WO2022166955A1 - Novel herbicide-resistant acetyl-coa carboxylase mutant and use thereof - Google Patents

Abstract

Provided are a mutant type acetyl-CoA carboxylase (ACC) protein, a nucleic acid and use thereof in plant breeding. The mutant type acetyl-CoA carboxylase (ACC) protein, compared with a parent acetyl-CoA carboxylase (ACC) protein, has mutations at any one or more of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID NO: 1: position 2125, position 2097, position 2139, position 2194, position 2186, position 2273, position 2168, position 1975, position 1954, position 1864, position 2211, position 2187, position 2123, and position 2126; and acetyl-CoA carboxylase (ACC) mutated plants have high herbicide resistance, and can be cultivated as herbicide-resistant plants.

Description

Novel herbicide-resistant acetyl-CoA carboxylase mutants and their applications technical field

The invention belongs to the fields of biotechnology and crop genetics and breeding, in particular to an acetyl-CoA carboxylase mutant protein, a nucleic acid, and a method and application for improving plants' antibodies to herbicides.

Background technique

Rice (Oryza sativa) is eaten by two-thirds of the world's population and is the main source of energy in the diet of at least half of them. Rice is a low-cost food that is easy, quick to prepare, and can be eaten with a variety of dishes.

The use of herbicides to control weeds or plants in crops has become an almost universal practice. As an important part of modern agricultural production system, herbicides are the most reliable and economical means of weeding technology in farmland. Since the use of 2,4-D in the 1940s, the herbicide industry has developed for more than 60 years, and a large number of selective herbicides have been successfully developed so far. Research on ACC enzyme inhibitors began in the 1970s. ACC herbicides are divided into four types, namely aryloxyphenoxypropionates.

(Aryloxyphenoxyp ropanoates, APP), oxime ether cyclohexanedione (Cyclohexanedione oximes, CHD), aryloxyphenyl cyclohexanedione (Aryloxyphenylcy-clohexanedione, APCHD) and triketone cyclohexanedione (Cyclict riketones, CTR) ). ACC herbicides can inhibit fatty acid synthesis in gramineous plants, have high selectivity, conduct in plants, and can control annual or perennial gramineous weeds in post-emergence.

It has the advantages of high efficiency, low toxicity, long application period, and safety to subsequent crops, so it occupies an important position in the herbicide market.

Acetyl CoA carboxylase (ACCase, ACC) is an important target of chemical herbicides and is a biotin-containing enzyme discovered in 1958. It catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA in vivo, provides substrates for the synthesis of fatty acids and many secondary metabolites, and is a key or rate-limiting enzyme in fatty acid biosynthesis. This carboxylase has a two-step reversible reaction, including the ATP-dependent carboxylation of the biotin group on the substrate domain by the enzymatic activity of biotin-carboxylase, and then the transfer of the carboxyl group from biotin by carboxyltransferase to obtain Acetyl-CoA substrate. Acetyl-CoA carboxylase is a key enzyme in plant biosynthesis of fatty acids, which occurs in chloroplasts and mitochondria, and ACC also plays a role in the formation of long-chain fatty acids and flavonoids and in malonylation that occurs in the cytoplasm.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a mutant acetyl-CoA carboxylase (ACC) protein or polynucleotide which can impart herbicide resistance to plants and its application.

Herein, ACCase or ACC refers to Acetyl CoA carboxylase (Acetyl CoA carboxylase).

Mutant acetyl-CoA carboxylase (ACC)

In one aspect, the present invention provides a mutant acetyl-CoA carboxylase (ACC), the amino acid sequence of the mutant acetyl-CoA carboxylase (ACC) and the parent acetyl-CoA carboxylase (ACC) In contrast, there are mutations at any or any of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No.1: 2125, 2097, 2139, 2194, 2186, Position 2273, Position 2168, Position 1975, Position 1954, Position 1864, Position 2211, Position 2187, Position 2123, Position 2126; There is a mutation at any or any of the following amino acid positions in the sequence: position 2125, position 2097, position 2139, position 2194, position 2186.

In one embodiment, the amino acid positions 2186 and 2187 are mutated simultaneously, or the amino acid positions 2123 and 2125 are mutated simultaneously, or the amino acid positions 2125 and 2126 are mutated simultaneously mutation at the same time.

In one embodiment, bit 2273, bit 2194, bit 2168, bit 1975, bit 1954, bit 1864, bit 2097, bit 2211, bit 2139, bit 2186, bit 2187, The 2123rd, 2125th and 2126th amino acid positions are S, G, R, S, P, I, W, E, I, C, Y, A, W, R, respectively.

In one embodiment, the amino acid at position 2273 is mutated to a non-S amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, T, C , P, H, R, I; preferably, F.

In one embodiment, the amino acid at position 2194 is mutated to a non-G amino acid, eg, A, V, L, Q, F, W, Y, D, N, E, K, M, S, T, C , P, H, R, I; preferably, S or A.

In one embodiment, the amino acid at position 2168 is mutated to a non-R amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , C, P, H, I; preferably, P.

In one embodiment, the amino acid at position 1975 is mutated to a non-S amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, T, C , P, H, R, I; preferably, F.

In one embodiment, the amino acid at position 1954 is mutated to a non-P amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , C, H, R, I; preferably, S.

In one embodiment, the amino acid at position 1864 is mutated to a non-I amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , C, P, H, R; preferably, V.

In one embodiment, the amino acid at position 2097 is mutated to an amino acid other than W, eg, A, V, G, L, Q, F, Y, D, N, E, K, M, S, T, C , P, H, R, I; preferably, S or G.

In one embodiment, the amino acid at position 2211 is mutated to a non-E amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, K, M, S, T, C , P, H, R, I; preferably, K.

In one embodiment, the amino acid at position 2139 is mutated to a non-I amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , C, P, H, R; preferably, V or N.

In one embodiment, the amino acid at position 2186 is mutated to a non-C amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , P, H, R, I; preferably, R or H.

In one embodiment, the amino acid at position 2187 is mutated to a non-Y amino acid, eg, A, V, G, L, Q, F, W, D, N, E, K, M, S, T, C , P, H, R, I; preferably, H.

