A method for improving plant genetic transformation and gene editing efficiency

Abstract

The present application belongs to the field of plant genetic engineering. Specifically, the present application relates to a method for improving the efficiency of plant genetic transformation and gene editing. More specifically, the present application relates to improving the regeneration efficiency of plant genetic transformation and / or improving the efficiency of plant gene editing by expressing a gene NiR that promotes plant nitrogen metabolism.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering. Specifically, this invention relates to a method for improving the efficiency of plant genetic transformation and gene editing. More specifically, this invention relates to improving the regeneration efficiency of plant genetic transformation and / or the efficiency of plant gene editing by expressing the gene NiR, which promotes plant nitrogen metabolism. Background of the Invention

[0002] Crop genetics and breeding have evolved through artificial selection breeding, hybridization breeding, mutation breeding, and molecular marker-assisted breeding using molecular techniques. As the genetic diversity of varieties gradually decreases, the bottleneck effect of traditional breeding is becoming increasingly apparent: conventional breeding techniques are no longer sufficient to develop breakthrough new varieties, failing to meet human needs and the development of sustainable agriculture. The rapid development of life sciences has propelled us from the stage of "reading" biological genetic information to the post-genomic era, where the precise "rewriting" and even "redesigning" of the genome are gradually becoming a reality. This biological technology, aimed at designing and creating new traits or organisms, shows enormous promise in disease treatment, medicine, manufacturing, and especially agriculture.

[0003] Genome editing technology is a revolutionary technique in life sciences, enabling precise, efficient, and specific rewriting of the genome, thus revolutionizing research and exploration across the entire life sciences. Gene editing refers to the manipulation of target genes, such as deletion, substitution, or insertion, to rewrite genetic information and obtain new functions or phenotypes, or even create new species. Developing efficient and precise breeding techniques for crops using gene editing will overcome the shortcomings of traditional breeding, enabling molecular design breeding through precise genome modification. This has significant strategic importance for the future development of agriculture.

[0004] Current gene editing technologies mainly include ZFN, TALEN, and the CRISPR / Cas system. Due to its high efficiency and flexibility, the CRISPR / Cas system is currently the simplest and most widely used gene editing technology. In the CRISPR / Cas system, the Cas protein, guided by artificially designed guide RNA, can target any location in the genome. Base editing systems are novel gene editing technologies developed based on the CRISPR system, divided into cytosine base editing systems and adenine base editing systems. These systems fuse cytosine deaminase and adenine deaminase with Cas9 single-stranded nickase, respectively. Under the targeting action of the guide RNA, the Cas9 single-stranded nickase produces a single-stranded DNA region. Therefore, the deaminase can efficiently deaminate the C and A nucleotides on the single-stranded DNA at the target location, converting them into U and I bases, which are then repaired into T and G bases during the cell's own repair process. Base editing technology overcomes the shortcomings of traditional DSB-mediated gene editing, enabling highly efficient and precise replacement of single bases. The powerful CRISPR / Cas system-mediated genome modification technology will provide strong technical support for plant genomics research and novel plant molecular design breeding, accelerating the cultivation of new crop varieties and achieving sustainable agricultural development.

[0005] A key step in plant gene editing is delivering gene-editing nuclease proteins or encoding nucleic acids into plant cells to edit the target gene. Currently, the delivery technologies for plant genome editing are mainly achieved through genetic transformation and tissue culture techniques, primarily including Agrobacterium-mediated transformation and gene gun methods. While significant progress has been made in plant transformation and genetic modification in recent years, the transformation and genetic modification of many agronomically important plants (such as octoploid strawberries) remain difficult and time-consuming. Brief description of the attached diagram

[0006] Figure 1 The image shows the carrier spectrum used in Embodiment 1 of this application.

[0007] Figure 2 The results show that NiR in strawberries can significantly improve the efficiency of strawberry callus regeneration.

[0008] Figure 3 The image shows the carrier spectrum used in Embodiment 2 of this application.

[0009] Figure 4 The results show that wheat NiR can significantly improve wheat callus regeneration efficiency.

[0010] Figure 5 The image shows the carrier spectrum used in Embodiment 4 of this application.

[0011] Figure 6The results show that NiR in tomatoes can significantly improve tomato regeneration efficiency. Detailed Implementation

[0012] I. Definition

[0013] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0014] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0015] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0016] The term "genome," as used in this article, encompasses not only chromosomal DNA located in the cell nucleus but also organelle DNA located in subcellular components of the cell, such as mitochondria and plastids.

[0017] In relation to a sequence, “exogenous” means a sequence that originates from a foreign species, or, if from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0018] The terms “nucleic acid sequence,” “polynucleotide,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “D” for A, T, or G, “I” for inosine, and “N” for any nucleotide.

[0019] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention 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 analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0020] As used in this invention, "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleic acid sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleic acid sequence can refer to the transcription of the nucleic acid sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0021] The "expression construct" of this invention can be a linear nucleic acid fragment (including DNA or RNA fragments), a circular plasmid, or a viral vector.

[0022] The "expression construct" of this invention may include a regulatory sequence and a nucleic acid sequence of interest operatively linked thereto. The regulatory sequence and the nucleic acid sequence of interest may be from different sources, or from the same source but arranged in a manner different from that typically found naturally.

[0023] The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that affects the transcription, RNA processing, or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences. A "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in a cell, regardless of whether it originates from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0024] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0025] "Introducing" a nucleic acid molecule (such as an expression construct) into a plant cell means presenting the nucleic acid molecule to the plant cell so that it enters the interior of the plant cell.

[0026] "Regeneration" refers to the process of growing a complete plant from one or more plant cells (e.g., plant protoplasts, callus, or explants).

