctacgagtgataattgaggaaaatcttgcatgtggttctgagaaggagaaggctacaaatgagaggcttgttgag

[0398] cctcttatgagcctactgaagtcatatgagggtgggagagaaagtcatgctcactttgttgtcaagtccctttttgaggagtatctctatgttgaag

[0399] aattgttcagtgatggaattcagtctgatgtgattgagcgtctgcgccttcaacatagtaaagacctacagaaggtcgtagacattgtgttgttccc

[0400] accagagtgttagaaataaaactaagctgatactaaaactcatggagagtctggtctatccaaatcctgctgcctacagggatcaattgattcgc

[0401] ttttcttcccttaatcacaaagcgtattacaagttggcacttaaagctagtgaacttcttgaacaaacaaaacttagtgagctccgtgcaagaatag

[0402] caaggagcctttcagagctggagatgtttactgagaaagcaagggtctctccatgcataagcgagaaattgccattaaggagagcatggaa

[0403] gatttagtcactgctccactgccagttgaagatgcgctcatttctttattgattgtagtgatacaactgttcaacagagagtgattgagacttatata

[0404] gctcgattataccagcctcatcttgtgaaggacagtatcaaaatgaaatggatagaatcgggtgttatgctttatgggaatttcctgaagggcatt

[0405] ttgatgcaagaaatggaggagcggttcttggtgacaaaagatggggtgccatggtcattgtcaagtctcttgaatcactttcaatggccattaga

[0406] tttgcactaaaggacatcacactacactagctctgagggcaatatgatgcatattgctttgttgggtgctgataataagatgcatataattcaag

[0407] aaagtggtgatgatgctgacaagaatagccaaacttcccttgatataaaggataatgtaaccgatctgcatgcctctggtgtgaaaaacaataagt

[0408] ttcattgttcaaagagatgaagcacggatgacaatgcgtcgtaccttcctttggtctgatgaaaagctttcttatgaggaagagccaattctccgg

[0409] catgtggaacctcctctttctgcacttcttgagttggacaagttgaaagtgaaaggaatacaatgaaatgaagtaccccatcacgggatcgtca

[0410] atggcatatctacacacttagaaatactgaaaaccccaaaatgttgcaccgggtatttttccgaacccttgtcaggcaacccagtgtatccaaca

[0411] agttttcttgggccagattggtgacatggaagttgggagtgctgaagaacctctgtcatttacatcaaccagcatattaagatctttgatgactgc

[0412] tatagagaattggagcttcacgcaattagaactggccattcacacatgtatttgcatgtattgaaagaacaaaagcttcttgatcttgttccagttt

[0413] cagggaatacagttttggatgttggtcaagatgaagctactgcatattcacttttaaaagaaatggctatgaagatacatgaacttgttggtgcaa

[0414] gaatgcaccatctttctgtatgccaatgggaagtgaaacttaagttggactgcgatggtcctgccagtggtacctggaggattgtaacaaccaat

[0415] gttactagtcacacttgcactgtggatatctaccgtgagatggagaagaataaagaatcacggaagttagtataccatcccgccactccggcggctg

[0416] gtcctctgcatggtgtggcactgaataatccatatcagcctttgagtgtcattgatctcaaacgctgttctgctaggaataatagaactacatactg

[0417] ctatgattttccactggcatttgaaactgcagtgaggaagtcatggtcctctagtacctctggtgcttctaaaggtgttgaaaatgcccaatgttat

[0418] gttaaagctacagagttggtatttgcggacaaacatgggtcatggggcactcctttagttcaaatggaccggcctgctgggctcaatgacattg

[0419] gtatggtagcttggaccttgaagatgtccactcctgaatttcctagtggtagggagattattgttgttgcaaatgatattacgttcagagctggatc

[0420] atttggcccaagggaagatgcattttttgaagctgttaccaacctagcctgtgagaagaaacttcctcttatttatttggcagcaaattctggtgctc

[0421] gaattggcatagcagatgaagtgaaatcttgcttccgtgttgggtggtctgatgatggcagccctgaacgtgggtttcagtacatttatctaagcg

[0422] aagaagactatgctcgtattggcacttctgtcatagcacataagatgcagctagacagtggtgaaattaggtgggttattgattctgttgtgggca

[0423] aggaagatggacttggtgtggagaatatacatggaagtgctgctattgccagtgcttattctagggcatataaggagacatttacacttacatttg

[0424] tgactggaagaactgttggaataggagcttatcttgctcgacttggcatccggtgcatacagcgtcttgaccagcctattattcttacaggctattc

[0425] tgcactgaacaagcttcttgggcgggaagtgtacagctcccacatgcagttgggtggtcccaaaatcatggcaactaatggtgttgtccatctta

[0426] ctgtttcagatgaccttgaaggcgtttctaatatattgaggtggctcagttatgttcctgcctacattggtggaccacttccagtaacaacaccgttg

[0427] gacccaccggacagacctgttgcatacattcctgagaactcgtgtgatcctcgagcggctatccgtggtgttgatgacagccaagggaaatg

[0428] gttaggtggtatgtttgataaagacagctttgtggaaacatttgaaggttgggctaagacagtggttactggcagagcaaagcttggtggaattc

[0429] cagtgggtgtgatagctgtggagactcagaccatgatgcaaactatccctgctgaccctggtcagcttgattcccgtgagcaatctgttcctcgt

[0430] gctggacaagtgtggtttccagattctgcaaccaagactgcgcaggcattgctggacttcaaccgtgaaggattacctctgttcatcctcgctaa

[0431] ctggagaggcttctctggtggacaaagagatctttttgaaggaattcttcaggctggctcgactattgttgagaaccttaggacatacaatcagc

[0432] ctgcctttgtctacattcccatggctgcagagctacgaggaggggcttgggttgtggttgatagcaagataaacccagaccgcattgagtgcta

[0433] tgctgagaggactgcaaaaggcaatgttctggaaccgcaagggttaattgagatcaagttcaggtcagaggaactccaggattgcatgagtc

[0434] ggcttgacccaacattaattgatctgaaagcaaaactcgaagtagcaaataaaaatggaagtgctgacacaaaatcgcttcaagaaaatatag

[0435] aagctcgaacaaaacagttgatgcctctatatactcagattgcgatacggtttgctgaattgcatgatacatccctcagaatggctgcgaaaggt

[0436] gtgattaagaaagttgtggactgggaagaatcacgatctttcttctataagagattacggaggaggatctctgaggatgttcttgcaaaagaaatt

[0437] agagctgtagcaggtgagcagttttcccaccaaccagcaatcgagctgatcaagaaatggtattcagcttcacatgcagctgaatgggatgat

[0438] gacgatgcttttgttgcttggatggataaccctgaaaactacaaggattatattcaatatcttaaggctcaaagagtatcccaatccctctcaagtc

[0439] tttcagattccagctcagatttgcaagccctgccacagggtctttccatgttactagataagatggatccctctagaagagctcaacttgttgaag

[0440] aaatcaggaaggtccttggttga

[0441] The nucleotide sequence of the indica rice ACCase gene is as follows (SEQ ID No. 5):

[0442] cggacacgaataaaaaaattaatttcataattcggctgaaaactaaaagatgaatcttttgagcctaattaatctgtcattagcacatggggttact

[0443] atagcatttatgcataatcatcgactaattaggcttaaaagatttgtctcacgattttccgctaatcatgtaattaattttttcttaatctcatgtcaaaga

[0444] ttcgatgtaacgtttttcggaaaaaaaattggaaaactaaaacaggcctaaattccattcagagtttcctctgccactgccgcagtcaaacaaac

[0445] cgtgcaaacagggacgggcagtcacacaccaagagagcgaaacgagcgcgcctgccggtcgcgaaaaccctgggaactcgtcacatct

[0446] ggtgcgccgggagcatcccggccgtccacggccccgcccatcctacagctggacgcgatccctgccgtccatccggcgcacctccctcgc

[0447] tgtcacgtgcacggaggaggagcgggggggggggggggggggggggggaggaggggggcggagtccccaatgccgatgcggccg

[0448] tgggaatttattattttaggccgcacagcccccatctctcccccacccctcgggatatccgccttctcccgcccgctccgcctccgcctccgcct