In one embodiment, the amino acid at position 2123 is mutated to a non-A amino acid, eg, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T, C , P, H, R, I; preferably, T.

In one embodiment, the amino acid at position 2125 is mutated to an amino acid other than W, eg, A, V, G, L, Q, F, Y, D, N, E, K, M, S, T, C , P, H, R, I; preferably, C or S.

In one embodiment, the amino acid at position 2126 is mutated to a non-R amino acid, eg, A, V, G, L, Q, F, W, Y, D, N, E, K, M, S, T , C, P, H, I; preferably, K.

In a preferred embodiment, the 2125th amino acid is mutated to C; the 2097th amino acid is mutated to G; the 2139th amino acid is mutated to V or N; the 2194th amino acid is mutated to A; The amino acid at position 2186 was mutated to H.

In one embodiment, the parent acetyl-CoA carboxylase (ACC) can be derived from any plant.

In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of: Poaceae, Legumes, Chenopodiaceae, Cruciferous plants.

In one embodiment, the parental acetyl-CoA carboxylase (ACC) is derived from one or more plants selected from the group consisting of Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, Soybeans, tomatoes, potatoes, quinoa, lettuce, canola, cabbage, strawberries.

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

In one embodiment, the parental acetyl-CoA carboxylase (ACC) has ACC activity, and the amino acid sequence of the parental ACC has at least 80%, at least 85%, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.

In a preferred embodiment, the amino acid sequence of the parental ACC has the sequence shown in SEQ ID No. 1.

In a preferred embodiment, the amino acid sequence of the parental ACC is shown in SEQ ID No. 1.

In another aspect, the present invention provides a mutant acetyl-CoA carboxylase (ACC), the mutant acetyl-CoA carboxylase (ACC) is selected from any of the following groups I-III:

I. A mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at any or any of the following amino acid positions: position 2273, position 2194, position 2168, position 1975, No. 1954, No. 1864, No. 2097, No. 2211, No. 2139, No. 2186, No. 2187, No. 2123, No. 2125, No. 2126;

II. Compared with the mutant ACC described in I, it has the mutation site described in I; and, compared with the mutant ACC described in I, 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% sequence identity of mutant ACC and retain herbicide resistance activity;

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

In other embodiments, the mutant acetyl-CoA carboxylases of the present invention also include other herbicide-resistance mutation sites.

It is clear to those skilled in the art that the structure of a protein can be altered without adversely affecting its activity and functionality, for example one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without affecting the activity and/or activity of the protein molecule. Three-dimensional structure adversely affects. Examples and embodiments of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, that is, another non-polar amino acid residue can be substituted with a non-polar amino acid residue, and polar and uncharged An amino acid residue that is substituted for another polar, uncharged amino acid residue, a basic amino acid residue for another basic amino acid residue, and an acidic amino acid residue for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions in which one amino acid is replaced by other amino acids belonging to the same group are within the scope of the present invention as long as the substitution does not result in inactivation of the biological activity of the protein. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably generated by substitution according to Table 1. In addition, the present invention also encompasses proteins that also contain one or more other non-conservative substitutions, so long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

Table 1

initial residue	representative substitution	Preferred substitution
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

Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be altered (deletion, substitution or substitution) without altering biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Amino acid substitutions can be made in non-conserved regions of ACC. Generally, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, as will be understood by those skilled in the art, functional variants may have minor conservative or non-conservative changes in conserved regions.

It is well known in the art that one or more amino acid residues can be altered (substituted, deleted, truncated or inserted) from the N and/or C terminus of a protein and still retain its functional activity. Accordingly, proteins in which one or more amino acid residues are altered from the N and/or C terminus of the ACC protein while retaining its desired functional activity are also within the scope of the present invention. These changes may include those introduced by modern molecular methods such as PCR, including PCR amplifications that alter or extend protein-coding sequences by including amino acid-coding sequences in oligonucleotides used in PCR amplifications.

It will be appreciated that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the ACC protein can be prepared by mutation of DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, e.g., single or multiple amino acid substitutions, using known mutagenesis, recombination and/or shuffling methods, in conjunction with relevant screening methods, Deletions and/or insertions.

Those skilled in the art will appreciate that these minor amino acid changes in the ACC proteins of the invention can occur (eg, naturally occurring mutations) or produced (eg, using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide can be altered, but the polypeptide can retain its activity. Smaller effects can be expected if mutations are present that are not in close proximity to the catalytic domain, active site, or other functional domain.

Those skilled in the art can identify the essential amino acids of ACC proteins according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics. The catalytic domain, active site, or other functional domain of a protein can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with putative key sites. point amino acid mutation.

fusion protein

In another aspect, the present invention provides a fusion protein comprising the mutant ACC protein of the present invention; further, the fusion protein further includes: a tag peptide such as a histidine tag, 6×His, or a plastid guide peptide , such as peptides directed into the chloroplast, or regulatory elements such as promoter sequences, terminator sequences, leader sequences, polyadenylation sequences, marker genes, and the like.

polynucleotide

In another aspect, the present invention provides a polynucleotide encoding the mutant ACC protein or an active fragment thereof.

In one embodiment, the polynucleotide is selected from the group consisting of a genomic sequence, a cDNA sequence, an RNA sequence, or a combination thereof.

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

In one embodiment, the polynucleotides additionally contain auxiliary elements selected from the group consisting of signal peptides, secretory peptides, tag sequences (such as 6His), nuclear localization signals (NLS) flanking the ORF of the mutein. ) or a combination thereof.

In one embodiment, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the mutant polypeptide.

In one embodiment, the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.

nucleic acid construct

In another aspect, the present invention provides a nucleic acid construct comprising the polynucleotide and regulatory elements operably linked thereto.

In one embodiment, the regulatory elements are selected from one or more of the group consisting of enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, marker genes.

carrier

The present invention also provides a vector comprising a nucleic acid sequence encoding the mutant ACCase or fusion protein of the present invention, preferably, the vector further includes an expression control element operably linked to the above nucleic acid sequence.

In one embodiment, the vectors include cloning vectors, expression vectors, shuttle vectors, and integration vectors.

