Gene combination for enhancing herbicide resistance and application thereof

Abstract

The present invention relates to the technical field of agricultural biology, and in particular, to a gene combination for enhancing herbicide resistance and an application thereof. The gene combination comprises a promoter and a gene coding region, wherein the promoter is a UBI2 gene promoter, and the gene coding region is an HPPD gene mutant coding region. Rice with significantly enhanced herbicide resistance is obtained by means of gene editing, and exhibits an unexpected resistance effect.

Description

Gene combination for improving herbicide resistance and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, and in particular, to a gene combination for improving herbicide resistance and application thereof. BACKGROUND

[0002] When weeds become resistant to a particular herbicide, that herbicide can no longer be used for weed control, and thus creating crop material with higher herbicide resistance has been a focus of research in the agricultural field. Improving herbicide resistance in crops can greatly benefit crop production. However, obtaining and incorporating into the rice genome a gene or combination of genes that improve herbicide resistance while maintaining favorable characteristics to preserve or improve fitness is challenging, unpredictable, time-consuming, and expensive, but is necessary to meet the world's increasing demand for food.

[0003] SUMMARY

[0004] To solve the above problems in the prior art, the present application provides a gene combination for improving herbicide resistance and application thereof.

[0005] The technical solution adopted by the present application is as follows:

[0006] A gene combination, comprising a promoter and a gene coding region, wherein the promoter is a ubi2 gene promoter, and the gene coding region is a hppd gene mutant coding region.

[0007] In one specific embodiment, the ubi2 gene promoter sequence is shown in SEQ ID NO: 1, and the hppd gene mutant coding region amino acid sequence is shown in SEQ ID NO: 2.

[0008] In another specific embodiment, the hppd gene mutant coding region nucleotide sequence is shown in SEQ ID NO: 3.

[0009] In one specific embodiment, the gene combination comprises SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 9.

[0010] In one specific embodiment, the nucleotide sequence comprises a sequence shown in SEQ ID NO: 4.

[0011] In another specific embodiment, the gene combination further comprises a sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 9.

[0012] In one embodiment, the gene combination is obtained by a gene editing system.

[0013] In another embodiment, the gene editing system is a Meganuclease, a Zinc finger nuclease, a TALEN or a CRISPR / Cas system.

[0014] In another embodiment, the gene editing system is a CRISPR / Cas9 or CRISPR / Cas12 system.

[0015] The present application also provides a recombinant genome comprising the gene combination.

[0016] The present application also provides a host cell comprising the gene combination or comprising the recombinant genome.

[0017] The present application also provides the use of the gene combination, the recombinant genome or the host cell in improving the resistance of plants to HPPD-inhibiting herbicides.

[0018] The present application also provides a primer pair for detecting the gene combination, the sequences of which are SEQ ID NO: 5 and SEQ ID NO: 6.

[0019] The present application also provides a method for breeding a gene-edited plant with or improved tolerance to HPPD-inhibiting herbicides, the method comprising the following steps:

[0020] (1) simultaneously generating DNA breaks at specific positions between the promoter and coding region of the hppd gene and between the promoter and coding region of the ubi2 gene in a plant cell, respectively, the DNA breaks being connected to each other through intracellular repair pathways, and screening a gene combination of the promoter of the ubi2 gene and the coding region of the hppd gene (i.e. a knock-up technology) by designing a primer pair; then mutating the G342 / R346 sites of the coding region of the hppd gene in the gene combination to G342D / R346H, respectively; or,

[0021] first mutating the G342 / R346 sites of the coding region of the hppd gene in a plant cell to G342D / R346H, respectively; then simultaneously generating DNA breaks at specific positions between the promoter and coding region of the hppd gene and between the promoter and coding region of the ubi2 gene in the plant cell, respectively, the DNA breaks being connected to each other through intracellular repair pathways, and screening a gene combination of the promoter of the ubi2 gene and the mutant coding region of the hppd gene by designing a primer pair;

[0022] (2) regenerating a plant from the plant cell.

[0023] In one embodiment, a plant comprising the combination of genes is obtained by the method.