[0449] ccgcctcgccgccgttgtcgacgccgcaaggatccaaacgccgccccgcgtcgccttctccgccttcttcttcttcttcttcttcttctccgaggg

[0450] tctctctcgggaaggtacagctgcccgcctccctgctcttatttatcgcgtgccgttcattccgtgccttctccagctccagatccgcgcgccgc

[0451] acgccgatccgcgcccggccggtgagtccgccgcgggtttccgccattgctgccgtttctgcgctgctagggggaggtttagatgtgggtgg

[0452] tgggagccgcgtgctgttgtctgggatgggagagagagggtttaagggtgggtgccggatgcggatgctcccgcccgggttttggtcctgct

[0453] tgtggactggcgaggggctaggggttgagttgtagggattcgggatggaaatggactggggagctcggatggggaggtaatgccacgccg

[0454] gtgaaatgcagctcgctgttgttggttctggttaatgcctgggtgttgctggctatgttactatgttgcatgcctacgcgagatactgccatgtgcc

[0455] attttgcgtgattgtgatattggtttgaggttttagctgcgctcatcatttcctcgcgttggctcagggtttaagctcctctttgttaagtgggacggta

[0456] tgcatatgcattgtgttcttttaggtttaaattctggttacggtgccacacaacaagtcgtagatgctcattcttgttcttttcatggggcttatggaatt

[0457] gatggcacatggcacacctttctcctttttccgtcatgcatgttagtgccatattgtctacgcaaaaaagaatattctgcatatttgtttactcactattt

[0458] ttttccaagagaattgggttttgccatgcttcaaaaggaacatttcaagctgaaaaagttctcccgatatggaattagctttagagttcaggggaa

[0459] aaaatatgttgttgctttgtgcttgtcaagtaaccacagttgcacaggttttgactgacctcatgctacttgacattattcaccatgataaagttttacc

[0460] ttttgtttttttcctttcactaggaacagaacgaattgtttgcataaccaatttgtagatttgacatggggaaatagcataactgaatatatctcaacct

[0461] attactaggcaaaataagaaatgaacctaagccttaacaggtttgcatcaaattatcctgtgcaaaatgaacaaaggtggcacacaatgtacaa

[0462] gcttgcatttcatttttggatcctcaaacgtcttaaagacttttgcataaagtatttgatgagtaacgagctggaactaatcattcttatatattttcatct

[0463] gtcagatttcacacatctggggatttatcttctctttgtatggcactacacatttgagaaaccgtgcaattctactgtttggtcagcaggacaacaat

[0464] gacatccacacatgtggcgacattgggagttggtgcccaggcacctcctcgtcaccagaaaaagtcagctggcactgcatttgtatcatctgg

[0465] gtcatcaagaccctcataccgaaagaatggtcagcgtactcggtcacttagggaagaaagcaatggaggagtgtctgattccaaaaagcttaa

[0466] ccactctattcgccaaggtgaccactagctactttacatatgctataatttgtgccaaacataaacatgcaatggctgctattatttaaacgttaatgt

[0467] tgaaatagctgctataggatacagcaaaaatatataattgactgggcaagatgcaacaattgtttttcactaaagttagttatcttttgctgtaaaag

[0468] acaactgttttttacataaaatggtattaataaccttgtaatattcaatgcaacatgttctcaagtaaaaaaaaacattgcctggttgtataagcaaat

[0469] gtgtcgttgtagacatcttattaaacctttttgtgatatctattaccgtagggaacaggggagctgtttaaatctgttatcatagagtaatatgagaaa

[0470] agtggattgtgcgactttggcatgtatacctgctcaatttcaaatatatgtctatgtgcaggtcttgctggcatcattgacctcccaaatgacgcag

[0471] cttcagaagttgatatttcacagtaaggactttatattttataataattattatataattttctgacatgttttgagaacctcaaaacatgtgattgcacct

[0472] tccttttttatgtctggttcagaaactgataagttttgacagtgtttaggatggatctttgatgcgcacagtgctttctaatgttttcatttttgaaagtaat

[0473] gttttaggaagaaatatctgattaaatttatactttatctttacaaaagtcaaatgcgttctgtatcaattgcggtttgtaatatggcaagaacatgcttt

[0474] cagaatttgttcatacaatgctttctttctattattatgtagaacaaatacctaatactttgttcaccttttatagtggacacctctcacagctttttcagta

[0475] agtgatgcaattttgtacatttgtaagatgtgttccagaaaccttttctcctgcaattctaatgtacccactcaaactggtatcaccaaagatctccat

[0476] ctgattgaaaaaaagctgcgtgaagtatgcttatttatgctaaccatacatgatttatactgttttatagtacaatgcttatttatgctaaccatacata

[0477] attttattctgttttctagtacattatttgtgcccctgaccataaatgatcctttcttttacagtggttccgaagatcccagggggcctacggtcccag

[0478] gttcctaccaaatgaatgggattatcaatgaaacacataatgggaggcatgcttcagtctccaaggttgttgagttttgtacggcacttggtggca

[0479] aaacaccaattcacagtgtattagtggccaacaatggaatggcagcagctaagttcatgcggagtgtccgaacatgggctaatgatacttttgg

[0480] atcagagaaggcaattcagctgatagctatggcaactccggaggatctgaggataaatgcagagcacatcagaattgccgatcaatttgtaga

[0481] ggtacctggtggaacaaacaacaacaactatgcaaatgtccaactcatagtggaggttagttcagctcatccctcaacacaacattttcgtttcta

[0482] tttaagttagggaaaaatctctacgaccctccaatttctgaacatccaattttcaccatcaactgcaatcacagatagcagagagaacaggtgttt

[0483] ctgctgtttggcctggttggggtcatgcatctgagaatcctgaacttccagatgcgctgactgcaaaaggaattgtttttcttgggccaccagcat

[0484] catcaatgcatgcattaggagacaaggttggctcagctctcattgctcaagcagctggagttccaacacttgcttggagtggatcacatgtgag

[0485] ccttgtcttctcttttttagcttatcatcttatcttttcggtgatgcattatcccaatgacactaaaccataggtggaagttcctctggagtgttgcttgg

[0486] actcaatacctgatgagatgtatagaaaagcttgtgttactaccacagaggaagcagttgcaagttgtcaggtggttggttatcctgccatgatta

[0487] aggcatcttggggtggtggtggtaaaggaataaggaaggtttgttcttcttgtagttatcaagagattgtttggattgcaagtgtttagtgcccata

[0488] gttaactctggtctttctaacatgagtaactcaactttcttgcaggttcataatgatgatgaggttaggacattatttaagcaagttcaaggcgaagt

[0489] acctggttccccaatatttatcatgaggctagctgctcaggtggggccttttatggaagttacaccttttcccttaatgttgagttattccggagttat

[0490] tatggttatgttctgtatgtttgatctgtaaattattgaaattcacctccattggttctccagattagcagacctacaattctacatatggtttatactttat

[0491] aaatactaggattttagggatcttcatatagtttatacatggtatttagatttcatttgtaaccctattgaagacatcctgattgttgtcttatgtagagtc

[0492] gacatcttgaagttcagttgctttgtgatcaatatggcaacgtagcagcacttcacagtcgagattgcagtgtacaacggcgacaccaaaaggt

[0493] ctgctgtctcagttaaatcacccctctgaatgatctacttcttgcctgctgcgttggtcagaggaataatggttgtattctactgaacagataatcga

[0494] ggaaggaccagttactgttgctcctcgtgagactgtgaaagagcttgagcaggcagcacggaggcttgctaaagctgtgggttatgttggtgc

[0495] tgctactgttgaatacctttacagcatggaaactggtgaatattatttctggaacttaatccacggctacaggtcggctcctttgacattcttcagg

[0496] aattaatttctgttgaccacatgatttacattgtcaaatggtctcacaggttgagcatcctgtcactgagtggatagctgaagtaaatttgcctgcgg

[0497] ctcaagttgctgttggaatgggtatacccctttggcagattccaggtaatgcttcttcatttagttcctgctctttgttaattgaatgagctcttataca

[0498] gaccatgagacacattctactgttaattcatagtatcccctgacttgttagtgttagagatacagagatgtatcacaaattcattgtatctcctcaag