In one embodiment, the vector may be a vector for gene editing of the ACC gene endogenous to the host cell.

In one embodiment, the expression vector further contains at least one origin of replication to achieve self-replication.

In one embodiment, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it is integrated.

Vectors can be of the type plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art.

Preferably, the vector in the present invention is a plasmid.

Edit carrier system

In another aspect, the present invention provides an editing vector system comprising one or more vectors comprising at least a leader sequence targeting a parental ACC.

The guide sequence contains part of the nucleotide sequence of the parental ACC, preferably at least 15bp of ACC nucleotide sequence, more preferably at least 20bp of ACC nucleotide sequence. In one embodiment, the editing vector further includes a gene editing enzyme. The gene editing enzymes include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), ZFN (Zinc Refers to nucleic acid technology, Zinc finger nuclease) editing tool nuclease.

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

Preferably, the Cas protein is operably linked to the first regulatory element.

In one embodiment, the gene editing enzyme is a Cas9 protein, and the vector further includes a Scaffold sequence that can specifically bind to the Cas9 protein. After the Scaffold sequence is operably linked to the guide sequence, the guide guide sequence (gRNA) is formed. Preferably, the gRNA is operably linked to the second regulatory element.

In other embodiments, the gene editing enzyme is a Cas12 protein, for example, Cas12a, Cas12b, Cas12i, and the vector further includes a unidirectional repeat sequence (Direct Repeat) that specifically binds to the Cas12 protein. The unidirectional repeat sequence is operably linked to the guide sequence and constitutes the guide guide sequence (gRNA). Preferably, the gRNA is operably linked to the second regulatory element.

Such 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 (such as transcription termination signals, such as polyadenylation signals and polyU sequences).

Preferably, the editing vector system further comprises a base editing element selected from adenine deaminase and/or cytosine deaminase.

In one embodiment, the editing vector also includes resistance genes for screening, the resistance genes include hyg, bar, kana, rif, spec, amp, and the resistance genes are well known to those skilled in the art .

Preferably, the Cas protein is nCas9 or other Cas9 proteins with nick activity. Wherein "n" represents nick, a Cas protein with only single-strand cleavage activity.

host cell

In another aspect, the present invention provides a host cell comprising the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), One or more of the fusion protein, vector and nucleic acid construct; or, the polynucleotide is integrated into the genome of the host cell.

In one embodiment, the host cell is a prokaryotic cell, such as E. coli.

In one embodiment, the host cells are plant cells including angiosperms and gymnosperms.

In one embodiment, the plants include monocotyledonous and dicotyledonous plants.

In one embodiment, the plants include herbaceous and woody plants.

In one embodiment, the plant comprises Arabidopsis, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, canola, cabbage, strawberry.

resistant plants

In another aspect, the present invention provides a herbicide-resistant plant, the plant comprising the mutant acetyl-CoA carboxylase (ACC), the encoding mutant acetyl-CoA carboxylase (ACC) One or more of the polynucleotide, the fusion protein, the vector and the nucleic acid construct; or the polynucleotide is integrated into the plant genome.

Methods of making mutant polypeptides

In another aspect, the present invention provides a method for preparing the mutant ACC polypeptide or an active fragment thereof, the method comprising the steps of:

(a) under conditions suitable for expression, culturing a host cell comprising the mutant ACC polypeptide, thereby expressing the mutant ACC polypeptide; preferably, the method further includes

(b) the step of isolating the mutant ACC polypeptide.

Methods of obtaining herbicide-resistant plants

In another aspect, the present invention provides a herbicide-resistant plant cell, plant seed, plant tissue, plant part, plant, wherein the plant cell, plant tissue, plant seed, plant part, plant contains the The mutant ACC polypeptide or its polynucleotide sequence.

In another aspect, the present invention provides a method for obtaining or preparing a herbicide-resistant plant cell, plant seed, plant tissue, plant part or plant, the method comprising in the plant cell, plant seed, plant tissue, plant The above-mentioned mutant ACC polypeptide or its polynucleotide sequence is introduced into part or plant.

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 polypeptides through an expression vector. The polypeptide is expressed, or the mutant polypeptide is integrated into the plant genome for expression.

In another preferred embodiment, the above-mentioned method comprises the following steps:

(1) Agrobacterium carrying an expression vector is provided, and the expression vector contains the DNA coding sequence of the mutant ACC polypeptide or its active fragment;

(2) contacting plant cells, plant tissues, and plant parts with the Agrobacterium in step (1), so that the DNA coding sequence of the mutant ACC polypeptide or its active fragment is transferred into plant cells, and integrated into the plant cells. on chromosomes; and

(3) Selecting plant cells into which the DNA coding sequence of the mutant ACC polypeptide or its active fragment has been transferred.

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; preferably, the mutant polypeptide can be introduced by means of gene editing.

In another preferred embodiment, the method comprises making the endogenous ACC coding sequence of plant cells, plant seeds, plant tissues and plant parts corresponding to positions 2273, 2194 and 2168 of SEQ ID No. 1 , any of the 1975th, 1954th, 1864th, 2097th, 2211th, 2139th, 2186th, 2187th, 2123rd, 2125th, 2126th amino acid positions or any number of mutated steps.

In another preferred embodiment, the method includes the following steps:

(1) introducing the aforementioned editing vector system into plant cells, plant seeds, plant tissues, and plant parts;

(2) Make the gene editing tool act on its endogenous ACC coding sequence, and make it corresponding to the 2273rd, 2194th, 2168th, 1975th, 1954th, The step of mutating any or any of the amino acid positions 1864, 2097, 2211, 2139, 2186, 2187, 2123, 2125, and 2126.

Further, the above method also includes the step of screening for mutant plant cells, plant tissues, plant parts, and optionally, isolating the gene editing tool.

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

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

In another preferred example, the plants include monocotyledonous plants and dicotyledonous plants.

In another preferred example, the plants include herbaceous plants and woody plants.

In another preferred example, the plants include Arabidopsis, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rape, cabbage, and strawberry.