[0024] The present application also provides a method of controlling weeds in a plant cultivation site, wherein the plant comprises a plant prepared by the method, the method comprising applying to the plant cultivation site a herbicidally effective amount of an HPPD-inhibiting herbicide.

[0025] In one embodiment, the HPPD-inhibiting herbicide is applied in combination with one or more additional herbicides.

[0026] In another embodiment, the plant is rice.

[0027] The present application has obtained a combination of genes with herbicide resistance by knock-up and point mutation, and obtained rice with significantly improved herbicide resistance, showing unexpected resistance effect.

[0028] DETAILED DESCRIPTION

[0029] Some of the terms used in the present specification are defined as follows.

[0030] In the present application, "HPPD-inhibiting herbicide" is a substance which is herbicidally active as such or in combination with other herbicides and / or additives which can modify its effect, which is able to act by inhibiting HPPD.Substances which are capable of exerting a herbicidal action by inhibiting HPPD are known per se in the art and include many classes, 1) triketones, for example, sulcotrione (CAS No.: 99105-77-8); mesotrione (CAS No.: 104206-82-8); bicyclopyrone (CAS No.: 352010-68-5); tembotrione (CAS No.: 335104-84-2); tefuryltrione (CAS No.: 473278-76-1); benzobicyclon (CAS No.: 156963-66-5); 2) diketonitriles, for example, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4- trifluoromethylphenyl)propane-1,3-dione (CAS No.: 143701 -75-1 ); 2-cyano-3- cyclopropyl-1-(2-methylsulfonyl-3,4-dichlorophenyl)propane-1,3-dione (CAS No.: 212829-55-5); 2-cyano-1-[4-(methylsulfonyl)-2-trifluoromethylphenyl]-3-(1- methylcyclopropyl)propane-1,3-dione (CAS No.: 143659-52-3); 3) isoxazoles, for example, isoxaflutole (CAS No.: 141 112-29-0); isoxachlortole (CAS No.: 141 112-06-3) clomazone (CAS No.: 81777-89-1 ); 4) pyrazoles, for example, topramezone (CAS No.: 210631 -68-8); pyrasulfotole (CAS No.: 365400-11 -9); pyrazoxyfen (CAS No.: 71561 -11 -0); pyrazolate (CAS No.: 58011 -68-0); benzofenap (CAS No.: 82692-44-2); pyraclonille (CAS No.: 1622908-18-2); Tolpyralate (CAS No.: 1101132-67-5); benzofluoracifop (CAS No.: 1992017-55-6); cyclopyraflubricide (CAS No.: 1855929-45-1 ); triazolopyrimidines (CAS No.: 191 1613-97-2); 5) benzophenones; 6) other classes: lancotrione (CAS No.: 1486617-21 -3); fenquinotrione (CAS No.: 1342891 -70-6).A plant which is "enhanced for tolerance to HPPD-inhibiting herbicides" or "enhanced for resistance to HPPD-inhibiting herbicides" is a plant whose tolerance or resistance to said HPPD-inhibiting herbicides is increased compared to a plant comprising a wild-type HPPD gene. An HPPD enzyme which is "enhanced for tolerance to HPPD-inhibiting herbicides" or "enhanced for resistance to HPPD-inhibiting herbicides" is an HPPD enzyme which exhibits an enzymatic activity which is at least 10% higher, preferably at least 15% higher, more preferably at least 20% higher than the wild-type HPPD enzyme at herbicide concentrations known to inhibit the activity of the corresponding wild-type HPPD enzyme protein. In the present invention, the two terms "HPPD-inhibiting herbicide tolerance" and "HPPD-inhibiting herbicide resistance" are used interchangeably and both refer to tolerance to HPPD-inhibiting herbicides and resistance to HPPD-inhibiting herbicides.

[0031] The term "genome" as used herein refers to the totality of the genetic material (genes and non-coding sequences) present in each cell of an organism or virus or organelle, and / or the complete set of chromosomes inherited as a unit (haploid) from one parent.

[0032] The term "gene editing" refers to strategies and techniques for targeted specific modification of any genetic information or genome of a living organism. The term thus includes editing of the coding regions of genes, but also editing of regions other than the coding regions of genes of the genome. It also includes editing or engineering of the nucleus, if present, and other genetic information of the cell.