[0499] gactgtaaaaatcctataattaaatttctgaaaatttgttcttttaagcagaaaaaaatctctaaattatctccctgtatacagagatcaggcgcttct

[0500] acggaatgaaccatggaggaggctatgacctttggaggaaaacagcagctctagcgactccatttaactttgatgaagtagattctaaatggcc

[0501] aaaaggccactgcgtagctgttagaataactagcgaggatccagatgatgggtttaagcctactggtggaaaagtaaaggtgcggtttcctga

[0502] tgttaggtgtatgaattgaacacattgctatattgcagctagtgaaatgactggatcatggttctcttattttcaggagataagtttcaagagtaaac

[0503] caaatgtttgggcctatttctcagtaaaggtagtcctcaatattgttgcactgccacattatttgagttgtcctaacaattgtgctgcaattgttagttt

[0504] caactatttgttgttctgtttggttgactggtaccctctctttgcagtctggtggaggcatccatgaattcgctgattctcagttcggtatgtaaagtta

[0505] aaagagtaatattgtctttgctatttatgtttgtcctcacttttaaaagatattgccttccattacaggacatgtttttgcgtatggaactactagatcgg

[0506] cagcaataactaccatggctcttgcactaaaagaggttcaaattcgtggagaaattcattcaaacgtagactacacagttgacctattaaatgtaa

[0507] ggactaaatatctgcttattgaaccttgctttttggttccctaatgccattttagtctggctactgaagaacttatccatcatgccatttctgttatcttaa

[0508] attcaggcctcagattttagagaaaataagattcatactggttggctggataccaggatagccatgcgtgttcaagctgagaggcctccatggta

[0509] tatttcagtcgttggaggggctttatatgtaagacaaactatgccactcattagcatttatgtgaagcaaatgcggaaaacatgatcaatatgtcgt

[0510] cttatttaaatttatttattttgtgctgcagaaaacagtaactgccaacacggccactgtttctgattatgttggttatcttaccaagggccagattcc

[0511] accaaaggtactattctgtttttcaggatatgaatgctgtttgaatgtgaaaaccattgaccataaatccttgtttgcagcatatatcccttgtctata

[0512] cgactgttgctttgaatatagatgggaaaaaatatacagtaagtgtgacattcttaatggggaaacttaatttgttgtaaataatcaatatcatattga

[0513] ctcgtgtatgctgcatcatagatcgatactgtgaggagtggacatggtagctacagattgcgaatgaatggatcaacggttgacgcaaatgtac

[0514] aaatattatgtgatggtgggcttttaatgcaggtaatatcttcttcctagttaaagaagatatatcttgttcaaagaattctgattattgatcttttaatgtt

[0515] ttcagctggatggaaacagccatgtaatttatgctgaagaagaggccagtggtacacgacttcttattgatggaaagacatgcatgttacaggta

[0516] atgatagccttgttctttttagttctagtcacggtgtttgcttgctatttgttgtatctatttaatgcattcactaattactatattagtttgcatcatcaagtt

[0517] aaaatggaacttctttcttgcagaatgaccatgacccatcaaagttattagctgagacaccatgcaaacttcttcgtttcttggttgctgatggtgct

[0518] catgttgatgctgatgtaccatatgcggaagttgaggttatgaagatgtgcatgcccctcttatcacccgcttctggtgtcatacatgttgtaatgtc

[0519] tgagggccaagcaatgcaggtacattcctacattccattcattgtgctgtgctgacatgaacatttcaagtaaatacctgtaacttgtttattattcta

[0520] ggctggtgatcttatagctaggctggatcttgatgacccttctgctgttaagagagctgagccgttcgaagatacttttccacaaatgggtctccc

[0521] tattgctgcttctggccaagttcacaaattatgtgctgcaagtctgaatgcttgtcgaatgatccttgcggggtatgagcatgatattgacaaggta

[0522] aacatcatgtcctcttgttttttcttttgtttatcatgcattcttatgttcatcatgtcctctggcaaatctagattccgctgtcgtttcacacagatttttctc

[0523] attctcataatggtgccaaacataaatatgctgctatattcatcaatgttttcactcgatttctaattttgcttttgagttttaaactttagtacaatccata

[0524] tctaatctcctttggcaacagtgaatccattatatatatttttattaaactgctttctttttcaggttgtgccagagttggtatactgcctagacactccg

[0525] gagcttcctttcctgcagtgggaggagcttatgtctgttttagcaactagacttccaagaaatcttaaaagtgaggtatattatggttgacaagata

[0526] gctagtctcatgctctaaggacttgtacatttcgccacataggttaattttccatatcaagttctaatgtacgatataaaagtagtactggcctaaaa

[0527] cagtattggtggttgactatctttgtgtgtaagatcaagtatttcttttcatgcttagtttgtcaatacttcacatttatcactgacttgtcgagctaaat

[0528] gagattttatttgatttctgtgctccattatttttgtatatatatatatatatttaactatgactatatgttatgcctcaaacgtttcaaactctttcagttgga

[0529] gggcaaatatgaggaatacaaagtaaaaatttgactctgggataatcaatgatttccctgccaatatgctacgagtgataattgaggtcagttattc

[0530] aatttgttgtgataatcactgccttaactgttcgttcttttaacaagcggttttataggaaaatcttgcatgtggttctgagaaggagaaggctacaa

[0531] atgagaggcttgttgagcctcttatgagcctactgaagtcatatgagggtgggagaaagtcatgctcactttgttgtcaagtccctttttgagg

[0532] agtatctctatgttgaagaattgttcagtgatggaattcaggttaacttactattcgcattaaacaaatcatcagttgttttatgataaagtcaaaatg

[0533] tttatatttcccattcttctgtggatcaaatatatcacggacatgatatagtttccttaggctatataatggttcttcatcaaataatattgcaggaaaca

[0534] gtatagcaaactatttgtatatactcgagatggaaattgttagaaacatcattgactaaatctgtcctttgttacgctgtttttgtagtctgatgtgattg

[0535] agcgtctgcgccttcaacatagtaaagacctacagaaggtcgtagacattgtgttgtcccaccaggtaaatttcttcatggtctgatgacttcact

[0536] gcgaatggttactgaactgtcttcttgttctgacaatgtgacttttctttgtagagtgttagaataaaactaagctgatactaaaactcatggagag

[0537] tctggtctatccaaatcctgctgcctacagggatcaattgattcgcttttcttcccttaatcacaaagcgtattacaaggtgaccaggataaacata

[0538] aataaacgtgaatttttcaatgaccttttcttctgacatctgaatctgatgaatttcttgcatattaatacagttggcacttaaagctagtgaacttcttg

[0539] aaaaaaaacttagtgagctccgtgcaagaatagcaaggagcctttcagagctggagatgtttactgaggaaagcaagggtctctccatg

[0540] cataagcgagaaattgccattaaaggagagcatggaagaatttagtcactgctccactgccagttgaagatgcgctcatttctttatttgattgtagtg

[0541] atacaactgttcaacagagagtgattgagacttatatagctcgattataccaggtatgaagaagaaagaccttttgaaattattatattaaacatatcc

[0542] tagtaaaacagcatgctcatcatttcttaaaaaagtttacagcacctgatgtttggttactgaccgcatcattaaaaataaagttacttgttgtgtggag

[0543] agatgtttttggaacttgtggcacatgcagtaacatgctactgctcgatatgtttgctaacttgacaacaatatttttcagcctcatcttgttaaaagga

[0544] foottcaaaatgaaatggatagaatcgggtgttatgctttatgggaatttcctgaagggcattttgatgcaagaaatggaggagcggttcttgg

[0545] tgacaaaagatggggtgccatggtcattgtcaagtctcttgaatcactttcaatggccattagatttgcactaaaggagacatcacactacactag

[0546] ctctgagggcaatatgatgcatattgctttgttgggtgctgataataagatgcatataattcaagaaaggtatgttcatatgctatgttggtgctgaa

[0547] atagttatatatgtagttagctggtggagttctggtaattaacctatcccattgttcagtggtgatgatgctgacagaatagccaaacttcccttgat

[0548] actaaaggataatgtaaccgatctgcatgcctctggtgtgaaaacaataagtttcattgttcaaagagatgaagcacggatgacaatgcgtcgta