Ways to Control Weeds

In another aspect, the present invention also provides a method for controlling the growth of weeds near a plant, comprising:

a) providing the above-mentioned herbicide-resistant plants;

b) applying an effective amount of a herbicide to the plant and weeds in its vicinity, thereby controlling the weeds in the vicinity of the plant.

The plant is preferably rice.

The herbicide is an ACCase inhibitor or an ACCase inhibitor herbicide, including Aryloxyphenoxypropanoates (APP), Cyclohexanedione oximes (CHD), aryloxyphenyl One or more of cyclohexanedione (Aryloxyphenylcy-clohexanedione, APCHD) and triketone cyclohexanedione (Cyclict riketones, CTR). Preferably, the herbicides include valprodofop-p-ethyl, diflufen, fenfluthofop, clethodim, cyclohexendione, cycloxydim, sethoxydim, pyrazolone, triclopyr ketone, phenpropion, propargoxy-ethyl, clodinafop-propargyl, chlorfenapyr, chlorfenapyr, fenflufen-ethyl, fenflufenafen, fenflufenafen, fenflufenafen, fenflufenafen , chlorfenapyr, fenflufop-ethyl, clofofop-ethyl, cyclofenazone, oxaprop, quizalofop, quizalofop-ethyl, quizalofop-ethyl, quizalofop-ethyl, trifluoro One or more of phenoxypropionic acid, pinoxaden, or a salt or ester thereof.

Preferably, the herbicide is one or more of valprodofop-ethyl, sethoxydim and clethodim.

use

In another aspect, the present invention provides the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the vector or the nucleic acid construct in Use in reagents or kits for the preparation of herbicide-resistant plants.

In another aspect, the present invention provides the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the vector or the nucleic acid construct in Use in weed control.

In another aspect, the present invention provides the mutant acetyl-CoA carboxylase (ACC), the gene encoding the mutant acetyl-CoA carboxylase (ACC), the fusion protein, the vector or the nucleic acid construct in Use in the preparation of herbicide-resistant plants.

herbicide

In one embodiment, the herbicide of the present invention is an ACCase-inhibiting herbicide, and the ACCase-inhibiting herbicide includes, but is not limited to, Aryloxyphenoxypropanoates (APP), oxime ether ring One or more of cyclohexanedione (Cyclohexanedione oximes, CHD), aryloxyphenylcy-clohexanedione (APCHD) and triketone cyclohexanedione (Cyclict riketones, CTR).

Preferably, the herbicides described in the present invention include, but are not limited to, halofopfop-p-ethyl, chlorfenapyr, fenfluthofop, clethodim, cyclohexendidione, cycloxydim, sethoxydim, pyran Triflufenazone, triclofenone, phenpropion, propargyl, clodinofop-ethyl, chloroprene, chlorfenapyr, fenflufen, fenflufen, thiazofen, diflufenapyr, Diflufenapyr One or more of furfuryl, trifluorophenoxypropionic acid, pinoxaden, or salts or esters thereof.

Preferably, the herbicide is one or more of valprodofop-p-ethyl, sethoxydim and clethodim.

General Definition

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

The terms "polynucleotide," "nucleotide sequence," "nucleic acid sequence," "nucleic acid molecule," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.

The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. Accordingly, the compositions and methods of the present invention also include homologues of the nucleotide sequences and polypeptide sequences of the present invention. "Homology" can be calculated by known methods including, but not limited to, Computational Molecular Biology (Lesk, A.M. ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects [Biocomputing: Informatics and Genome Projects] (Smith, D.W. ed.) Academic Press [Academic Press], New York (1993); Computer Analysis of Sequence Data, Part I [Computer Analysis of Sequence Data, Part I Section] (edited by Griffin, A.M. and Griffin, H.G.) Humana Press [Humana Press], New Jersey (1994); Sequence Analysis in Molecular Biology] (edited by von Heinje, G.) Academic Press [Academic Press] (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.) Stockton Press [Stockton Press], New York (1991).

The specific amino acid position (numbering) within the protein of the present invention is determined by aligning the amino acid sequence of the target protein with SEQ ID No. 1 using standard sequence alignment tools, such as Smith-Waterman algorithm or CLUSTALW2 The algorithm aligns two sequences, wherein the sequences are considered aligned when the alignment score is the highest. Alignment scores can be calculated as described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. Default parameters are preferably used in the ClustalW2(1.82) algorithm: protein gap opening penalty=10.0; protein gap extension penalty=0.2; protein matrix=Gonnet; protein/DNA endgap=-1; protein/DNAGAPDIST=4. The AlignX program (part of the vectorNTI group) is preferably used, with default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty of 0.05) as determined by comparing the amino acid sequence of the protein to SEQ ID No. 1 The positions of specific amino acids within the proteins of the present invention.

The term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, as defined in a polynucleotide having a defined nucleotide sequence (ie, rRNA, tRNA and mRNA) or a defined amino acid sequence and its Templates for the synthesis of other polymers and macromolecules during biological processes that generate biological properties. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system.

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.

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues thing. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

The term "mutein" or "mutant protein" refers to a protein that has one or more substitutions, insertions, deletions and/or additions of amino acid residues as compared to the amino acid sequence of the parent protein.

In the present invention, amino acid residues can be represented by single letters or three letters, 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), Glutamate (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), Arginine (Arg, R).

The term "AxxB" means that amino acid A at position xx is changed to amino acid B, eg S1975F means that S at position 1975 is changed to F. For double or multiple mutations, each mutation is separated by "/", e.g., A2123T/W2125C means, with respect to the amino acid sequence of SEQ ID No. 1, A at position 2123 is substituted with T and W at position 2125 replaced by C.

The term "regulatory element" also known as "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 (eg, transcription termination signals such as polyadenylation signals and poly U sequences), which are described in detail in Goeddel, "Gene Expression Technology". : GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In certain instances, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, lymphocytes). In certain instances, regulatory elements may also direct expression in a timing-dependent manner (eg, in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; CMV enhancer; R-U 5' fragment in the LTR of HTLV-1 ((Mol. Cell. Biol., p. 8( 1) Vol, pp. 466-472, 1988); the 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).