[0033] The term "CRISPR / Cas" can be a CRISPR-based nuclease or a nucleic acid sequence encoding the same, including but not limited to: 1) Cas9, including SpCas9, ScCas9, SaCas9, xCas9, VRER-Cas9, EQR-Cas9, SpG-Cas9, SpRY-Cas9, SpCas9-NG, NG-Cas9, NGA-Cas9 (VQR), etc., 2) Cas12, including LbCpf1, FnCpf1, AsCpf1, MAD7, KingCas12 (SEQ ID NO: 3 in CN116732003A), etc., or any variant or derivative of the foregoing CRISPR-based nucleases, preferably wherein the at least one CRISPR-based nuclease comprises a mutation compared to the corresponding wild-type sequence, such that the obtained CRISPR-based nuclease recognizes a different PAM sequence. The term "CRISPR" refers to a sequence-specific genetic manipulation technique that relies on the clustered regularly interspaced short palindromic repeat sequence approach, which is different from RNA interference in down-regulating gene expression at the transcriptional level.

[0034] " Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to an RNA- guided nuclease comprising a Cas9 protein or fragment thereof (e.g., a protein comprising the active DNA cleavage domain of Cas9 and / or the gRNA binding domain of Cas9). Cas9 is a component of the CRISPR / Cas (clustered regularly interspaced short palindromic repeat and associated system) genome editing system that can target and cleave a DNA target sequence to form a DNA double-strand break (DSB) under the guidance of a guide RNA.

[0035] As used herein, the term "organism" includes animals, plants, fungi, bacteria, and the like.

[0036] As used herein, the term "host cell" includes plant cells, animal cells, fungal cells, bacterial cells, and the like.

[0037] As used herein, the term "plant" includes whole plants, and any progeny, cells, tissues, or parts thereof. The term "plant part" includes any part of a plant, including, for example and without limitation: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and related explants). A plant tissue or plant organ can be a seed, callus, or any other group of plant cells that is organized into a structural or functional unit. Some plant cells or tissue cultures are capable of regenerating plants having the physiological and morphological characteristics of the plants from which the cells or tissue were derived, and of regenerating plants having essentially the same genotype as the plant from which the cells or tissue were derived. In contrast, some plant cells are not capable of regenerating a plant. Regenerable cells in a plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.

[0038] Plant parts include harvestable parts and parts that can be used to propagate progeny plants. Plant parts that can be used for propagation include, for example and without limitation: seeds; fruits; cuttings; seedlings; tubers; and rootstocks. Harvestable parts of a plant can be any useful part of a plant, including, for example and without limitation: flowers; pollen; seedlings; tubers; leaves; stems; fruits; seeds; and roots.

[0039] A plant cell is a structural and physiological unit of a plant. As used herein, a plant cell includes a protoplast and a protoplast with a partial cell wall. A plant cell can be in the form of an isolated single cell or a cell aggregate (e.g., a loose callus and a cultured cell), and can be part of a higher order tissue unit (e.g., a plant tissue, a plant organ, and a whole plant). Thus, a plant cell can be a protoplast, a cell that produces gametes, or a cell or collection of cells that is capable of regenerating into a whole plant. Accordingly, in embodiments herein, a seed comprising a plurality of plant cells and capable of regeneration into a whole plant is considered a "plant part."

[0040] The term "wild type" refers to a nucleic acid molecule or protein that can be found occurring in nature.

[0041] In the present application, the term "locus" includes the site where the plants of the present application are cultivated, e.g., soil, and also includes, for example, plant seeds, plant seedlings, and established plants. The term "weed-controlling effective amount" refers to an amount of herbicide sufficient to affect the growth or development of a target weed, e.g., to prevent or inhibit the growth or development of the target weed, or to kill the weed. Advantageously, the weed-controlling effective amount does not significantly affect the growth and / or development of the plants of the present application. One skilled in the art can determine such a weed-controlling effective amount through routine experimentation.