[0549] ccttcctttggtctgatgaaaagctttcttatgaggaagagccaattctccggcatgtggaaacctcctctttctgcacttcttgagttggtacgtgat

[0550] atcatcaaaatgataatgttttggtatggcattgattatcttctatgctctttgtatttattcagcctattgtggatacaggacaagttgaaagtgaaag

[0551] gatacaatgaaatgaagtataccccatcacgggatcgtcaatggcatatctacacacacttagaaatactgaaaacccccaaaatgttgcaccgggt

[0552] atttttccgaacccttgtcaggcaacccagtgtatccaacaagttttcttcgggccagattggtgacatggaagttgggagtgctgaagaacctc

[0553] tgtcatttacatcaaccagcatattaagatctttgatgactgctatagaggaattggagcttcacgcaattagaactggccattcacacatgtatttg

[0554] catgtattgaaagaacaaaagcttcttgatcttgttccagtttcagggtaagtgcgcatatttctttgggaacatatgcttgcttatgaggttggtct

[0555] tctcaatgatcttcttatcttactcaggaatacagttttggatgttggtcaagatgaagctactgcatattcacttttaaaagaaatggctatgaagat

[0556] acatgaacttgttggtgcaagaatgcaccatctttctgtatgccaatgggaagtgaaacttaagttggactgcgatggtcctgccagtggtacct

[0557] ggaggattgtaacaaccaatgttactagtcacacttgcactgtggatgtaagtttaatcctctagcattttgttttctttggaaaagcatgtgattttaa

[0558] gccggctggtcctcatacccagacctagtgatctttatatagtgtagacatttttctaactgcttttaattgttttagatctaccgtgagatggaagat

[0559] aaagaatcacggaagttagtataccatcccgccactccggcggctggtcctctgcatggtgtggcactgaataatccatatcagcctttgagtg

[0560] tcattgatctcaaacgctgttctgctaggaataatagaactacatactgctatgattttccactggtgagttgactgctcccttatattcaatgcatta

[0561] ccatagcaaattcatattcgttcatgttgtcaaaataagccgatgaaaattcaaaactgtaggcatttgaaactgcagtgaggaagtcatggtcct

[0562] ctagtacctctggtgcttctaaaggtgttgaaaatgcccaatgttatgttaaagctacagagttggtatttgcggacaaacatgggtcatggggca

[0563] ctcctttagttcaaatggaccggcctgctggggctcaatgacattggtatggtagcttggaccttgaagatgtccactcctgaatttcctagtggta

[0564] gggagattatgttgttgcaaatgatattacgttcagagctggatcatttggcccaagggagaatgcattttttgaagctgttaccaacctagcctg

[0565] tgagaagaaacttctctcttatttatttggcagcaaattctggtgctcgaattggcatagcagatgaagtgaaatcttgcttccgtgttgggtggtct

[0566] gatgatggcagccctgaacgtgggtttcagtacatttatctaagcgaagaagactatgctcgtattggcacttctgtcatagcacataagatgca

[0567] gctagacagtggtgaaattaggtgggttattgattctgttgtgggcaaggaagatggacttggtgtggagaatatacatggaagtgctgctattg

[0568] ccagtgcttattctagggcatataaggagacatttacacttacatttgtgactggaagaactgttggaataggagcttatcttgctcgacttggcat

[0569] ccggtgcatacagcgtcttgaccagcctattattcttacaggctattctgcactgaacaagcttcttgggcgggaagtgtacagctcccacatgc

[0570] agttgggtggtcccaaaatcatggcaactaatggtgttgtccatcttactgtttcagatgaccttgaaggcgtttctaatatattgaggtggctcagt

[0571] tatgttcctgcctacattggtggaccacttccagtaacaacaccgttggacccaccggacagacctgttgcatacattcctgagaactcgtgtga

[0572] tcctcgagcggctatccgtggtgttgatgacagccaagggaaatggttaggtggtatgtttgataaagacagctttgtggaaacatttgaaggtt

[0573] gggctaagacagtggttactggcagagcaaagcttggtggaattccagtgggtgtgatagctgtggagactcagaccatgatgcaaactatc

[0574] cctgctgaccctggtcagcttgattcccgtgagcaatctgttcctcgtgctggacaagtgtggtttccagattctgcaaccaagactgcgcaggc

[0575] attgctggacttcaaccgtgaaggattacctctgttcatcctcgctaactggagaggcttctctggtggacaaagagatctttttgaaggaattctt

[0576] caggctggctcgactattgttgagaaccttaggacatacaatcagcctgcctttgtctacattcccatggctgcagagctacgaggaggggctt

[0577] gggttgtggttgatagcaagataaacccagaccgcattgagtgctatgctgagaggactgcaaaaggcaatgttctggaaccgcaagggtta

[0578] attgagatcaagttcaggtcagaggaactccaggattgcatgagtcggcttgacccaacattaattgatctgaaagcaaaactcgaagtagca

[0579] aataaaaatggaagtgctgacacaaaatcgcttcaagaaaatatagaagctcgaacaaaacagttgatgcctctatatactcagattgcgatac

[0580] ggtttgctgaattgcatgatacatccctcagaatggctgcgaaaggtgtgattaagaaagttgtggactgggaagaatcacgatctttcttctata

[0581] agagattacggaggaggatctctgaggatgttcttgcaaaagaaattagagctgtagcaggtgagcagttttcccaccaaccagcaatcgagc

[0582] tgatcaagaaatggtattcagcttcacatgcagctgaatgggatgatgacgatgcttttgttgcttggatggataaccctgaaaactacaaggatt

[0583] atattcaatatcttaaggctcaaagagtatcccaatccctctcaagtctttcagattccagctcagatttgcaagccctgccacagggtctttccat

[0584] gttactagataaggtaattagcttactgatgcttatataaattctttttcattacatatggctggagaactatctaatcaaataatgattataattccaat

[0585] cgttctttttatgccattatgatcttctgaaatttccttctttggacacttattcagatggatccctctagaagagctcaacttgttgaagaaatcaggaaggtccttggttgaatcatatgatgccaaaactattattggaggcacaaatagcttgtggaccctgtcggattgttggtgagtgtatattggatttgttagttctgccagatgaaagtgcaagtctgatgattcatgataccgtcagttggcaagaacaccggttaacctgagtgcttgtttacaaatggtcctttatgacaatcgttgtttcgcgctagttccgtgatctactatcatctgttagacgctgtaattagtgagtctccgcggatccacagtatacggttgagctgttgattcaattttggacacgaataatatgattttgtaggcataaatgcgtctgtatgtgaaataaattgtctgttgagttaacacacaagatgacaatatgtttgtgctctactgctattgtccatgaatactgattgcggaatcaaccacatgcattata;

[0586] The amino acid sequence of adenosine deaminase is as follows (SEQ ID No. 6):

[0587] sevefsheywmrhaltlakrarderevpvgavlvlnnrvigegwnraiglhdptahaeimalrqgglvmqnyrlidatlyvtfepcvmcagamihsrigrvvfgwrnskrgaagslmnvlnypgmnhrveitegiladecaallcdfyrmprqvfnaqkkaqssin;

[0588] The amino acid sequence of Cas12i3 is as follows (SEQ ID No. 7):

[0589]

[0590] 2. Screening and identification of rice genetic transformation and edited plants

[0591] Using japonica rice (Wanzhijing 006) as experimental material, the base editor iABE was transformed with Agrobacterium-mediated transformation to obtain gene-edited plants. Transformed seedlings were screened using a medium containing clethodim herbicide at a concentration of 2 mg / L (SET-2). Alternatively, transformed seedlings were planted in a cultivation area, and both the transformed seedlings and wild-type control plants were sprayed with clethodim herbicide at a concentration of 2.0 g / L (corresponding to a field application rate of 40 g / mu). Seedling survival was assessed after 10 days.

[0592] Edited plants resistant to herbicides were obtained by screening on a medium containing clethodim. Identification of the edited plants by PCR and sequencing revealed multiple mutation types in the ACC protein. This was caused by one or more A mutations to G in the codons of amino acid Asn(N) at position 1878 and amino acid Ile(I) at position 1879 of the target sequence. Figure 2 As shown; here, amino acid N at position 1878 and amino acid I at position 1879 are the amino acid positions obtained from the first amino acid sequence encoded by the wild-type ACCase gene of japonica rice, i.e., SEQ ID No. 1 as the parent sequence.