[0027] II. Improved plant regeneration, transformation, and gene editing

[0028] In one aspect, the present invention provides a method for improving plant cell regeneration efficiency, the method comprising a) overexpressing NiR in the cells of the plant, for example, by introducing an expression construct containing a coding nucleic acid sequence of NiR into the plant cells. In some embodiments, the method further comprises b) regenerating an intact plant from the plant cells obtained in a).

[0029] In another aspect, the present invention provides a method for improving the transformation efficiency of exogenous nucleic acid sequences of interest in plants or for transforming exogenous nucleic acid sequences of interest into plants, the method comprising:

[0030] (a) Overexpressing NiR in the cells of the plant, for example, by introducing an expression construct containing a nucleic acid sequence encoding NiR into the cells of the plant;

[0031] (b) Introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cells;

[0032] (c) Regenerate a complete plant from the plant cells.

[0033] In some implementations, steps (a) and (b) are performed in parallel (simultaneously). In some implementations, step (a) is performed first, followed by step (b). In some implementations, step (b) is performed first, followed by step (a).

[0034] In another aspect, the present invention provides a method for improving gene editing efficiency in plants or for performing gene editing in plants, the method comprising:

[0035] (a) Overexpressing NiR in the cells of the plant, for example, by introducing an expression construct containing a nucleic acid sequence encoding NiR into the cells of the plant.

[0036] (b) Introducing at least one expression construct containing at least one exogenous sequence of interest into the plant cells, wherein the at least one exogenous sequence of interest encodes a component of a gene editing system;

[0037] (c) Regenerate a complete plant from the plant cells.

[0038] In some implementations, steps (a) and (b) are performed in parallel (simultaneously). In some implementations, step (a) is performed first, followed by step (b). In some implementations, step (b) is performed first, followed by step (a).

[0039] In some embodiments of the method of the present invention, overexpression of NiR in the plant cells can also be achieved by modifying the expression regulatory sequence of the endogenous NiR gene in the plant cells. For example, overexpression of NiR in the plant cells can be achieved by gene editing the expression regulatory sequence of the endogenous NiR gene in the plant cells. Therefore, in some embodiments, the method includes introducing a gene editing system (including the introduction of one or more expression constructs encoding components of the gene editing system) targeting the expression regulatory sequence of the endogenous NiR gene into the plant cells, resulting in overexpression of the endogenous NiR gene in the plant cells.

[0040] In some embodiments of the method of the present invention, overexpression of NiR in the plant cells further includes increasing the biological activity of the endogenously expressed NiR protein in the plant cells. In some embodiments, a mutation leading to increased biological activity may be introduced into the endogenous NiR protein, for example, by gene editing. Therefore, in some embodiments, the method includes introducing a gene editing system (including the introduction of one or more expression constructs encoding components of the gene editing system) targeting an endogenous NiR coding sequence into the plant cells, resulting in increased biological activity of the endogenously expressed NiR protein in the plant cells.

[0041] The present invention also provides a kit for carrying out the method of the present invention, comprising at least an expression construct containing a coding nucleic acid sequence of NiR, or an expression construct of one or more components of a gene editing system, said gene editing system targeting an expression regulatory sequence or coding sequence of endogenous NiR, and capable of causing overexpression of endogenous NiR genes in plant cells or increased biological activity of endogenously expressed NiR proteins in plant cells.

[0042] The present invention also provides an expression construct comprising a coding nucleic acid sequence of NiR, or an expression construct comprising one or more components of a gene editing system encoding a regulatory sequence or coding sequence of endogenous NiR, for use in improving plant cell regeneration efficiency during plant transformation, improving the transformation efficiency of exogenous nucleic acid sequences of interest in plants, or improving gene editing efficiency in plants, wherein the gene editing system targeting the expression regulatory sequence or coding sequence of endogenous NiR can lead to overexpression of endogenous NiR genes in plant cells or increase the biological activity of endogenously expressed NiR proteins in plant cells.

[0043] Nitrite reductase (NiR) is a widely distributed enzyme in microorganisms and plants that catalyzes the reduction of nitrite. In plants, NiR is responsible for reducing nitrite to ammonium and is a crucial enzyme in nitrogen assimilation. The inventors have surprisingly discovered that overexpression of NiR in plant cells significantly improves the efficiency of regenerating complete plants and also significantly improves the efficiency of converting exogenous nucleic acid sequences of interest into plants. Even more unexpectedly, when exogenous nucleic acid sequences of interest encode gene editing systems, gene editing efficiency can be significantly improved.

[0044] In some embodiments, the NiR is a NiR or a functional variant thereof derived from a plant, such as a NiR or a functional variant thereof derived from wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, potato, or Arabidopsis thaliana. In some embodiments, the NiR is a NiR or a functional variant thereof derived from a plant species to be regenerated, transformed, or gene-edited. In some embodiments, the NiR is a NiR or a functional variant thereof derived from a plant species different from the plant species to be regenerated, transformed, or gene-edited.

[0045] In some embodiments, the NiR is strawberry NiR or a functional variant thereof. Exemplary strawberry NiR or a functional variant thereof contains, for example, the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0046] In some embodiments, the NiR is wheat NiR or a functional variant thereof. Exemplary wheat NiR or a functional variant thereof may contain, for example, the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3.

[0047] In some embodiments, the NiR is tomato NiR or a functional variant thereof. Exemplary tomato NiR or a functional variant thereof contains, for example, the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5.

[0048] In some embodiments, the coding nucleic acid sequence of the NiR and the at least one exogenous nucleic acid sequence of interest are placed in the same expression construct. In some embodiments, the coding nucleic acid sequence of the NiR and the at least one exogenous nucleic acid sequence of interest are placed in different expression constructs. In some embodiments, the coding nucleic acid sequence of the NiR is placed in one expression construct, and the at least one exogenous nucleic acid sequence of interest is placed in another expression construct.