[0042] The term "gene" includes a nucleic acid segment that expresses a functional molecule, such as but not limited to, a particular protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0043] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into the RNA. A DNA polynucleotide can encode an RNA that is translated into a protein (mRNA), or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or an RNA that targets DNA; also known as "non-coding" RNA or "ncRNA").

[0044] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0045] The terms "polynucleotide," "nucleotide," and "nucleic acid" are used interchangeably herein and include DNA, RNA, or hybrids thereof, which can be double-stranded or single-stranded.

[0046] The terms "nucleotide sequence" and "nucleic acid sequence" both refer to the order of bases in DNA or RNA.

[0047] As is well understood by those skilled in the art, due to the degeneracy of the genetic code, there are multiple 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 additional nucleic acid sequences that encode the same protein, and thus the present application encompasses nucleic acid sequences that encode 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 for codon usage preferred by the host organism to better express the gene.

[0048] The term "identity" refers to sequence similarity with a native nucleic acid sequence. Identity can be assessed by eye or by computer software. Using computer sequence alignment software, identity between two or more sequences can be expressed as a percentage (%) that can be used to assess identity between related sequences. "Portions of sequences" means at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a given sequence.

[0049] For the terms used in the specification relating to amino acid substitutions, the first letter represents the naturally occurring amino acid at a particular position of a particular sequence, the number following represents the position relative to SEQ ID NO: 11, and the second letter represents the different amino acid that replaces the natural amino acid. For example, G342D means that the glycine at position 342 is replaced with an aspartic acid relative to the amino acid sequence of SEQ ID NO: 11. For amino acid substitutions where the first letter is not present, it means that the natural amino acid at the position corresponding to SEQ ID NO: 11 is replaced with the amino acid represented by the letter following the number relative to the amino acid sequence of the wild-type protein. For double or multiple mutations, each mutation is separated by a " / " or a "+". For example, G342D / R346H means that the glycine at position 342 is replaced with an aspartic acid and the arginine at position 346 is replaced with a histidine relative to the amino acid sequence of SEQ ID NO: 11, both mutations being present in the particular mutant HPPD protein.

[0050] It is also clear to those skilled in the art that the structure of a protein can be changed 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 adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples of conservative amino acid substitutions and embodiments are clear to those skilled in the art. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group, i.e., a non-polar amino acid residue is replaced with another non-polar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. A conservative substitution in which one amino acid is replaced with another amino acid belonging to the same group is within the scope of the present application, provided that the substitution does not impair the biological activity of the protein.

[0051] Accordingly, the mutant proteins of the present application can comprise one or more additional mutations, such as conservative substitutions, in the amino acid sequence in addition to the mutations described above. In addition, the present application also encompasses mutant proteins comprising one or more additional non-conservative substitutions, provided that the non-conservative substitutions do not significantly affect the desired functions and biological activities of the proteins of the present application.

[0052] 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 still retaining its functional activity. Accordingly, in another aspect, the present application also relates to fragments of the mutant proteins of the present application that have one or more amino acid residues deleted from the N and / or C terminus while retaining their desired functional activities, which are also within the scope of the present application and are referred to as biologically active fragments. In the present application, a "biologically active fragment" refers to a portion of a mutant protein of the present application that retains the biological activity of the mutant protein of the present application. For example, a biologically active fragment of a mutant protein can be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, such as 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C terminus, but which still retains the biological activity of the full-length protein.

[0053] As used herein, "expression cassette", "expression vector", and "expression construct" refer to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in a plant. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (e.g., to produce mRNA or a functional RNA) and / or translation of the RNA into a precursor or mature protein.

[0054] An "expression construct" of the present application can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (e.g., mRNA).

[0055] The "expression construct" of the present application can comprise regulatory sequences and nucleotide sequences of interest of different origins, or regulatory sequences and nucleotide sequences of interest of the same origin but arranged in a manner different from that normally found in nature.

[0056] The "high expression gene" of the present application refers to a gene that is expressed in a higher amount than ordinary genes in a particular tissue.

[0057] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically generated for the purpose of expressing and / or propagating the insert or for the purpose of constructing other recombinant nucleotide sequences. The insert can be operably or inoperably linked to a promoter sequence and can be operably or inoperably linked to DNA regulatory sequences.