[0593] Identification revealed that the ACC protein editing types in these edited plants were: N1878D single mutation (the 1878th amino acid of the first amino acid sequence encoded by the ACC protein of the edited plant was mutated to D), N1878S single mutation (the 1878th amino acid of the first amino acid sequence encoded by the ACC protein of the edited plant was mutated to S), N1878G single mutation (the 1878th amino acid of the first amino acid sequence encoded by the ACC protein of the edited plant was mutated to G), I1879V single mutation (the 1879th amino acid of the first amino acid sequence encoded by the ACC protein of the edited plant was mutated to V), and N1878D single mutation. The edited plants have the following double mutations: / I1879V (the ACC protein of the edited plant has amino acid 1878 changed to D and amino acid 1879 changed to V), N1878S / I1879V (the ACC protein of the edited plant has amino acid 1878 changed to S and amino acid 1879 changed to V), and N1878G / I1879V (the ACC protein of the edited plant has amino acid 1878 changed to G and amino acid 1879 changed to V). In other words, the ACCase genotypes of the edited plants are ACC... N1878D ACC N1878S ACC N1878G ACCI1879V ACC N1878D / I1879V ACC N1878S / I1879V and ACC N1878G / I1879V These edited plants were self-pollinated to obtain homozygous edited plants.

[0594] Using the same method described above, indica rice was used as the experimental material to obtain herbicide-resistant edited plants. The edit type was N1791S / I1792V double mutation (the ACC protein of the edited plant had amino acid N mutated to S at position 1791 and amino acid I mutated to V at position 1792). That is, the ACCase genotype of the edited plant was ACC. N1791S / I1792V The edited plants were self-pollinated to obtain homozygous edited plants; the amino acid N at position 1791 and amino acid I at position 1792 are the amino acid positions obtained from the amino acid sequence encoded by the wild-type ACCase gene of indica rice, i.e., SEQ ID No.3 as the parent sequence.

[0595] The amino acid sequences encoded by the ACCase gene in japonica rice (SEQ ID No. 1 and SEQ ID No. 3) were compared with those encoded by the ACCase gene in indica rice (SEQ ID No. 3). The results are as follows: Figure 3 As shown. Overall, the amino acid sequence from amino acid 156 onwards in the first amino acid sequence encoded by the japonica rice ACCase gene is completely identical to the amino acid sequence from amino acid 69 onwards in the second amino acid sequence encoded by the japonica rice ACCase gene (i.e., the amino acid sequence encoded by the indica rice ACCase gene). In other words, the amino acid sequence from amino acid 156 onwards in the sequence described in SEQ ID No. 1 is completely identical to the amino acid sequence from amino acid 69 onwards in the sequence described in SEQ ID No. 3. Furthermore, amino acids N at position 1878 and I at position 1879 of the first amino acid sequence encoded by the japonica rice ACCase gene are the same sites as amino acids N at position 1791 and I at position 1792 of the second amino acid sequence encoded by the japonica rice ACCase gene; and amino acids N at position 1878 and I at position 1879 of the first amino acid sequence encoded by the japonica rice ACCase gene are homologous sites to amino acids N at position 1791 and I at position 1792 of the indica rice ACCase gene. That is, amino acid N at position 1878 of SEQ ID No. 1 and amino acid N at position 1791 of SEQ ID No. 3 are the same amino acid site, and amino acid I at position 1879 of SEQ ID No. 1 and amino acid I at position 1792 of SEQ ID No. 3 are the same amino acid site.

[0596] Example 2: Herbicide resistance and other trait tests of japonica rice (Wanzhijing 006) plants.

[0597] Wild-type japonica rice plants WT (Wanzhijing 006 wild-type plant) and japonica rice edited plants (edited plants ACC) obtained in Example 1 were grown in a greenhouse. N1878D ACC N1878S ACC N1878G ACC I1879V ACC N1878D / I1879V ACC N1878S / I1879V Then spray with water without herbicide or with 240 ml / mu of the herbicide tebuconazole (12.5%, EC). Observe the plant growth and herbicide resistance after 14 days. The results are as follows: Figure 4 As shown, the wild-type japonica rice plants (WT) sprayed with water and all edited plants grew normally without phytotoxicity; all wild-type japonica rice plants (WT) sprayed with haloxyfop-R-methyl died, and the edited plants (ACC) sprayed with haloxyfop-R-methyl died. N1878D ACC N1878S ACC N1878G ACC I1879V ACC N1878D / I1879V Compared to wild-type plants, they exhibited some herbicide resistance; the ACC of plants treated with chlorpyrifos showed improvement. N1878S / I1879V It can grow normally without withered leaves, showing stronger herbicide resistance.

[0598] Wild-type japonica rice plants WT (Wanzhijing 006 wild-type plant) and japonica rice edited plants (edited plants ACC) obtained in Example 1 were planted in the field. I1879V ACC N1878D / I1879V ACC N1878S / I1879V ACC N1878G / I1879V ), respectively applied 750ga.i.ha -1 Assess the phytotoxicity level 14 days after applying haloxyfop-R-methyl (12.5%, EC) or herbicide-free water (according to the "National Standard of the People's Republic of China for Field Efficacy Tests of Pesticides," crop phytotoxicity is divided into five levels: Level 0 - no phytotoxicity; Level 1 - slight phytotoxicity; Level 2 - moderate phytotoxicity; Level 3 - severe phytotoxicity; Level 4 - crop mortality). Results are as follows: Figure 5 As shown, wild-type japonica rice plants (WT) and all edited plants grew normally without herbicide damage under water treatment; wild-type japonica rice plants (WT) died after application of clethodim, indicating level 4 herbicide damage; while edited plants (ACC)... I1879V ACC N1878D / I1879V ACC N1878S / I1879V and ACC N1878G / I1879V Different herbicide resistances were observed: among them, the edited plants exhibited ACC... I1879V Green new leaves are emerging, but older leaves are withered, indicating level 2 pesticide damage; edit plant ACC. N1878D / I1879V It exhibits some degree of phytotoxicity, classified as level 2.5; (Edit plant ACC) N1878S / I1879Vand ACC N1878G / I1879V There was almost no pesticide damage; older leaves and newer leaves were normal, indicating a pesticide damage level of 0.1. The pesticide damage level is shown in the table below. (That is, edit the plant's ACC...) I1879V and ACC N1878D / I1879V It exhibits some herbicide resistance; edited plant ACCN 1878S / I1879V and ACC N1878G / I1879V It has high herbicide resistance.

[0599] WT <![CDATA[ACC I1879V ]]> <![CDATA[ACC N1878D / I1879V ]]> <![CDATA[ACC N1878S / I1879V ]]> <![CDATA[ACC N1878G / I1879V ]]> Spraying chloroquine 4 2 2.5 0.1 0.1 Spray with clean water that does not contain herbicides. 0 0 0 0 0

[0600] The above results indicate that single mutations of the ACC protein in japonica rice relative to the N1878D, N1878S, or N1878G sequence of SEQ ID No. 1, or double mutations of the above mutation sites (N1878D, N1878S, or N1878G) and the I1879V site, can confer herbicide resistance to plants.

[0601] Editing plant ACC N1878S / I1879V Field growth tests were conducted, and the results were as follows: Figure 6 As shown, compared with the wild-type japonica rice plant WT (Wanzhijing 006 wild-type plant), the ACC of the edited plant... N1878S / I1879V There were no significant differences in yield-related traits (e.g., number of effective spikes, spike length, single spike filling rate, and thousand-grain weight).

[0602] Example 3: Herbicide resistance in edited indica rice plants

[0603] Wild-type indica rice plants (WT) and edited indica rice plants (ACC) obtained in Example 1 were grown in a greenhouse. N1791S / I1792V When rice plants reach the three-leaf stage, they are sprayed with water without herbicides or with herbicides such as quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, clethodim, or clodinafop-P-ethyl. The phytotoxicity symptoms, fresh weight, and fresh weight inhibition rate are investigated 15 days after spraying. The fresh weight inhibition rate is calculated as: (CK fresh weight - treatment fresh weight) / CK fresh weight * 100%.