[0049] In some implementations, the coding nucleic acid sequence of the NiR and / or the at least one exogenous nucleic acid sequence of interest is operatively linked to a transcriptional regulatory element.

[0050] The "at least one exogenous nucleic acid sequence of interest" can be any nucleic acid sequence that needs to be transformed into a plant. For example, the exogenous nucleic acid sequence of interest can be a nucleic acid sequence that encodes traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products. The nucleic acid sequence of interest can also include those nucleic acid sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, as well as those nucleic acid sequences that affect fruit size, sucrose load, etc.

[0051] In some preferred embodiments, the "at least one exogenous nucleic acid sequence of interest" encodes a component of the gene editing system, thereby enabling gene editing in plants. The gene editing system can target endogenous genes or their expression regulatory sequences that are important for traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products, thereby modifying the expression or activity of the endogenous genes. For example, the gene editing system can target endogenous genes or their expression regulatory sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, or endogenous genes or their expression regulatory sequences that affect fruit size, sucrose load, etc., thereby modifying the expression or activity of the endogenous genes.

[0052] Gene editing, also known as genome editing, involves the insertion, deletion, or substitution of nucleotides in an organism's genome using sequence-specific nucleases or their derivatives. Gene editing typically works by inducing site-specific double-strand breaks (DSBs) at desired locations in the genome, followed by the introduction of the desired DNA insertion, deletion, or substitution during DSB repair. However, gene editing can also encompass base editing techniques that do not involve DSBs, transcriptional activation or repression, and epigenetic modification techniques, provided they are sequence-specific.

[0053] This invention does not particularly limit the gene editing system used. For example, gene editing systems suitable for use in this invention include, but are not limited to, zinc finger nucleases (ZFNs), large-scale nucleases (MGNs), transcription activator-like effector nucleases (TALENs), and CRISPR (Clustered regularly interspaced short palindromic repeats) systems.

[0054] Zinc finger nucleases (ZFNs) are artificial restriction enzymes prepared by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. A single ZFN typically contains 3-6 individual zinc finger repeats, each of which can recognize a unique sequence, for example, 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

[0055] Meganucleases are typically homing endonucleases that recognize nucleic acid sequences of 14-40 bases in length. The long recognition sequence gives meganucleases high specificity, thus reducing off-target effects.

[0056] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences. They are typically prepared by fusing the DNA-binding domain of a transcription activator-like effector (TALE) with its DNA-cleaving domain. Once engineered, TALEs can bind to almost any desired DNA sequence.

[0057] A CRISPR system typically comprises two components that can form a sequence-specific complex: a CRISPR nuclease or a variant thereof, and a corresponding guide RNA. Therefore, for a CRISPR system, the "at least one exogenous nucleic acid sequence of interest" described in this invention may include the nucleic acid sequence encoding a CRISPR nuclease or a variant thereof, and / or the nucleic acid sequence encoding a corresponding guide RNA.

[0058] In some preferred embodiments, the gene editing system is a CRISPR system. A large number of different CRISPR gene editing systems are known in the art, and all can be used in this invention. For example, suitable CRISPR gene editing systems can be found in… http: / / www.addgene.org / crispr / CRISPR gene editing systems encompass systems that alter the genome sequence, as well as systems used for transcriptional regulation that do not alter the genome sequence.

[0059] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in the naturally occurring CRISPR system. "CRISPR nuclease variants" include modified forms of natural CRISPR nucleases, artificial mutants (including nicking enzyme mutants), catalytically active fragments, or fusions with other functional proteins / peptides. Various artificial functional variants of CRISPR nucleases are known in the art, such as highly specific variants or nicking enzyme variants, or fusion proteins of them with cytidine deaminases or adenosine deaminases. CRISPR nucleases or their variants can recognize, bind to, and / or cleave target nucleic acid structures by interacting with corresponding guide RNAs. Those skilled in the art will understand how to select suitable CRISPR nucleases or their variants to achieve the objectives of this invention.

[0060] The CRISPR nuclease or variant thereof used in the CRISPR gene editing system of the present invention may be selected, for example, from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1 (Cas12a), C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.

[0061] In some embodiments, the CRISPR nuclease or a variant thereof includes the Cas9 nuclease or a variant thereof. CRISPR gene editing systems based on the Cas9 nuclease or a variant thereof are also referred to herein as CRISPR-Cas9 gene editing systems. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from *Streptococcus pyogenes*.

[0062] Cas9 nuclease variants may include Cas9 nickase (nCas9), in which one of the two subdomains (HNH nuclease subdomain and RuvC subdomain) of the DNA cleavage domain of the Cas9 nuclease is inactivated to form the nickase. In some embodiments, the Cas9 nickase can be combined with two gRNAs targeting upstream and downstream of the sequence to be edited to achieve deletion of the sequence to be edited, or to achieve replacement of the sequence to be edited in the presence of a donor sequence.

[0063] In some embodiments, the CRISPR nuclease or a variant thereof may further include a Cpf1 (Cas12a) nuclease or a variant thereof, such as a highly specific variant. The Cpf1 nuclease may be a Cpf1 nuclease from different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceaebacterium ND2006. CRISPR gene editing systems based on Cpf1 nucleases or variants thereof are also referred to herein as CRISPR-Cpf1 systems.

[0064] In some embodiments, the CRISPR nuclease variant may also include a base editor. A base editor is typically a fusion protein containing a deaminase and a CRISPR nuclease variant lacking DNA cleavage activity.

[0065] As used in this invention, "CRISPR nuclease variants lacking DNA cleavage activity" include, but are not limited to, Cas9 nick nuclease (nCas9), cas9 nuclease with dead nuclease (dCas9), or casf1 nuclease with dead nuclease (dCpf1). Cas9 nuclease with dead nuclease (dCas9) or casf1 nuclease with dead nuclease (dCpf1) completely lacks DNA cleavage activity. Various CRISPR nuclease variants lacking DNA cleavage activity are known in the art.