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

A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, ie, a protein with an NLS is transported to the nucleus. Typically, NLSs contain 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 proteins.

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

The term "vector" includes elements that allow the vector to integrate into the host cell genome or to replicate autonomously within the cell independent of the genome. The vector may contain any elements that ensure self-replication. It usually carries genes that are not part of the central metabolism of the cell, and is usually in the form of double-stranded DNA. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector 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 can be used.

The term "ACC inhibitor herbicides" refers to a class of herbicides that inhibit fatty acid synthesis in gramineous plants, have high selectivity and conduct in plants, and can control annual or perennial gramineous weeds after emergence. It has the advantages of high efficiency, low toxicity, long application period, and safety to subsequent crops, so it occupies an important position in herbicides.

ACC inhibitor herbicides are divided into 4 types, namely Aryloxyphenoxypropanoates (APP), Cyclohexanedione oximes (CHD), aryloxyphenylcyclohexane Aryloxyphenylcy-clohexanedione (APCHD) and triketone cyclohexanedione (Cyclict riketones, CTR), the results are shown below.

The herbicides include but are not limited to: haloxypyr, chlorfenapyr, bufendidione, clethodim, cyclohexendizone, cycloxydim, clethodim, clethodim, chlorpyrifos ketone, triclofenone, phenpropion, propargyl, clodinofop-ethyl, chlorfenapyr, chlorfenapyr, fenoxafen, fenflufenafen, fenoxafen, diflufenafen, fenox Diflufenapyr, Chlorfenapyr, Chlorfenapyr, Clofenafen, Cyclofop-ethyl, Oxalop-ethyl, Quizalofop-ethyl, Quizalofop-ethyl, Quizalofop-ethyl, Quizalofop-ethyl Furfuryl ester, trifluorophenoxypropionic acid, pinoxaden.

"Herbicide resistance" or "herbicide resistance" refers to the genetic ability of a plant to survive and reproduce after exposure to doses of herbicide that are normally 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 specified, herbicide "resistance" is heritable and allows plants to grow and reproduce in the presence of a herbicide that is typical of a herbicidal effective treatment of a given plant, as currently disclosed in the Herbicide Handbook at the time of filing version suggested. As will be appreciated by those skilled in the art, plants can still be considered "resistant" even if some degree of plant damage due to herbicide exposure is evident. As used herein, the term "tolerant" or "tolerance" includes "resistant" or "resistant" plants as defined herein, as well as the tolerance of a particular plant to the wild-type of the same genotype at the same herbicide dosage Improved capacity for various degrees of herbicide-induced injury, typically ethyl, in plants.

In one embodiment, the mutant polypeptide is at least 1-fold higher, for example, at least 1.5-fold higher, preferably at least 2-fold higher, preferably at least 2-fold higher, compared to the parent polypeptide, to the maximum ACC-inhibiting herbicide tolerance concentration At least 3 times, preferably at least 4 times, preferably at least 5 times, preferably at least 6 times, preferably at least 10 times.

In one embodiment, the plant containing the mutant polypeptide can tolerate at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, or at least 10-fold the recommended amount of an ACC-inhibiting herbicide concentration.

The terms "parental ACC polypeptide" and "parental ACC polypeptide" refer to the polypeptide from which the ACC mutant polypeptide is derived. In a preferred embodiment, the parental ACC polypeptide is a nucleic acid molecule that can be found in nature or Proteins (polypeptides) whose nucleotides can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., whose amino acid sequences can be derived from nucleotide sequences. The amino acid sequence of the wild-type ACC polypeptide, for example, is shown in SEQ ID No. 1; in certain embodiments, the parent ACC polypeptide may be one or more amino acid residues altered from the wild-type ACC polypeptide, A polypeptide that does not affect its enzymatic activity.

The terms "mutated ACC protein", "mutated ACC protein", "mutated ACC", "mutated ACC enzyme", "mutated protein", "mutated polypeptide", "polypeptide of the present invention", "protein of the present invention" and the like are interchangeable. Use instead.

The term "host organism" is understood to mean any unicellular or multicellular organism into which a mutant ACC protein-encoding nucleic acid can be introduced, including, for example, bacteria such as E. coli, fungi such as yeast (eg Saccharomyces cerevisiae), molds (eg Aspergillus), plant cells and plants etc.

The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, cabbage, Arugula, leeks, asparagus, lettuce (eg, head lettuce, leaf lettuce, romaine), bok choy, yellow taro, melons (eg, melon, watermelon, crenshaw ), white melon, romaine melon), canola crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), spinach, carrots, napa, Okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, zucchini, zucchini, squash), radishes, dried bulb onions, rutabagas, Purple eggplant (also called eggplant), salsify, endive, shallots, endive, garlic, spinach, green onions, zucchini, greens, beets (sugar beets and fodder beets), sweet potatoes, chard, Wasabi, tomatoes, turnips, and spices; fruits and/or trailing crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quinces, almonds, chestnuts, hazelnuts, pecans, Pistachios, walnuts, citrus, blueberries, boysenberry, cranberries, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple , tropical fruits, pomes, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, jasmine, corn/maize (fodder corn, sweet corn, popcorn), hops, lotus Barley, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oily plants (rapese, mustard, poppy, olive, sunflower, coconut, castor oil plants, cocoa beans, groundnuts), Arabidopsis, fibrous plants (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or flower bed plants, such as flowering plants, cacti, succulents and/or ornamentals, and trees such as forests (broadleaf and evergreen, such as conifers), fruit trees, ornamental Trees, and nut-bearing trees, as well as shrubs and other seedlings.

The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, shoots, seedlings, fruits , nucleus, ear, root, root tip, anther, etc.

The term "plant cell" is to be understood as any cell from or found in a plant, which is capable of forming, for example: undifferentiated tissue such as callus, differentiated tissue such as embryos, components of plants, plants or seeds.