[0604] In this embodiment, wild-type indica rice plants (WT) and edited plants (ACC) N1791S / I1792V There are six spraying treatments: CK, 1X, 2X, 4X, 8X, and 16X. CK refers to spraying with water without herbicide. 1X (1x), 2X (2x), 4X (4x), 8X (8x), and 16X (16x) refer to different multiples of the herbicides quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, clethodim, and clodinafop-P-ethyl, respectively. The concentrations of the herbicides quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, clethodim, and clodinafop-P-ethyl at the 1X dosage are shown in the table below.

[0605] Serial Number test reagents 1× (1x) dose (ml / acre) 1 10% quizalofop-P-ethyl 40 2 10.8% High-efficiency flupyradifurone 30 3 12.5% ​​tebufenozide 90 4 24% Clethodim 25 5 10% cyclohexane 30

[0606] Fifteen days after herbicide application, the wild-type indica rice plants (WT) and edited plants (ACC) showed [results]. N1791S / I1792V The symptoms of phytotoxicity, fresh weight, and fresh weight inhibition rate are shown below:

[0607] Symptoms of herbicide damage from spraying quizalofop-P-ethyl

[0608] Dosage ratio Indica rice - WT <![CDATA[ACC N1791S / I1792V ]]> CK Normal growth Normal growth 1X The whole plant died The heart leaves turned white and then turned green again. 2X The whole plant died Heart leaves turn white 4X The whole plant died White heart leaves and stunted growth 8X The whole plant died Heart leaves wither and old leaves turn yellow 16X The whole plant died The whole plant died

[0609] Fresh weight and fresh weight inhibition rate of the herbicide quizalofop-P-ethyl

[0610]

[0611] Symptoms of phytotoxicity after spraying the herbicide flupyradifurone

[0612] Dosage ratio Indica rice - WT <![CDATA[ACC N1791S / I1792V ]]> CK Normal growth Normal growth 1X Heart leaves withered Normal growth 2X The whole plant died Heart leaves turn white 4X The whole plant died The heart leaves are white or curled 8X The whole plant died White, curled leaves and stunted growth 16X The whole plant died The whole plant died

[0613] Fresh weight and fresh weight inhibition rate of the herbicide haloxyfop-R-methyl

[0614]

[0615]

[0616] Symptoms of phytotoxicity after spraying the herbicide tebufenozide

[0617] Dosage ratio Indica rice - WT <![CDATA[ACC N1791S / I1792V ]]> CK Normal growth Normal growth 1X Heart leaves wither and growth is stunted Normal growth 2X The whole plant died Normal growth 4X The whole plant died White heart leaves and stunted growth 8X The whole plant died Heart leaves withered 16X The whole plant died Heart leaves withered

[0618] Fresh weight and fresh weight inhibition rate of the herbicide tebufenozide

[0619]

[0620] Symptoms of herbicide clethodim damage

[0621] Dosage ratio Indica rice - WT <![CDATA[ACC N1791S / I1792V ]]> CK Normal growth Normal growth 1X The whole plant died White heart leaves and stunted growth 2X The whole plant died Heart leaves withered 4X The whole plant died Heart leaves withered 8X The whole plant died The whole plant died 16X The whole plant died The whole plant died

[0622] Fresh weight and fresh weight inhibition rate after spraying the herbicide clethodim

[0623]

[0624] Symptoms of herbicide damage from spraying clodinafop-propargyl

[0625]

[0626]

[0627] Fresh weight and fresh weight inhibition rate after spraying the herbicide clodinafop-propargyl

[0628]

[0629] Wild-type indica rice plants (Indica-WT) and edited plants ACC N1791S / I1792V Resistance results to herbicides quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, haloxyfop-R-methyl, and clodinafop-P-ethyl are as follows: Figure 7 As shown.

[0630] Combining the above-mentioned wild-type indica rice plants WT and edited plants ACC N1791S / I1792V Symptoms of phytotoxicity, fresh weight, fresh weight inhibition rate and Figure 7 The herbicide resistance shown can be used to conclude that:

[0631] Wild-type indica rice plants (WT) and edited plants (ACC) sprayed with water. N1791S / I1792V All are growing normally and there is no pesticide damage.

[0632] Wild-type indica rice plants sprayed with 1X, 2X, 4X, 8X, and 16X of quizalofop-P-ethyl all died (WT); edited plants sprayed with 1X of quizalofop-P-ethyl died (ACC). N1791S / I1792V The heart leaves turned white and then turned green again, showing no signs of pesticide damage; the plants were sprayed with 2X and 4X quizalofop-p-ethyl. N1791S / I1792V The heart leaves are white, indicating slight herbicide damage; the plant was sprayed with 8X and 16X quizalofop-P-ethyl. (Edited plant ACC) N1791S / I1792V Dead; visible plant ACC edit N1791S / I1792V It exhibits strong herbicide resistance to quizalofop-P-ethyl.

[0633] Wild-type indica rice plants sprayed with 1X of high-efficiency flupyridine haloxyfop-R-methyl exhibited withered central leaves, stunted growth, and severe herbicide damage (WT). All wild-type indica rice plants sprayed with 2X, 4X, 8X, and 16X of high-efficiency flupyridine haloxyfop-R-methyl (WT) died (WT). Edited plants sprayed with 1X of high-efficiency flupyridine haloxyfop-R-methyl (ACC) showed... N1791S / I1792V Normal growth, no pesticide damage; ACC of plants sprayed with high-efficiency flupyridine 2X and 4X was edited. N1791S / I1792V The heart leaves are white, indicating mild pesticide damage; the plants were sprayed with high-efficiency fluopyram 8X. N1791S / I1792V The plants were stunted and growing slowly, exhibiting some pesticide damage; the plants were sprayed with high-efficiency flupyradifurone 16X. N1791S / I1792V Death; visible editing of plant ACC N1791S / I1792V It exhibits strong herbicide resistance to haloxyfop-R-methyl.

[0634] Wild-type indica rice plants sprayed with 1X, 2X, 4X, 8X, and 16X of clethodim all died (WT); plants sprayed with 1X of clethodim died (ACC). N1791S / I1792V The heart leaves are white, indicating mild herbicide damage; the ACC of plants treated with 2X and 4X clethodim was edited. N1791S / I1792V Some leaves withered, indicating some herbicide damage; the ACC of the plants was edited after spraying with 8X and 16X clethodim. N1791S / I1792VDeath; visible editing of plant ACC N1791S / I1792V It exhibits strong herbicide resistance to clethodim.

[0635] Wild-type indica rice plants sprayed with 1X clethodim exhibited withered central leaves, stunted growth, and severe phytotoxicity (WT); wild-type indica rice plants sprayed with 2X, 4X, 8X, and 16X clethodim all died (WT); and plants sprayed with 1X and 2X clethodim showed ACC... N1791S / I1792V The plants grew normally, with no withered leaves and no pesticide damage; the ACC of the plants treated with chlorpyrifos 4X was edited. N1791S / I1792V The heart leaves are turning white, indicating some phytotoxicity; spraying with 8X and 16X chlorpyrifos edited the plant ACC. N1791S / I1792V The heart leaves are withered, the plant is stunted, and it shows severe pesticide damage; (The plant's ACC is visible in the image.) N1791S / I1792V It exhibits strong herbicide resistance to clopyralid.

[0636] Wild-type indica rice plants sprayed with 1X of azoxystrobin showed severe WT (whole heart leaf) damage, stunted growth, and significant herbicide damage; all wild-type indica rice plants sprayed with 2X, 4X, 8X, and 16X of azoxystrobin died; and plants sprayed with 1X of azoxystrobin showed ACC (accumulated chorionic acid) damage. N1791S / I1792V Normal growth, no herbicide damage; ACC of plants sprayed with 2X cyclohexane was edited. N1791S / I1792V The heart leaves are white, indicating mild herbicide damage; the plant ACC was edited after spraying with 4X, 8X, and 16X of cyclohexane. N1791S / I1792V Some leaves were withered, and the plant was stunted, indicating some degree of pesticide damage; (The plant's condition was thus compromised.) N1791S / I1792V It exhibits strong herbicide resistance to clodinafop-propargyl.