[0058] "Regulatory sequences" and "regulatory elements" are used interchangeably herein to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Plant expression regulatory elements refer to nucleotide sequences capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest in a plant.

[0059] Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0060] A "promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. A promoter refers to a specific DNA regulatory region located upstream from the coding sequence of a gene and having the function of mediating and regulating the initiation of transcription of a target gene and the expression level. The region contains core promoter elements (such as TATA box, initiator Inr, etc.) and adjacent regulatory sequences (such as GC box, CAAT box, etc.), which can form a pre-initiation complex (PIC) by binding RNA polymerase, general transcription factors (GTFs), and specific transcription factors (TFs), thereby precisely controlling the temporal and spatial specificity of gene expression. In genetic engineering, the promoter is operably linked to the open reading frame (ORF) of the target gene to achieve controllable expression of the exogenous gene in the host cell. In some embodiments of the present application, the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it is derived from plant cells. The promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0061] As used herein, the term "promoter" generally refers to a DNA molecule that is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter can be initially isolated from a DNA sequence in the 5' untranslated region (5' UTR) of a genomic copy of a gene, up to 1-4000 bp upstream. Alternatively, a promoter can be a synthetically produced or manipulated DNA molecule. A promoter can also be chimeric, i.e., a promoter produced by the fusion of two or more heterologous DNA molecules. Promoters useful in embodiments of the present application include SEQ ID NO: 3, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52, or fragments or variants thereof. In particular embodiments of the present application, these promoters described herein, and any variants or derivatives thereof, are further defined as comprising promoter activity, i.e., are capable of functioning as a promoter in a host cell (e.g., in a transgenic plant). In still further particular embodiments, a fragment can be defined as exhibiting the promoter activity that the starting promoter from which it was derived has, or a fragment can comprise a "minimal promoter" (which provides a basal level of transcription and consists of a TATA box or equivalent sequence for recognition and binding of the RNA polymerase II complex to effect initiation of transcription).

[0062] In one embodiment, fragments of the promoter sequences disclosed herein are provided. Promoter fragments can comprise promoter activity as described above, and can be used individually or in combination with other promoters and promoter fragments, such as in a constructed chimeric promoter. In particular embodiments, promoter fragments comprising at least about 50, 95, 150, 250, 500, 750, 1000, 1500, 2000, 3000, or at least about 4000 contiguous nucleotides or longer of a polynucleotide molecule having promoter activity disclosed herein are provided.

[0063] The promoter can be an isolated nucleic acid, wherein the sequence of the isolated nucleic acid comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52, or at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment thereof.

[0064] The term "strong promoter" is a term well known and widely used in the art, and many strong promoters are known in the art or can be identified through routine experimentation. The activity of the strong promoter is higher than the activity of the promoter operably linked to the nucleic acid molecule to be overexpressed in the wild type organism, e.g. a promoter having a higher activity than the promoter of an endogenous gene. Preferably, the activity of the strong promoter is about 2%, 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more than 1000% higher than the activity of the promoter operably linked to the nucleic acid molecule to be overexpressed in the wild type organism. The skilled person knows how to determine the activity of a promoter and to compare the activity of different promoters.

[0065] A "herbicide" in the present application means an active ingredient which kills or controls or is detrimental to the growth of a plant. "Herbicide tolerance" or "herbicide resistance" in the present application means that a plant continues to grow even though a herbicide is used which would kill a normal or wild type plant or resist the growth of a plant or make the ability of the plant to grow weaker or stop the growth of a plant as compared to a wild type plant.

[0066] The term "controlling weeds" will be understood to mean killing and / or retarding or inhibiting the normal growth of weeds. In the broadest sense, weeds are understood to mean all plants which are known to grow where they are not wanted, for example at (crop) plant cultivation sites. Weeds according to the present application include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum.Monocotyledonous weeds include, but are not limited to, weeds of the following genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. In addition, weeds of the present invention can include, for example, crop plants growing in an undesirable location. For example, if corn plants are not desired in a field of soybean plants, a volunteer corn plant present in a field of primarily soybean plants can be considered a weed.