The term "gene editing" technology includes CRISPR technology, TALEN technology, ZFN technology. CRISPR technology refers to clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats), which are derived from the immune system of microorganisms. Among them, gene editing tools include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, etc.). Gene editing tools referred to in TALEN technology are restriction enzymes that can cleave specific DNA sequences and include a TAL effector DNA binding domain and a DNA cleavage domain. Gene editing tools referred to in ZFN technology are also restriction enzymes that can cut specific DNA sequences, including a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that by constructing nucleotides encoding gene editing tools and other regulatory elements in a suitable vector, and then transforming cells, the editing of the intracellular genome can be achieved, and the types of editing include gene knockout, insertion , base editing.

As used herein, the term "gene editing enzyme" refers to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), ZFN (zinc finger nucleic acid technology, Zinc finger nuclease) and other editing tools nuclease. Preferably, the gene editing enzyme is CRISPR enzyme, also known as Cas protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein. Described Cas protein refers to the protein family, which can have different structures according to different sources, such as SpCas9 derived from Streptococcus pyogenes, SaCas9 derived from Staphylococcus aureus; Features (eg, domains) are subclassified, eg, the Cas12 family includes Cas12a (aka Cpf1), Cas12b, Cas12c, Cas12i, and others. The Cas protein can have double-chain or single-chain or no cleavage activity. The Cas protein of the present invention can be wild-type or a mutant thereof. The mutation type of the mutant includes amino acid substitution, substitution or deletion. The mutant can change or not change the enzyme cleavage activity of the Cas protein. . Preferably, the Cas protein of the present invention has only single-chain cleavage activity or no cleavage activity, and is a mutant of the wild-type Cas protein. Preferably, the Cas protein of the present invention is Cas9, Cas12, Cas13 or Cas14 with single-chain cleavage activity. In a preferred embodiment, the Cas9 proteins of the present invention include SpCas9n (D10A), nSpCas9NG, SaCas9n, ScCas9n, XCas9n, wherein "n" represents nick, that is, a Cas protein with only single-chain cleavage activity. Mutation of known Cas proteins to obtain Cas proteins with single-chain or no cleavage activity is a routine technical means in the art. As known to those skilled in the art, various Cas proteins with nucleic acid cleavage activity have been reported in the prior art, the known proteins or their modified variants can all achieve the functions of the present invention, which are incorporated herein by reference for protection scope.

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 a fragment of the mutant ACC protein that has one or more amino acid residues deleted from the N and/or C terminus of the mutant ACC protein, while retaining its desired functional activity (eg, containing the mutant of the present invention). amino acid fragments), which are also within the scope of the present invention, are referred to as biologically active fragments. In the present invention, "biologically active fragment" refers to a part of the mutant ACC protein of the present invention, which retains the biological activity of the mutant ACC protein of the present invention. For example, a biologically active fragment of a mutant ACC protein may be one or more (eg, 1-50, 1-25, 1-10, or 1-5 deleted) at the N and/or C terminus of the protein. 1, 2, 3, 4, or 5) amino acid residues that still retain the biological activity of the full-length protein.

In addition, the muteins of the present invention can also be modified. Modified (usually without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of muteins in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the mutein or in further processing steps. This modification can be accomplished by exposing the muteins to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are mutant proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

It will be clear to those skilled in the art that, due to the degeneracy of the genetic code, there are many different nucleic acid sequences that can encode the amino acid sequences disclosed herein. It is within the ability of one of ordinary skill in the art to generate other nucleic acid sequences encoding the same protein, and thus the present invention encompasses nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, in order to achieve high expression of a heterologous gene in a target host organism, such as a plant, the gene can be optimized for better expression using codons preferred by the host organism.

The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercial cDNA libraries or cDNAs prepared by conventional methods known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order. The obtained nucleotide sequence can be cloned into a vector, then transferred into cells, and then isolated from the propagated host cells by conventional methods to obtain large quantities of relevant sequences. The mutation site of the present invention can also be introduced by artificial synthesis.

More than one copy of a polynucleotide of the invention can be inserted into a host cell to increase the yield of the gene product. An increase in the copy number of a polynucleotide can be achieved 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 comprising amplified copies of the selectable marker gene and thus additional copies of the polynucleotide can be selected by artificially culturing the cells in the presence of an appropriate selectable agent.

Methods well known to those skilled in the art can be used to construct vectors containing the ACC mutant polypeptide-encoding DNA sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence is operably linked to an appropriate promoter in the vector to direct mRNA synthesis. The vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Vectors suitable for use in the present invention include commercially available plasmids such as but not limited to: pBR322 (ATCC37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) pQE70 , pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, 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), among others.

The present invention also provides host cells comprising a nucleic acid sequence, nucleic acid construct or expression vector encoding an ACC mutant polypeptide of the present invention. Introduction of a vector comprising the encoding of the present invention into a host cell allows the vector to exist as part of a chromosomal integrant or as a self-replicating extrachromosomal vector as described earlier, or the vector may perform gene editing of the ACC gene endogenous to the host cell. The host cell can be any host cell familiar to those skilled in the art, including prokaryotic cells and eukaryotic cells.

The nucleic acid sequences, nucleic acid constructs or expression vectors of the invention can be introduced into host cells by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid mediated gene delivery, and calcium phosphate transfection transfection, DEAE-dextran-mediated transfection, lipofection or electroporation, etc.

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, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

As used herein, the terms "guide RNA", "mature crRNA", "guide sequence" are used interchangeably and have the meanings commonly understood by those skilled in the art. In general, a guide RNA may comprise a direct repeat and a guide sequence, or consist essentially of or consist of a direct repeat and a guide sequence (also referred to as a spacer in the context of an endogenous CRISPR system) )composition.

In certain instances, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the leader 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%. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

The main advantages of the present invention:

1. The present invention screened out a group of mutant ACC proteins.

2. The plants containing the mutant ACC protein of the present invention have obvious herbicide resistance compared with wild-type plants.

Description of drawings

Figure 1. The evoAPOBEC1-SpRYCas9n base editor.

Figure 2. ABE8e-SpRYCas9n base editor.

Figure 3. Sequence structure of the ACCase gene.

Figure 4. Callus transformed with the CBE base-edited library differentiated into resistant seedlings (arrows).

Figure 5. Callus transformed with the ABE base-edited library differentiated into resistant seedlings (arrows).