[0066] As used in this invention, "deaminase" refers to an enzyme that catalyzes deamination reactions. In some embodiments of this invention, the deaminase refers to a cytosine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments of this invention, the deaminase refers to an adenine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the formation of inosine (I) from adenosine or deoxyadenosine (A). Various suitable cytosine deaminases or adenine deaminases that accept single-stranded DNA as a substrate are known in the art. Suitable cytosine deaminases include, but are not limited to, for example, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, and human APOBEC3A deaminase. In some preferred embodiments, the cytosine deaminase is human APOBEC3A. Examples of suitable adenine deaminases include, but are not limited to, the DNA-dependent adenine deaminases disclosed by Nicole M. Gaudelli et al. (doi: 10.1038 / nature24644, 2017).

[0067] By fusing a CRISPR nuclease variant lacking DNA cleavage activity with a deaminase (forming a so-called "base editor"), base editing of target nucleotide sequences, such as C-to-T or A-to-G conversions, can be achieved. Various base editors are known in the art, and those skilled in the art know how to select a suitable base editor to achieve the objectives of this invention. CRISPR gene editing systems based on base editors are also called base editing systems.

[0068] As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to RNA molecules capable of forming a complex with a CRISPR nuclease or a variant thereof and targeting the target sequence by means of a certain degree of similarity to the target sequence. For example, the gRNA used by the Cas9 nuclease or a variant thereof typically consists of partially complementary crRNA and tracrRNA molecules forming a complex, wherein the crRNA contains a guide sequence that is sufficiently similar to the target sequence to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single guide RNAs (sgRNAs) can be designed that contain features of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or a variant thereof typically consists only of mature crRNA molecules, which may also be referred to as sgRNA. Designing a suitable gRNA based on the CRISPR nuclease or a variant thereof and the target sequence to be edited is within the capabilities of those skilled in the art.

[0069] The sequence-specific nucleases used for gene editing in this invention, such as zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR nucleases or variants thereof, may also include subcellular localization signals (such as nuclear localization signals), peptide linkers, detectable tags, and other elements. For example, the base editor in a CRISPR base editing system typically includes one or more nuclear localization signals (NLS) to facilitate its entry into the cell nucleus and achieve editing of chromosomal DNA.

[0070] The expression constructs of the present invention can be introduced into plant cells by one of a variety of methods known in the art, including but not limited to gene gun method, PEG-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation.

[0071] In some embodiments, the plant cells described in this invention are cells suitable for regenerating complete plants through tissue culture. Examples of suitable plant cells include, but are not limited to, protoplast cells, callus cells, immature embryonic cells, and explant cells.

[0072] Methods for regenerating transformed whole plants by culturing transformed protoplasts, callus, immature embryos, or explants are known in the art. During said regeneration process, the transformants may also be screened based on selectivity markers carried on the introduced expression construct. In some embodiments, the regeneration is carried out without selection pressure.

[0073] In some embodiments, the expression constructs of the present invention are transiently transformed into plant cells. Transient transformation refers to introducing the construct into the cell to function but without integrating it into the cellular genome. This is particularly useful for gene editing because it can produce non-GMO modified plants.

[0074] Plants suitable for transformation or gene editing using the methods of this invention can be monocotyledonous or dicotyledonous plants. Examples of such plants include, but are not limited to, wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, and potato. The methods of this invention are particularly suitable for genetic transformation or gene editing in plant varieties or genotypes that have been previously difficult to transform. In some specific embodiments, the plant is wheat, for example, Xiaoyan 54 or Jimai 22. In some specific embodiments, the plant is strawberry, for example, the strawberry plant can be different varieties of strawberry plants, such as Benihoppe, Tochiotome, etc.

[0075] In order to achieve effective expression in plants, in some embodiments of the present invention, the coding nucleic acid sequence or the nucleic acid sequence of interest is codon-optimized for the plant species.

[0076] Codon optimization refers to the modification of nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example, in the Codon Usage Database (“Codon Usage Database”) available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See Nakamura. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0077] In some implementations, the coding nucleic acid sequence for NiR is selected from SEQ ID NO:2, 4, and 6.

[0078] In one aspect, the present invention provides plants and their offspring obtained by the method of the present invention.

[0079] Example

[0080] A further understanding of the invention can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Obviously, various modifications and variations can be made to the invention without departing from its spirit; therefore, such modifications and variations are also within the scope of protection claimed in this application.

[0081] Example 1: Overexpression of nitrite reductase NiR improves the efficiency of genetic transformation and gene editing in strawberries.

[0082] 1. Carrier Construction

[0083] Nitrite reductase in rice can improve the regeneration efficiency of dominant varieties. This study selected octoploid strawberry NiR for research, and tested whether it has an improving effect on the genetic transformation and gene editing of strawberry through different constructions.

[0084] First, the target gene FaNiR, which is highly homologous to rice NiR, was identified using BLAST (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2). The coding sequence of FaNiR was placed under the control of the ZmUBI promoter and cloned into the gene editing vector pHUE411-GFP, where FaNiR and Cas9 are expressed via P2A splitting. The resulting vector was named pHUE411-GFP-FaNiR. The vector map is shown below. Figure 1 As shown.

[0085] 2. Transformation of strawberry plants

[0086] The vector constructed above was used to transform strawberry callus tissue using Agrobacterium. The results of strawberry callus regeneration seedlings are as follows: Figure 2 As shown in the figure, overexpression of endogenous NiR in strawberries can significantly improve the efficiency of callus regeneration in strawberry seedlings.