[0637] The above results indicate that the mutations at amino acid positions 1791 and 1792 of the ACC protein in indica rice relative to the sequence of SEQ ID No. 3 can confer strong resistance to ACC-inhibiting herbicides (e.g., quizalofop-P-ethyl, haloxyfop-R-methyl, clethodim, clethodim, and clodinafop-P-ethyl).

[0638] In summary, compared to SEQ ID No. 1, a single point mutation at amino acid 1878 or a double-site mutation at amino acids 1878 and 1879 in the rice ACC protein; or compared to SEQ ID No. 3, a single point mutation at amino acid 1791 or a double-site mutation at amino acids 1791 and 1792 in the rice ACC protein can confer strong herbicide resistance to ACCase inhibitor herbicides in rice.

[0639] Example 4: Homology and conservation of the ACCase gene in different plants

[0640] The ACCase gene shows high homology in different monocotyledonous and dicotyledonous plants, and the sequence similarity of its encoded amino acid sequences is also high. In wheat (monocotyledonous), the amino acid sequence encoded by the ACCase gene is shown in SEQ ID No. 10; in maize (monocotyledonous), it is shown in SEQ ID No. 11; in soybean (dicotyledonous), it is shown in SEQ ID No. 12; and in Arabidopsis thaliana (dicotyledonous), it is shown in SEQ ID No. 13. The sequence similarity between the amino acid sequences encoded by the ACCase genes of wheat, maize, soybean, and Arabidopsis thaliana and the amino acid sequence encoded by the ACCase gene of rice (monocotyledonous) (SEQ ID No. 3) is 84%, 84.2%, 69.5%, and 67.8%, respectively.

[0641] The amino acid sequence encoded by the wheat ACCase gene is as follows (SEQ ID No. 10):

[0642]

[0643] The amino acid sequence encoded by the maize ACCase gene is as follows (SEQ ID No.11):

[0644] msqlglaataskalpllpnrqrspagttfpssalprpsnrrkshtrslrdggnevsdakkhsqsvrqglagiidipsdavsevdishgpkdprgptdsyqmngiinethngrhasgsmvvefcaalggktpihsilvanngmaatkfmrsvrtwandtfgsekaihliamatpedmrinaehirladqfvevpggtnnnnyanvqliveiaervgvsavwpgwghasenpelpdaltakgivflgppatsmnalgdkvgsaliaqaagvptlawsgshvevpleccldaipeemyrkacvttteeavascqvvgypamikaswggggkgirkvhnddevralfkqvqgevpgspifimrlasqsrhlevqllcdqygnvaalhsrdcsvqrrhqkiieegpvtvapretvkaleqaarrlakavgyvgaatveylysmetgeyyflelnprlqvehpvtewiaevnlpaaqvavgmgiplwqipeirrfygmdygggydiwrktaalaapfnfdevdslwpkghcvavritsedpddgfkptggkvkeisfkskpnvwayfsvksgggihefadsqfghvfayglsrpaaitnmslalkeiqirgeihsnvdytvdllnasdfrenkihtgwldtriamrvqaerppwyisvvggalyktvttnaatvseyvsyltkgqippkhislvnstvnlniegskytietvrtghgryklrmndstveanvqslcdggllmqldgnshviyaeeeaggtrlqingktcllqndhdpskllaetpckllrflvadgahvgadvpyaevevm

[0645] kmcmpllspasgvihcmmsegqalqagdliarldlddpsavkraepfdgmfplmdlpvaassqvhkryaaslnaarmvlagyehni

[0646] nevvqdlvccldnpelpflqwdelmsvlatrlprnlkseledkykeyklnfyhgknkdfpskllrdiveenlaygsekekatnerlvepl

[0647] mnllksyeggreshahfvvkslfeeyltveelfsdgiqsdvietlrhqhskdlqkvvdivlshqgvrnkaklvtalmeklvypnpgayrdl

[0648] lvrfsslnhkryyklalkaselleqtklselcssiarslsdlgmhkgemtikdsmedlvsaplpvedalislfdysdptvqqkvivtyisrlyq

[0649] phlvkdsiqmkfkesgaivfwefseghvdtrngqgailggkrwgamvvlrslesastaimaalkdsvqynnsevntmhivllnaetes

[0650] nisgtssddqaqhrmekltkilkdssvasdlqaaglkviscivqrdagrmpmrhtflwfdekncyeeehilrhvepplsallelgklkvkg

[0651] ynemkytpsrdrqwhiytlrntenpkmlhrvffrtivrqpnagnkftsaqvsdtglgcpeeslsftsnsilrslmtaieelelhairtghshm

[0652] flcilkeqklldlvpfsgstivdvgqdeatacsllrsmalkihelvgarmhhlsvcqwevklkldcdgpasgtwrvvttnvtshtctidiyre

[0653] vedtesqkllyhsatssagpmhgvalnnpyqplsvidlkrcsarnnrttycydfplafetalqkswqsncssvpegsensksyvkstelvf

[0654] aekhgswgtpiipmerpaglndigmvawilemstpefpngrqiivvanditfragsfgpredaffeavtnlacerklpliylaansgarig

[0655] iadevkscfrvgwsderspergfqyiylteedyarisssviahklqldngeirwiidsvvgkedglgvenihgsaaiasaysrayeetftltf

[0656] vtgrtvgigaylarlgirciqrldqpiiltgfsalnkllgrevysshmqlggpkimatngvvhltvsddlegvsnilrwlsyvpaniggplpit

[0657] kpldppdrpvayipentcdpraairgvddsqgkwlggmfdkdsfvetfegwaktvvtgraklggipvgviavetqtmmqlipadpgq

[0658] ldshersvpragqvwfpdsatktaqalldfnreglplfilanwrgfsggqrdlfegilqagsaivenlrtynqpafvyipmagelrggawv

[0659] vidskinpdriecyaertakgnvlepqglieikfrseelqdcmgrldpelinlkaklqdakhgngslpdieslqksieartkqllplytqiavr

[0660] faelhdtslrmaakgvikkvvdweesrsffykrlrrrisedllakeirriigdnfthqsamelinewylasqattgstagwddddafvawkdspenykgyiqelraqkvsqslsdladsssdlqafsqglstlldkmdpsqrvkfvqevkkvlg;

[0661] The amino acid sequence encoded by the soybean ACCase gene is as follows (SEQ ID No. 12):

[0662] madigrrngyansvlpnrppaaisevdefcnalggnrpihsilianngmaavkfirsvrswayetfgsekaillvamatpedmrinaehi

[0663] riadqfvevpggtnnnnyanvqlilemaeithvdavwpgwghasenpelpdalkakgivflgppaismaalgdkigssliaqaaevpt

[0664] lpwsgshvkippesslitipdeiyreacvytteeavascqvvgypamikaswggggkgirkvhnddevralfkqvqgevpgspifimk

[0665] vasqsrhlevqllcdqygnvaalhsrdcsvqrrhqkiieegpitvapietvkkleqaarrlaisvnyvgaatveylysmetgeyyflelnprl

[0666] qvehpvtewiaeinlpaaqvaigmgvplwqipeirrfygvehgggydawrktsvlatpfdfdkaqstrpkghcvavrvtsedpddgfk

[0667] ptsgkvqelnfkskpnvwayfsvksgggihefsdsqfghvfafgesralaianmvlglkeiqirgeirtnvdytidllnasdyrenkihtg

[0668] wldsriamrvraerpawylsvvggalykasassaalvsdyvgylekgqippkhislvhsqvslniegskytidmirggsgsyrlrmnqs

[0669] eieaeihtlrdggllmqldgnshviyaeeeaagtrllidgrtcllqndhdpsklvaetpckllrylvaddshvdadtpyaevevmkmcmp

[0670] llspasgiihfkmsegqamqageliarldlddpsavrkaepftgsfpvlgpptaisgkvhqkcaaslnaarmilsgyehnidevvqsllnc

[0671] ldspelpflqwqeclavlatrlpkelkneleskykefegisssqivdfpakllkgiieahlsscpdkekgaqerlvepllslvksyeggresh

[0672] ahiivqslfdeylsveelfsdniqadvierlrlqykkdllkivdivlshqgiksknklilqlmdklvypnpvayrdqlirfsllnhtnyselalk