Figure 6. Transformation of the CBE base-edited library produces resistant seedlings that root normally (arrows).

Figure 7. Transformation of the ABE base-edited library produces resistant seedlings that root normally (arrows).

Implementation

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the content of the solution of the present invention fall within the protection scope of the present invention.

Example 1. Base editing vector optimization, library construction and screening of herbicide-resistant mutation sites

1. Construct the rice evoAPOBEC1-SpRYCas9n (CBE) and ABE8e-SpRYCas9n (ABE) base editors with high efficiency and wide editing range.

The base editor can realize C/G->T/A(CBE) or A/T->G/C(ABE) base conversion within a certain sequence window, while evoAPOBEC1 and ABE8e are in the second generation Based on the optimization of CBEmax and ABEmax base editors, the efficiency of targeted base editing has been further improved. SpRYCas9 is improved from SpCas9. SpCas9 mainly recognizes the PAM sequence of NGG, while SpRYCas9 greatly reduces the requirement for the PAM motif, and almost any three bases can be used as the PAM sequence, but the recognition efficiency of NRN is higher than that of NYN ( R=G/A, Y=C/T). In order to further improve the efficiency of rice base editing and expand the scope of base editing, we designed a fusion of a new deaminase and Cas9 protein, based on the previously developed Anc689BE4max-nCas9 and ABEmax-nCas9 base editors , by replacing the deaminase and Cas9 proteins, new evoAPOBEC1-SpRYCas9n (CBE) and ABE8e-SpRYCas9n (ABE) base editors were formed (Fig. 1, Fig. 2).

2. To construct a base editing library targeting specific domains of the endogenous ACCase gene in rice.

The rice endogenous ACCase gene (LOC_Os05g22940, the encoded amino acid sequence is shown in SEQ ID No. 1) consists of 35 exons, of which the CT domain located in exon 34 is presumed to be the target binding of exogenous herbicides the main domain (Figure 3). We set some specific segments in the CT domain as the target region for base editing, import the DNA sequence into the CRISPR-GE website, and design the sg (sgRNA) target with NRN (R=R=G/A) as the PAM motif point, a total of hundreds of sg were generated. We sent these sg sequences to Sangon Bioengineering Co., Ltd. (Shanghai) for synthesis, and then cloned them into evoAPOBEC1-SpRYCas9n and ABE8e-SpRYCas9n vectors respectively to form two base editing libraries targeting the endogenous ACCase gene in rice.

3. Rice genetic transformation and screening of herbicide-resistant mutants

The base editing library constructed above was transformed into the callus of the japonica rice variety Xiushui 134 by Agrobacterium, and the transgenic positive callus was screened with hygromycin. At the stage of positive callus differentiation, we added a herbicide targeting ACCase: 2 mg/L haloxyfop-R-methyl (Sigma) to the medium. On this medium, the callus transferred into the empty vector cannot differentiate into seedlings, while the callus transferred into the CBE or ABE base editing library, when the ACCase gene achieves base editing resulting in amino acid mutation, and the mutation site When the resistance to the high-efficiency flufenoxaden can be developed, the corresponding mutant plants can differentiate normally to form seedlings (Fig. 4, Fig. 5).

Similarly, we also added 2 mg/L of high-efficiency flufenoxapyr in the culture of rooting and strong seedlings. The rice seedlings transferred into the empty vector could not take root on this medium, while the callus transferred into the CBE or ABE base editing library The resulting seedlings were able to root normally (Fig. 6, Fig. 7).

4. Genotyping of resistant mutants

We extracted the leaf DNA of the screened resistant plants, and identified the target region of the ACCase gene by PCR and Sanger sequencing. The results showed that these plants had one or more base substitutions in the target region relative to the wild-type sequence, resulting in one or more base substitutions in the target region. Multiple amino acid mutations indicated that these mutations conferred resistance to the herbicide carboxyfenclox. We screened 12 rice edited plants, and their corresponding ACCase resistance mutation sites are summarized in Table 2.

Table 2. Mutation sites of ACCase gene resistance in different edited rice

It can be seen from the above table that the single amino acid site mutation of rice ACC enzymes S2273F, G2194S, R2168P, S1975F, P1954S, I1864V, W2097S or E2211K respectively caused rice to be resistant to the herbicide Betafenapyr; I2139 was mutated to V or N causes rice to develop resistance to haloxyfen-pyr; mutations C2186R and Y2187H, A2123T and W2125C, and W2125S and R2126K also lead to resistance to haloxyfen-ethyl in rice, respectively .

For the different mutation sites obtained above, in order to study the effect of mutating the same amino acid site into different residues on rice herbicide resistance, we obtained other single amino acid site mutations by single base editing. The mutant forms of rice ACCase involved are as follows: W2125C, G2194A, I2139V, I2139N, W2097G, W2097L or C2186H.

Example 2. Anti-herbicide performance test of the rice ACCase mutant obtained in Example 1

In the present embodiment, high-efficiency fenfluoxop-ethyl and sethoxydime are used for the herbicide resistance at the E0 generation tissue culture stage, the herbicide resistance at the E1 generation seedling stage, and the greenhouse effect of the different rice ACCase mutant plants obtained in Example 1. The main agronomic traits of different rice mutant plants during field planting were tested, and the main agronomic traits included plant height, number of tillers, and seed setting rate.

Among them, in the tissue culture stage of the E0 generation, the dosage of oxafenoxamethoxine was 2 mg/L; at the seedling stage of the E1 generation, the amount of oxafenoxop-ethyl or sethoxypyr was sprayed with Use 1 and 2 times the recommended dosage of Datian respectively.

Note: herbicide resistance: ++ means strong resistance; + means weak resistance. Among them, the strong resistance in the seedling resistance of the E1 generation means that the plant still grows well under the herbicide concentration twice the recommended dosage, but this does not mean that it can only tolerate the herbicide concentration twice the recommended dosage. It can also tolerate herbicides with 3 times, 4 times or even higher concentrations of the recommended dosage; weak resistance in the seedling resistance of E1 generation means that the plants do not perform well at the recommended dosage of 2 times the herbicide concentration, but at the recommended dosage Grows well at 1 times the herbicide concentration.