[0067] Unless defined otherwise, all technical and scientific terms and any course terminology used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0068] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing in this indication is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates, which can need to be independently confirmed.

[0069] Unless specifically stated or implied, as used herein the terms "a," "an," and "the" refer to "at least one." All patents, patent applications, and publications cited or referred to in this disclosure are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 shows a schematic diagram of rice HPPD knock-up and point mutation.

[0071] Figure 2 shows the results of herbicide resistance test of each material T1 generation at seedling stage under 480g a.i / ha pyrasulfotole treatment. 1: wild type Kinsho 818; 2: HPPD G342D / R346H editing; 3: UBI promoter + HPPD WT knock-up; 4: UBI promoter + HPPD G342D / R346H knock-up.

[0072] Sequence listing DETAILED DESCRIPTION

[0073] The present application is further illustrated by the following examples. The following description is by way of example only, but the scope of the present application should not be limited thereto.

[0074] Example 1, PE-mediated rice knock-up HPPD mutant editing

[0075] In order to further improve the resistance of rice to HPPD inhibitor herbicides, the UBI promoter + HPPD WT knock-up editing material (UBI promoter + HPPD CDS partial sequence is SEQ ID NO: 10, i.e. SEQ ID NO: 18 in CN112779266A, other sequencing conditions are as follows: Ubi promoter-UBI interface sequence is shown in SEQ ID NO: 7, Ubi promoter-HPPD interface sequence is shown in SEQ ID NO: 8, and HPPD promoter-HPPD interface sequence is shown in SEQ ID NO: 9) described in CN112779266A was subjected to secondary editing, i.e. using PE editing to edit the rice HPPD G342D+R346H site (schematic diagram is shown in Figure 1), and identification and herbicide resistance test were carried out.

[0076] The specific steps are as follows:

[0077] 1. Construction of gene editing vector and Agrobacterium-mediated genetic transformation of rice

[0078] After designing and synthesizing the pegRNA expression frame for editing the rice HPPD G342D / R346H site, the editing vector was constructed.

[0079] The constructed PE editing vector is respectively introduced into UBI promoter+HPPD WT and wild type Jinjian 818 receptor material by Agrobacterium-mediated rice transformation, after callus recovery culture, it is moved to screening medium for three rounds of screening, after screening is completed, the callus with good growth state is picked to differentiate, 1 cm of seedlings can be obtained after 15 days, the differentiated seedlings are moved to rooting medium for rooting culture, after the seedlings with rooting are completed, they are moved to the flowerpot filled with soil and placed in the greenhouse for culture, and the rice gene editing seedlings are obtained.

[0080] 2. Molecular detection of edited rice HPPD G342D / R346H site (T0 generation)

[0081] After the transformed seedlings are moved into the greenhouse for culture, genomic DNA is extracted, the DNA is used as a template for PCR amplification detection, and T0 generation rice strains of wild type Jinjian 818 and edited HPPD gene G342D+R346H site (HPPD double mutant amino acid sequence is shown as SEQ ID NO: 2, and nucleotide sequence is shown as SEQ ID NO: 3) on the basis of high-knock HPPD material (UBI promoter+HPPD WT) are successfully obtained, and HPPD G342D / R346H edited material and UBI promoter+HPPD G342D / R346H high-knock material (wherein the full length of the hppd double mutant gene driven by the UBI promoter is shown as SEQ ID NO: 4, and the amplification primers for identifying the UBI promoter driving the hppd double mutant gene are primer-F-SEQ ID NO: 5 and primer-R-SEQ ID NO: 6) are obtained.

[0082] 3. Herbicide resistance test of T0 generation rice

[0083] The obtained T0 generation correctly edited rice seedlings are sprayed with pyrazolynate to test their resistance to HPPD herbicides, 7 days after the pesticide is applied, the HPPD G342D / R346H edited control rice seedlings begin to show whitening symptoms, and the rice UBI promoter+HPPD G342D / R346H high-knock edited seedlings remain normal green. After 4 weeks of pesticide application, the HPPD G342D / R346H edited rice seedlings die, while the rice UBI promoter+HPPD G342D / R346H high-knock edited seedlings continue to remain green and grow normally. The test results show that the tolerance of UBI promoter+HPPD G342D / R346H high-knock material to pyrazolynate is significantly improved compared with HPPD G342D / R346H edited rice material.