[0087] Example 2: Overexpression of nitrite reductase NiR improves the efficiency of wheat genetic transformation and gene editing.

[0088] This embodiment selects wheat NiR for research to test whether it has an enhancing effect on the genetic transformation and gene editing of wheat.

[0089] 1. Carrier Construction

[0090] First, a wheat NiR sequence with the highest homology to the Kasalath NiR of easily regenerating indica rice was found through sequence alignment in NCBI. After optimization, expression was driven using ZmUBI. The construct is as follows: Figure 3 As shown.

[0091] 2. Analysis of transformation and editing efficiency using gene gun to transform immature wheat embryos.

[0092] The constructed vectors were transformed using the wheat gene gun transformation system. UBI-A3A was co-transformed with TaU6-sgRNA and different booster expression vectors (GFP as a control). The wheat ALS gene was selected as the editing site. Transformants were regenerated into plants through tissue culture. After herbicide resistance screening, resistant seedlings were further tested for mutants using PCR / RE methods. The results are as follows: Figure 4 As shown in the table below.

[0093] Gene gun plasmid Number of bombardment guns Total number of embryos Number of regenerated seedlings Number of mutant seedlings UBI-GFP-A3A 5 690 129 5 UBI-TaNiR-A3A 5 601 269 15

[0094] The results indicate that, compared to the control UBI-GFP-A3A, the addition of wheat NiR to UBI-NiR-A3A significantly improved the regeneration efficiency of wheat transformation, reaching 2.08 times. Unexpectedly, the number of mutants obtained increased significantly, reaching three times that of the control, far exceeding the increase in regeneration efficiency. This demonstrates that the plant nitrogen metabolism regulator nitrite reductase NiR can act as an important regulatory protein, promoting the efficiency of plant genetic transformation, and more importantly, improving the efficiency of genome editing.

[0095] Example 3: Improvement of Agrobacterium tumefaciens-mediated regeneration and transformation efficiency by NiR

[0096] 1. Construction of the tomato SlNiR vector

[0097] The above results indicate that plant growth factor NiR can improve the regeneration efficiency and gene editing efficiency of tissue culture in monocotyledonous wheat. Whether plant NiR can increase the regeneration efficiency of tissue-cultured dicotyledonous plants remains questionable. The inventors selected the endogenous NiR gene from the dicotyledonous plant tomato for research to see if it improves the plant regeneration efficiency of tomato. The inventors selected the tomato SlNiR gene as the test subject, started it with Cas9 using the 35S promoter, separated and linked by P2A, and started the sgRNA targeting the SlSP1 gene using the U6-26 promoter. The construct is as follows: Figure 5 As shown in the figure (pKSE401-NiR).

[0098] 2. The effect of tomato NiR on the regeneration efficiency of tomato callus tissue.

[0099] The vector constructed above was used to transform tomatoes with Agrobacterium, and the results on the tomato regeneration efficiency were as follows: Figure 6 And as shown in the table below:

[0100] deal with Number of explants Number of regenerated buds Number of transgenic buds Number of mutants <![CDATA[ p KSE401(CK)]]> 84 73 30 1 pKSE401-NiR 83 108 86 6

[0101] The above results indicate that endogenous NiR in tomatoes can significantly improve the efficiency of callus regeneration. In addition, testing the gene editing efficiency of endogenous genes in regenerated plants can also significantly improve their efficiency.

[0102] The sequence involved in this article

[0103] SEQ ID NO:1 Strawberry NiR amino acid sequence

[0104] MSSFGFLSPPPISSSAPTRQLSAATTPAAAPIVAPDVAAERLEPRVEERQGYWVLKEKFRQGINVQEKVKIQREPMKLYMEGGIEELAKLPFEELDKAKDTKDDIDVRLKWLGLFHRRKHHYGRFMMRLKLPNGVTTSAQTRYLASVIRRYGKDGCADVTTRQNWQIRGIVLADVPDILKGLAGVGLTSLQSGMDNVRNPVGNPLAGIDPHEIVDTRPYTNLLSQYITGNSLGNPTITNLPRKWNVCVIGSHDLYEHPHINDLAYMPATKDGKFGFNLLVGGFFSPKRCAEAVPLDAWVSTEDMIPVCKAILEAYRDLGFRGNRQKTRMMWLIDELGIEGFRSEVVKRMPFKELERASTEDLVQKQWERRDYIGVHPQKQEGLSFVGLHIPVGRIQADDMDELARIADEYGTGELRLTVEQNIIIPNVENTKLQALLQEPLLKEKFSPEPSILMKGLVACTGNQFCGQAIIETKQRALKVTEEVERHVSVTRPVRMHWTGCPNTCGQVQVADIGFMGCMARDENGKPVEGVDVFLGGRIGSDSHLGDIYKKSVPCKDLVPLVIDILVNHFGAVPREREEGEE

[0105] SEQ ID NO:2 Strawberry NiR Coding Sequence

[0106]

[0107] SEQ ID NO:3 Amino acid sequence of wheat NiR

[0108] MALFTEGGIKELAKLPMEQIDADKLTKEDVDVRLKWLGLFHRRKQQYGRFMMRLKLPNGVTTSEQTRYLAGVIEKYGKEGCADVTTRQNWQIRGVTLPDVPEILEGLRSVGLTSLQSGMDNVRNPVGSPLAGIDPLEIVDTRPYTNLLSSYITNNSEGNLAITNLPRKWNVCVIGTHDLYEHPHINDLAYMPAEKDGKFGFNLLVGGFISPKRWGEALPLDAWVPGDDIIPVCKAVLEAFRDLGTRGNRQKTRMMWLIDELGMEAFRSEIEKRMPNGVLERAAAEDLIDKKWERRDYLGVHPQKQEGLSFVGLHVPVGRLQAADMFELARLADEYGSGELRLTVEQNIVLPNVKNEKVEALLAEPLLQKFSAHPSLLMKGLVACTGNQFCGQAIIETKARALQVTRDVEARVSVPRAVRMHWTGCPNSCAQVQVADIGFMGCLTKNSSGKIVEAADIFVGGRVGSDSHLTGVYKKAVPCEDLVPLVADLLVERFGAVPREREEDEE