Phenotypes in greenhouse and field: √ means the phenotype of the mutant plant is consistent with the wild type; - means the plant phenotype is slightly worse than the wild type; × means the plant phenotype is seriously affected, including dwarf plants and significantly lower seed setting rate.

It can be seen from Table 3 that although some mutant plants showed resistance to herbicides at the tissue culture stage, they could no longer show resistance at the seedling stage, and their agronomic traits were also seriously affected in greenhouse and field cultivation. For example, Mutations of I1879L and G2194N. Among them, the mutants of W2125C, G2194S, G2194A, I2139V, I2139N, W2097G or C2186H with better herbicide resistance and agronomic traits; I2139N, W2097G or C2186H have practical application value for breeding herbicide-resistant rice. In addition, from Table 3, it can also be known that even the mutation of the same amino acid site, when it is mutated to a different amino acid, has a huge impact on the plant; However, the mutation of W2097G had no effect on the agronomic traits of the plants.

For the above-mentioned mutants of W2125C, G2194S, G2194A, I2139V, I2139N, W2097G or C2186H with better herbicide resistance and agronomic traits, use other ACC inhibitor herbicides for herbicide tolerance tests (for example, pyridoxine). Chlorhefop), the above mutants also showed a tolerance effect comparable to that of haloxypyr or sethoxyfop.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (17)

1. A mutant acetyl-CoA carboxylase (ACC), the mutant acetyl-CoA carboxylase (ACC) compared with the amino acid sequence of the parent acetyl-CoA carboxylase (ACC), in corresponding to SEQ There is a mutation at any or any of the following amino acid positions in the amino acid sequence shown in ID No.1: position 2125, position 2097, position 2139, position 2194, position 2186, position 2273, position 2168 , 1975th, 1954th, 1864th, 2211th, 2187th, 2123rd, 2126th.
2. The mutant ACC according to claim 1, characterized in that there is a mutation at any one or any of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1: position 2125, position 2097 , 2139th, 2194th, 2186th.
3. A mutant acetyl-CoA carboxylase (ACC), the mutant acetyl-CoA carboxylase (ACC) is selected from any of the following groups I-III:

   I. A mutant ACC obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at any or any of the following amino acid positions: position 2125, position 2097, position 2139, position 2194, No. 2186, No. 2273, No. 2168, No. 1975, No. 1954, No. 1864, No. 2211, No. 2187, No. 2123, No. 2126;

   II. Compared with the mutant ACC described in I, it has the mutation site described in I; and, compared with the mutant ACC described in I, 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% sequence identity of mutant ACC and retain herbicide resistance activity;

   III. Compared with the mutant ACC described in I, it has the mutation site described in I; and, compared with the mutant ACC described in I, it has one or more amino acid substitutions, deletions or additions and retains herbicide resistance activity; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Amino acid substitutions, deletions or additions.
4. The mutant ACC according to any one of claims 1-3, wherein,

   The amino acid at position 2273 is mutated to a non-S amino acid,

   The amino acid at position 2194 is mutated to a non-G amino acid,

   The amino acid at position 2168 is mutated to a non-R amino acid,

   The amino acid at position 1975 is mutated to a non-S amino acid,

   The amino acid at position 1954 is mutated to a non-P amino acid,

   The amino acid at position 1864 is mutated to a non-I amino acid,

   The amino acid at position 2097 is mutated to a non-W amino acid,

   The amino acid at position 2211 is mutated to an amino acid other than E,

   The amino acid at position 2139 is mutated to a non-I amino acid,

   The amino acid at position 2186 is mutated to a non-C amino acid,

   The amino acid at position 2187 is mutated to an amino acid other than Y,

   The amino acid at position 2123 is mutated to an amino acid other than A,

   The amino acid at position 2125 is mutated to a non-W amino acid,

   The amino acid at position 2126 is mutated to a non-R amino acid.
5. The mutant ACC according to claim 4, wherein the 2125th amino acid is mutated to C, the 2097th amino acid is mutated to G, the 2139th amino acid is mutated to V or N, the Amino acid 2194 is mutated to A, and amino acid 2186 is mutated to H.
6. The mutant ACC according to any one of claims 1-3, wherein the parent ACC is derived from a monocotyledonous plant or a dicotyledonous plant.
7. A polynucleotide, characterized in that the polynucleotide encodes the mutant ACC of any one of claims 1-6.
8. A nucleic acid construct, characterized in that the nucleic acid construct contains the polynucleotide of claim 7.
9. The nucleic acid construct of claim 8, further comprising a regulatory element operably linked therewith.
10. The nucleic acid construct according to claim 9, wherein the regulatory element is selected from one or any of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polynucleoside Acid sequences, marker genes.
11. A host cell, wherein the host cell comprises the mutant ACC of any one of claims 1-6, or the polynucleotide of claim 7, or the nucleic acid construct of claim 8 .
12. A method for imparting resistance to herbicides in plants or for preparing plants having herbicide resistance, the method comprising introducing any of claims 1 to 6 in plant cells, plant seeds, plant tissues, plant parts or plants A step of the mutant ACC.
13. The method of claim 12, wherein the method comprises expressing the mutant ACC of any one of claims 1-6 in plant cells, plant seeds, plant tissues, plant parts or plants step.
14. The method of claim 13, wherein the method comprises the step of mutating the plant's endogenous ACC to introduce the mutant ACC.
15. The mutant ACC of any one of claims 1-6, the polynucleotide of claim 7, the nucleic acid construct of claim 8, or the host cell of claim 11 are prepared to have herbicide resistance. Sexual use in plants.
16. A plant cell, plant seed, plant tissue, plant part or plant comprising the mutant ACC of any one of claims 1-6, the polynucleotide of claim 7, the nucleic acid of claim 8 The construct, or the host cell of claim 11.
17. A method for controlling weeds in farmland, characterized in that:

    a) providing a herbicide-resistant plant prepared by any of the methods of claims 12-14,

    b) applying an effective amount of a herbicide to the plant and weeds in its vicinity, thereby controlling the weeds in the vicinity of the plant.

Images