[0673] asqlleqtklselrsniarslselemftedgenidtpkrksaindrmedlvsapfavedalvglfdhsdhtlqrrvvesyirrlyqpylvkgsa

[0674] rmqwhrsgliatwefydeyierkngvedqslsktveekhsekkwgvmviikslqflpaiitaalreatnnphealtsgsvepvnygnm

[0675] mhiglvginnqmsllqdsgdedqaqerinklakilkeqevgstiraagvgvisciiqrdegrapmrhsfhwseeklyyaeepllrhlepp

[0676] lsiyleldklkayenirytpsrdrqwhlytvvdhkpqpiqrmflrtlvrqpttnegfssyqrldaetsrtqlamsftsrsifrslmaameelel

[0677] nahnvniksehahmylyiireqqiddlvpypkrinieagkeeitveavleelareihssvgvrmhrlgvvvweiklwmaacgqanga

[0678] wrvivnnvtghtctvhlyrekedtithkvvyssvsvkgplhgvavnenyqplgvidrkrlsarknsttycydfplafetaleqswaiqqpg

[0679] fqrakdknllkvtelkfadkegswgtplvpvenypglndvgmvawfmemctpefpsgrtilvvandvtfkagsfgpredaffravtdl

[0680] actkklpliylaansgarlgvaeevkscfrvgwseesnpengfqyvyltpednarigssviahelklesgetrwvidtivgkedglgvenls

[0681] gsgaiagaysrayketftltyvtgrtvgigaylarlgmrciqrldqpiiltgfsalnkllgrevysshmqlggpkimatngvvhltvsddleg

[0682] vssilkwlsyipshvggalpivkpldpperpveyfpenscdpraaisgtldgngrwlggifdkdsfvetlegwartvvtgraklggipvgv

[0683] vavetqtvmqiipadpgqldshervvpqagqvwfpdsatktaqaildfnreelplfilanwrgfsggqrdlfegilqagstivenlrtykqp

[0684] ifvyipmmgelrggawvvvdsrinsdhiemyadrtakgnvlepegmieikfrtrellesmgrldqqlitlkaklqeakssrnivafeslq

[0685] qqiksrerqllpvytqiatkfaelhdtslrmaakgvirevldwrnsrsvfyqrlhrrigeqslinsvrdaagdqlshasamnllkewylnsdiakgredawlddeaffrwkdipsnyenklkelrvqkvllqltnigdsaldlqalpqglaallskleplgrvkltdelrkvlg;

[0686] The amino acid sequence encoded by the Arabidopsis ACCase gene is as follows (SEQ ID No. 13):

[0687] magsvngnhsavgpginyetvsqvdefckalrgkrpihsilianngmaavkfirsvrtwayetfgtekaillvgmatpedmrinaehiri

[0688] adqfvevpggtnnnnyanvqlivemaevtrvdavwpgwghasenpelpdaldakgiiflgppassmaalgdkigssliaqaadvptl

[0689] pwsgshvkippnsnlvtipeeiyrqacvytteeaiascqvvgypamikaswggggkgirkvhnddevralfkqvqgevpgspifimk

[0690] vasqsrhlevqllcdkhgnvsalhsrdcsvqrrhqkiieegpitvappetvkkleqaarrlaksvnyvgaatveylysmdtgeyyflelnp

[0691] rlqvehpvtewiaeinlpaaqvavgmgiplwqipeirrfygiehgggydswrktsvvafpfdfdkaqsirpkghcvavrvtsedpddg

[0692] fkptsgrvqelsfkskpnvwayfsvksgggihefsdsqfghvfafgesralaianmvlglkeiqirgeirtnvdytidllhasdyrdnkiht

[0693]

[0694] The amino acid sequences encoded by the ACCase genes in rice, wheat, maize, soybean, and Arabidopsis thaliana are as follows: Figure 8 As shown, the amino acid sequences encoded by the ACCase gene in rice, wheat, maize, soybean, and Arabidopsis are relatively conserved, especially in monocotyledonous plants (rice, wheat, and maize). In particular, amino acids 1781-1799 (vgkedglgvenihgsaaia) in the sequence shown in SEQ ID No. 3 are highly conserved across species. Furthermore, amino acids 1791 and 1792 of the sequence shown in SEQ ID No. 3 in Example 1 are also located within this conserved region.

[0695] In addition, according to Figure 8 It is known that amino acid N at position 1791 of the amino acid sequence encoded by the rice ACCase gene (SEQ ID No. 3) corresponds to amino acid N at position 1768 of the amino acid sequence encoded by the wheat ACCase gene (SEQ ID No. 10), amino acid N at position 1783 of the amino acid sequence encoded by the maize ACCase gene (SEQ ID No. 11), amino acid N at position 1718 of the amino acid sequence encoded by the soybean ACCase gene (SEQ ID No. 12), and amino acid N at position 1713 of the amino acid sequence encoded by the Arabidopsis thaliana ACCase gene (SEQ ID No. 13). The amino acid I at position 1792 of the rice ACCase gene (SEQ ID No. 3) corresponds to the amino acid I at position 1769 of the wheat ACCase gene (SEQ ID No. 10), the amino acid I at position 1784 of the maize ACCase gene (SEQ ID No. 11), the amino acid L at position 1719 of the soybean ACCase gene (SEQ ID No. 12), and the amino acid L at position 1714 of the Arabidopsis thaliana ACCase gene (SEQ ID No. 13).

[0696] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for conferring herbicide resistance to plants or a method for preparing herbicide-resistant plants, said method comprising the step of introducing a mutant acetyl-CoA carboxylase into plant cells, plant seeds, plant tissues, or plants; characterized in that, Compared with the parental acetyl-CoA carboxylase, the mutated acetyl-CoA carboxylase has the following amino acid sequences: amino acid 1878 of the amino acid sequence shown in SEQ ID No. 1 is mutated to S; and amino acid 1879 is mutated to V; or amino acid 1791 of the amino acid sequence shown in SEQ ID No. 3 is mutated to S; and amino acid 1792 is mutated to V. The herbicide is haloxyfop-R-methyl, haloxyfop-R-methyl, or clopyralid. The plant in question is rice.

2. The method according to claim 1, characterized in that, The method includes the step of expressing the gene encoding a mutated acetyl-CoA carboxylase in plant cells, plant seeds, plant tissues, or plants.

3. The method according to claim 2, characterized in that, The method includes the step of mutating the gene encoding endogenous acetyl-CoA carboxylase in plants to introduce the mutated acetyl-CoA carboxylase.

4. Use of an acetyl-CoA carboxylase mutant, a nucleotide encoding the mutant, a nucleic acid construct containing the nucleotide, or a host cell containing the nucleotide in the preparation of herbicide-resistant plants; The herbicide is haloxyfop-R-methyl, haloxyfop-R-methyl, or clopyralid. The plant in question is rice; The acetyl-CoA carboxylase mutant corresponds to the mutation of amino acid position 1878 to S and amino acid position 1879 to V in the amino acid sequence shown in SEQ ID No. 1; or the mutation of amino acid position 1791 to S and amino acid position 1792 to V in the amino acid sequence shown in SEQ ID No.

3.

5. Use of an acetyl-CoA carboxylase mutant, a nucleotide encoding the mutant, a nucleic acid construct containing the nucleotide, or a host cell containing the nucleotide in the preparation of a reagent or kit; said reagent or kit being used to prepare herbicide-resistant plants; The herbicide is haloxyfop-R-methyl, haloxyfop-R-methyl, or clopyralid. The plant in question is rice; The acetyl-CoA carboxylase mutant corresponds to the mutation of amino acid position 1878 to S and amino acid position 1879 to V in the amino acid sequence shown in SEQ ID No. 1; or the mutation of amino acid position 1791 to S and amino acid position 1792 to V in the amino acid sequence shown in SEQ ID No.

3.

6. A method for preparing herbicide-resistant hybrid plants, the method comprising the step of hybridizing the plants prepared by the method according to any one of claims 1-3 with other plants.

7. A method for controlling weeds in farmland, characterized in that: a) Planting the plants prepared by the method according to any one of claims 1-3 in farmland. b) Apply an effective amount of herbicide to the plant and the weeds nearby to control the weeds near the plant; The herbicide is haloxyfop-R-methyl, haloxyfop-R-methyl, or clodinafop-R-methyl.

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