[0109] SEQ ID NO:4 Coding sequence of wheat NiR

[0110]

[0111] SEQ ID NO: 5 Tomato NiR Amino Acids

[0112] MASFSIKFLAPSLPNPTRFSKSSIVKLNATPPQTVAAAGPPEVAAERLEPRVEEKDGYWILKEQFRQGINPQEKVKIEKEPMKLFMENGIEELAKIPIEEIDQSKLTKDDIDVRLKWLGLFHRRKNQYGRFMMRLKLPNGVTTSAQTRYLASVIRKYGEEGCADITTRQNWQIRGVVLPDVPEILKGLEEVGLTSLQSGMDNVRNPVGNPLAGIDPEEIVDTRPYTNLLSQFITGNSRGNPAVSNLPRKWNPCVVGSHDLYEHPHINDLAYMPAIKDGRFGFNLLVGGFFSAKRCDEAIPLDAWVPADDVVPVCKAILEAFRDLGFRGNRQKCRMMWLIDELGVEGFRAEVVKRMPQQELERASPEDLVQKQWERRDYLGVHPQKQEGYSFIGLHIPVGRVQADDMDDLARLADEYGSGELRLTVEQNIIIPNIENSKIDALLKEPILSKFSPDPPILMKGLVACTGNQFCGQAIIETKARSLKITEEVQRQVSLTRPVRMHWTGCPNTCAQVQVADIGFMGCLTRDKDKKTVEGADVFLGGRIGSDSHLGEVYKKAVPCDELVPLIVDLLIKNFGAVPREREETED