[0084] Example II, test the resistance of T1 generation rice edited HPPD G342D / R346H site in high-knock background and wild type background to herbicides

[0085] T1 generation of UBI promoter + HPPD G342D / R346H knock-up material and HPPD G342D / R346H edited material were tested for herbicide resistance in seedling stage, wild type Ginj 818 and UBI promoter + HPPD WT knock-up material were used as controls, after the seedling height reached the three-leaf-one-heart stage, pyraflufen was sprayed in the field, the drug concentration was set at 60g, 120g, 240g, 480g a.i / ha four gradients.

[0086] The investigation results after 7 days of drug application showed that the wild type control rice seedlings showed obvious phytotoxicity at a dose of 60g a.i / ha. With the increase of herbicide concentration, at a dose of 120g a.i / ha, the wild type control rice plants soon died, and the HPPD G342D / R346H edited and UBI promoter + HPPD WT knock-up T1 generation edited seedlings also showed obvious phytotoxicity. At a dose of 480g a.i / ha, the UBI promoter + HPPD G342D / R346H knock-up seedlings could still keep green and continue to grow, and the UBI promoter + HPPD WT knock-up and HPPD G342D / R346H edited materials had basically whitened. After 28 days of drug application, part of the UBI promoter + HPPD WT knock-up and HPPD G342D / R346H edited materials could recover growth, but the plant growth was obviously inhibited, while the UBI promoter + HPPD G342D / R346H knock-up edited seedlings grew normally, as shown in Figure 2, and the specific phytotoxicity symptom evaluation was shown in Table 1 and Table 2. The results showed that the UBI promoter + HPPD G342D / R346H knock-up had significantly improved herbicide resistance compared with the UBI promoter + HPPD WT knock-up and the HPPD G342D / R346H edited material alone.

[0087] Table 1 Herbicide phytotoxicity symptom grading standard

[0088] Table 2 Herbicide phytotoxicity symptom evaluation table

[0089] Example Three, verification of the resistance of OsUbi2 promoter combined with OsHPPD G342D / R346H in transgenic rice

[0090] To quickly validate the tolerance of HPPD inhibitor herbicides in plants with the combination of rice OsUbi2 promoter and OsHPPD G342D / R346H, two vectors overexpressing rice OsHPPD G342D / R346H mutant and wild type WT with OsUbi2 promoter and one vector overexpressing rice OsHPPD G342D / R346H mutant with CaMV 35S promoter (enhanced) were constructed for rice genetic transformation.

[0091] T0 generation of rice seedlings overexpressing rice OsHPPD G342D / R346H and OsHPPD WT with OsUbi2 promoter and OsHPPD G342D / R346H mutant with CaMV 35S promoter (enhanced) were sprayed with different concentrations of pyroxasulfone for resistance test. Compared with wild type of Jin Gang 818, the three transgenic rice lines all have certain tolerance / resistance to pyroxasulfone and can grow normally under the treatment of 60 g a.i / ha; but under the treatment of 240 g a.i / ha herbicide, the leaves of transgenic lines of OsUbi2 promoter overexpressing rice OsHPPD WT are dead, and the transgenic lines of CaMV 35S promoter (enhanced) overexpressing rice OsHPPD G342D / R346H also show obvious phytotoxicity; under the condition of higher application concentration of 480 g a.i / ha, rice overexpressing OsHPPD G342D / R346H mutant with OsUbi promoter still shows resistance, and the leaves of the other two transgenic rice lines are dead, which has no difference from the wild type control, indicating that overexpression of OsHPPD G342D / R346H with OsUbi2 promoter can significantly enhance the tolerance of rice to pyroxasulfone.

[0092] In addition, the resistance of transgenic OsUbi2 promoter combined with OsHPPD G342D / R346H to other HPPD herbicides was also verified in rice, and the results showed that rice containing OsUbi2 promoter combined with OsHPPD G342D / R346H significantly improved the resistance to different HPPD herbicides.