[0113] SEQ ID NO: 6 Tomato SlNiR Coding Sequence

[0114] sequence list <110> Shanghai Blue Cross Medical Science Research Institute <120> A method to improve the efficiency of plant genetic transformation and gene editing <130> I2022TC6709CB <150> 202110321591.2 <151> 2021-03-25 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 582 <212> PRT <213> Fragaria x ananassa <400> 1 Met Ser Ser Phe Gly Phe Leu Ser Pro Pro Pro Ile Ser Ser Ser Ala 1 5 10 15 Pro Thr Arg Gln Leu Ser Ala Ala Thr Thr Pro Ala Ala Ala Pro Ile 20 25 30 Val Ala Pro Asp Val Ala Ala Glu Arg Leu Glu Pro Arg Val Glu Glu 35 40 45 Arg Gln Gly Tyr Trp Val Leu Lys Glu Lys Phe Arg Gln Gly Ile Asn 50 55 60 Val Gln Glu Lys Val Lys Ile Gln Arg Glu Pro Met Lys Leu Tyr Met 65 70 75 80 Glu Gly Gly Ile Glu Glu Leu Ala Lys Leu Pro Phe Glu Glu Leu Asp 85 90 95 Lys Ala Lys Asp Thr Lys Asp Asp Ile Asp Val Arg Leu Lys Trp Leu 100 105 110 Gly Leu Phe His Arg Arg Lys His His Tyr Gly Arg Phe Met Met Arg 115 120 125 Leu Lys Leu Pro Asn Gly Val Thr Thr Ser Ala Gln Thr Arg Tyr Leu 130 135 140 Ala Ser Val Ile Arg Arg Tyr Gly Lys Asp Gly Cys Ala Asp Val Thr 145 150 155 160 Thr Arg Gln Asn Trp Gln Ile Arg Gly Ile Val Leu Ala Asp Val Pro 165 170 175 Asp Ile Leu Lys Gly Leu Ala Gly Val Gly Leu Thr Ser Leu Gln Ser 180 185 190 Gly Met Asp Asn Val Arg Asn Pro Val Gly Asn Pro Leu Ala Gly Ile 195 200 205 Asp Pro His Glu Ile Val Asp Thr Arg Pro Tyr Thr Asn Leu Leu Ser 210 215 220 Gln Tyr Ile Thr Gly Asn Ser Leu Gly Asn Pro Thr Ile Thr Asn Leu 225 230 235 240 Pro Arg Lys Trp Asn Val Cys Val Ile Gly Ser His Asp Leu Tyr Glu 245 250 255 His Pro His Ile Asn Asp Leu Ala Tyr Met Pro Ala Thr Lys Asp Gly 260 265 270 Lys Phe Gly Phe Asn Leu Leu Val Gly Gly Phe Phe Ser Pro Lys Arg 275 280 285 Cys Ala Glu Ala Val Pro Leu Asp Ala Trp Val Ser Thr Glu Asp Met 290 295 300 Ile Pro Val Cys Lys Ala Ile Leu Glu Ala Tyr Arg Asp Leu Gly Phe 305 310 315 320 Arg Gly Asn Arg Gln Lys Thr Arg Met Met Trp Leu Ile Asp Glu Leu 325 330 335 Gly Ile Glu Gly Phe Arg Ser Glu Val Val Lys Arg Met Pro Phe Lys 340 345 350 Glu Leu Glu Arg Ala Ser Thr Glu Asp Leu Val Gln Lys Gln Trp Glu 355 360 365 Arg Arg Asp Tyr Ile Gly Val His Pro Gln Lys Gln Glu Gly Leu Ser 370 375 380 Phe Val Gly Leu His Ile Pro Val Gly Arg Ile Gln Ala Asp Asp Met 385 390 395 400 Asp Glu Leu Ala Arg Ile Ala Asp Glu Tyr Gly Thr Gly Glu Leu Arg 405 410 415 Leu Thr Val Glu Gln Asn Ile Ile Ile Pro Asn Val Glu Asn Thr Lys 420 425 430 Leu Gln Ala Leu Leu Gln Glu Pro Leu Leu Lys Glu Lys Phe Ser Pro 435 440 445 Glu Pro Ser Ile Leu Met Lys Gly Leu Val Ala Cys Thr Gly Asn Gln 450 455 460 Phe Cys Gly Gln Ala Ile Ile Glu Thr Lys Gln Arg Ala Leu Lys Val 465 470 475 480 Thr Glu Glu Val Glu Arg His Val Ser Val Thr Arg Pro Val Arg Met 485 490 495 His Trp Thr Gly Cys Pro Asn Thr Cys Gly Gln Val Gln Val Ala Asp 500 505 510 Ile Gly Phe Met Gly Cys Met Ala Arg Asp Glu Asn Gly Lys Pro Val 515 520 525 Glu Gly Val Asp Val Phe Leu Gly Gly Arg Ile Gly Ser Asp Ser His 530 535 540 Leu Gly Asp Ile Tyr Lys Lys Ser Val Pro Cys Lys Asp Leu Val Pro 545 550 555 560 Leu Val Ile Asp Ile Leu Val Asn His Phe Gly Ala Val Pro Arg Glu 565,570,575 Arg Glu Glu Gly Glu Glu 580 <210> 2 <211> 1749 <212> DNA <213> Strawberry x pineapple <400> 2 atgcgtcat ttgggttcct atctcctcct ctatatcct cctcggctcc cacgaggcag 60 ctctccgcag ccactactcc ggctgcggca ccgattgtcg ctccggacgt ggctgcggag 120 aggctggagc cgagggtgga ggagacaa gggtattggg tgttgaagga gaagttttcga 180 caagggatta atgttcagga gaaggtcaag attcagaggg agcctatgaa gctatatatg 240 gaaggcggga ttgaagagct tgctaagttg cttttgagg agcttgacaa agctaaggat 300 actaaggatg atattgatgt taggctcaag tggcttggtc tcttccacag aagaaagcat 360 cactatggta gatttatgat gaggctgaag cttccgaatg gggtcacaac aagtgcacag 420 acaagatatc tagcgagtgt gatacggaga tatggcaagg atggttgtgc agatgtgaca 480 accaggcaaa attggcaaat ccgcggaatt gtgctggcag atgtgcctga tattctgaag 540 ggtcttgctg gagtaggatt gactagtctg cagagtggga tggacaatgt gagaaaccca 600 gttgggaatc cacttgcagg cattgacccg catgaaattg ttgacacaag accttacacg 660 aacttgttgt cccaatacat tactggcaat tcactcggca atccaactat aacaaacttg 720 ccaaggaagt ggaatgtgtg tgtgataggg tctcatgatc tttatgagca tccccacatc 780 aatgatcttg cgtacatgcc tgcaacaaag gatgggaaat ttggattcaa tttactcgtt 840 ggaggcttct tcagtcccaa aagatgtgca gaggcagttc ctcttgatgc atgggtctca 900 actgaagata tgatcccagt ttgcaaagct attctagagg ctttaagaga ccttggattc 960 agaggcaacc gacaaaagac aagaatgatg tggttgatcg atgaactagg catagaagga 1020 ttcagatcag aggttgttaa gagaatgccc tttaaggaac tggaaagagc atctactgaa 1080 gacttggtac agaagcaatg ggaaggaga gactatatag gtgtccatcc acagaaacaa 1140 gaaggtctaa gctttgtggg tcttcacatt cccgtaggca ggatccaagc agatgacatg 1200 gatgagctag