[0093] All publications and patent applications mentioned in the specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0094] While the foregoing application has been described in some detail for purposes of clarity and the specific embodiments, it will be apparent to those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. A genetic combination comprising a promoter and a gene coding region, wherein the promoter is a ubi2 gene promoter, and the gene coding region is a hppd gene mutant coding region. 2.The genetic combination of claim 1, wherein the ubi2 gene promoter sequence is shown as SEQ ID NO: 1, and the hppd gene mutant coding region amino acid sequence is shown as SEQ ID NO: 2; preferably, the hppd gene mutant coding region nucleotide sequence is shown as SEQ ID NO:

3. 3.The genetic combination of claim 1 or 2, comprising SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO:

9. 4.The genetic combination of any one of claims 1-3, wherein the nucleotide sequence comprises a sequence shown as SEQ ID NO: 4; more preferably, the genetic combination further comprises a sequence shown as SEQ ID NO: 7 and / or SEQ ID NO:

9. 5.The genetic combination of any one of claims 1-4, which is obtained by a gene editing system; preferably, the gene editing system is a Meganuclease, a Zinc finger nuclease, a TALEN or a CRISPR / Cas system; more preferably, the gene editing system is a CRISPR / Cas9 or CRISPR / Cas12 system. 6.A recombinant genome comprising the genetic combination of any one of claims 1-5. 7.A host cell comprising the genetic combination of any one of claims 1-5 or comprising the recombinant genome of claim 6. 8.Use of the genetic combination of any one of claims 1-5, the recombinant genome of claim 6 or the host cell of claim 7 in improving plant HPPD inhibitor herbicide resistance. 9.A primer pair for detecting the genetic combination of any one of claims 1-5, wherein the sequences are shown as SEQ ID NO: 5 and SEQ ID NO:

6.

10. A method of breeding a genetically edited plant having or enhanced ability to tolerate HPPD-inhibiting herbicides, characterized in that, The method comprises the following steps: (1) simultaneously generating DNA breaks at specific positions between the promoter and coding region of the hppd gene and between the promoter and coding region of the ubi2 gene in plant cells, respectively, wherein the DNA breaks are connected to each other through intracellular repair pathways, and the genetic combination of the ubi2 gene promoter and the hppd gene coding region is screened by designing a primer pair; then the G342 / R346 sites of the hppd gene coding region in the genetic combination are respectively mutated to G342D / R346H; or, First, the G342 / R346 sites in the coding region of the hppd gene in the plant cell are respectively mutated into G342D / R346H; then, DNA breaks are simultaneously generated at specific positions between the promoter and the coding region of the hppd gene and between the promoter and the coding region of the ubi2 gene in the plant cell, the DNA breaks are connected to each other through the intracellular repair pathway, and the combination of the promoter of the ubi2 gene and the coding region of the hppd gene mutant is screened by designing primers; (2) the plant cells are regenerated into plants; Preferably, the plant comprising the combination of genes according to any one of claims 1-5 is obtained.

11. A method of controlling weeds in a plant growing locus, wherein the plant comprises a plant produced by the method of claim 10, the method comprising applying to the plant growing locus a herbicidally effective amount of an HPPD-inhibiting herbicide; preferably, the HPPD-inhibiting herbicide is applied in combination with one or more additional herbicides.

12. The use of claim 8 or the method of claim 10 or 11, wherein, The HPPD-inhibiting herbicide is selected from at least one of the following active ingredients: 1) triketones: sulcotrione, mesotrione, bicyclopyrone, benzobicylon, pyrasulfotole, bicyclopyrone; 2) diketonitriles: 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)propane-1,3-dione, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-3,4-dichlorophenyl)propane-1,3-dione, 2-cyano-1-[4-(methylsulfonyl)2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane 1,3-dione; 3) isoxazoles: isoxaflutole, benoxacor, isoxachlortole; 4) pyrazoles: pyrazolynate, pyrasulfotole, benzofluor, pyrazoxyfen, tolpyralate, benzofluor, topralate, benzofluor, benzofluor; 5) benzophenones; 6) others: lancotrione, fenquinotrione.

13. The use of claim 8 or the method of claim 10 or 11, wherein, The plant is rice.

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