ctcgtatagc tgacgagtat ggcactgggg aactccgtct cactgtggag 1260 caaaacatta ttatccccaa tgttgagaac acgaaactcc aagcattgct tcaagaaccg 1320 cttctgaaag aaaaattttc acctgaacct tccattctca tgaaggact agtagcttgc 1380 actggcaacc agttttgcgg ccaagccatc atcgagacaa agcaacgggc cttgaaggtg 1440 actgaggagg tggaaaggca tgtgtcagtg actcggccag tgaggatgca ttggacgggg 1500 tgccccaata cctgcgggca ggtgcaagtg gcagatatag gtttcatggg gtgtatggca 1560 agggatgaga atggaaagcc tgttgaaggg gttgatgtct tcttgggagg aagaatagga 1620 agtgactctc atttgggaga tatttataag aagagtgtcc cttgtaagga cttggtgccc 1680 ttagttattg acatcttggt aaatcacttt ggggctgtcc caagggagag ggaagaaggg 1740 gaagagtaa 1749 <210> 3 <211> 506 <212> PRT <213> Triticum aestivum <400> 3 Met Ala Leu Phe Thr Glu Gly Gly Ile Lys Glu Leu Ala Lys Leu Pro 1 5 10 15 Met Glu Gln Ile Asp Ala Asp Lys Leu Thr Lys Glu Asp Val Asp Val 20 25 30 Arg Leu Lys Trp Leu Gly Leu Phe His Arg Arg Lys Gln Gln Tyr Gly 35 40 45 Arg Phe Met Met Arg Leu Lys Leu Pro Asn Gly Val Thr Thr Ser Glu 50 55 60 Gln Thr Arg Tyr Leu Ala Gly Val Ile Glu Lys Tyr Gly Lys Glu Gly 65 70 75 80 Cys Ala Asp Val Thr Thr Arg Gln Asn Trp Gln Ile Arg Gly Val Thr 85 90 95 Leu Pro Asp Val Pro Glu Ile Leu Glu Gly Leu Arg Ser Val Gly Leu 100 105 110 Thr Ser Leu Gln Ser Gly Met Asp Asn Val Arg Asn Pro Val Gly Ser 115 120 125 Pro Leu Ala Gly Ile Asp Pro Leu Glu Ile Val Asp Thr Arg Pro Tyr 130 135 140 Thr Asn Leu Leu Ser Ser Tyr Ile Thr Asn Asn Ser Glu Gly Asn Leu 145 150 155 160 Ala Ile Thr Asn Leu Pro Arg Lys Trp Asn Val Cys Val Ile Gly Thr 165 170 175 His Asp Leu Tyr Glu His Pro His Ile Asn Asp Leu Ala Tyr Met Pro 180 185 190 Ala Glu Lys Asp Gly Lys Phe Gly Phe Asn Leu Leu Val Gly Gly Phe 195 200 205 Ile Ser Pro Lys Arg Trp Gly Glu Ala Leu Pro Leu Asp Ala Trp Val 210 215 220 Pro Gly Asp Asp Ile Ile Pro Val Cys Lys Ala Val Leu Glu Ala Phe 225 230 235 240 Arg Asp Leu Gly Thr Arg Gly Asn Arg Gln Lys Thr Arg Met Met Trp 245 250 255 Leu Ile Asp Glu Leu Gly Met Glu Ala Phe Arg Ser Glu Ile Glu Lys 260 265 270 Arg Met Pro Asn Gly Val Leu Glu Arg Ala Ala Ala Glu Asp Leu Ile 275 280 285 Asp Lys Lys Trp Glu Arg Arg Asp Tyr Leu Gly Val His Pro Gln Lys 290 295 300 Gln Glu Gly Leu Ser Phe Val Gly Leu His Val Pro Val Gly Arg Leu 305 310 315 320 Gln Ala Ala Asp Met Phe Glu Leu Ala Arg Leu Ala Asp Glu Tyr Gly 325 330 335 Ser Gly Glu Leu Arg Leu Thr Val Glu Gln Asn Ile Val Leu Pro Asn 340 345 350 Val Lys Asn Glu Lys Val Glu Ala Leu Leu Ala Glu Pro Leu Leu Gln 355 360 365 Lys Phe Ser Ala His Pro Ser Leu Leu Met Lys Gly Leu Val Ala Cys 370 375 380 Thr Gly Asn Gln Phe Cys Gly Gln Ala Ile Ile Glu Thr Lys Ala Arg 385 390 395 400 Ala Leu Gln Val Thr Arg Asp Val Glu Ala Arg Val Ser Val Pro Arg 405 410 415 Ala Val Arg Met His Trp Thr Gly Cys Pro Asn Ser Cys Ala Gln Val 420 425 430 Gln Val Ala Asp Ile Gly Phe Met Gly Cys Leu Thr Lys Asn Ser Ser 435 440 445 Gly Lys Ile Val Glu Ala Ala Asp Ile Phe Val Gly Gly Arg Val Gly 450 455 460 Ser Asp Ser His Leu Thr Gly Val Tyr Lys Lys Ala Val Pro Cys Glu 465 470 475 480 Asp Leu Val Pro Leu Val Ala Asp Leu Leu Val Glu Arg Phe Gly Ala 485 490 495 Val Pro Arg Glu Arg Glu Glu Asp Glu Glu 500 505 <210> 4 <211> 1521 <212> DNA <213> Triticum aestivum <400> 4 atggcgctct tcacggagggg cggcatcaag gagctcgcca agctgccat ggagcagatc 60 gacgccgaca agctcaccaa ggaggacgtc gacgtgcggc tcaagtggct cggccctctc 120 caccgccgca agcagcagta tggcggttc atgatgcggc tgaagctgcc caacggcgtg 180 acgacgagcg agcagacgcg ctacctggcc ggcgtgatcg agagtacgg caggagggg 240 tgcgccgacg tgacgacccg gcagaactgg cagatccgcg gcgtgacgct gccggacgtg 300 ccggagatcc tggaggct ccgctccgtt ggctcacca gcctgcagag cggcatggac 360 aacgtgcgca accccgtcgg cagcccgctc gccggcatcg accccctcga gatcgtcgac 420 acgcgcccct acaccacct cctctcctcc tacatcacca aciactccga gggcaacctc 480 gccatcacca accttcctag gaagtggaac gtgtgcgtga tcggtaccca tgacctgtac 540 gagcacccgc acatcaacga cctggcgtac atgccggccg agaaggacgg caagttcggg 600 ttcaacctgc tggtgggcgg gttcatcagc cccaagaggt ggggcgaggc cctgccgctc 660 gacgcctggg tccccggcga cgacatcatc ccggtctgca aggccgtcct cgaggctttc 720 cgcgacctcg gcaccagggg caaccgccag aagacgcgca tgatgtggct catcgacgag 780 ctcgggatgg aggcgttccg gtcggagatc gagaagagga tgccgaacgg cgtgctggag 840 cgcgcggcgg ccgaggacct gatcgacaag aagtgggagc ggcgcgacta cctcggcgtg 900 cacccgcaga agcaggaggg gctctccttc gtgggcctgc acgtgcccgt cggccggctg 960 caggccgccg acatgttcga gctggcccgc ctcgccgacg agtacggctc cggcgagctc 1020 cgcctcaccg tggagcagaa catcgtcctc cccaacgtca agaacgagaa ggtggaagct 1080 ctgctcgccg agccgctgct gcagaagttc tcggcgcacc cgtcgctgct gatgaagggg 1140 ctggtggcgt gcacgggcaa ccagttctgc gggcaggcca tcatcgagac caaggcgcgg 1200 gcgctgcagg tgacgcgcga cgtggaggcg cgcgtgtccg tgcccagggc ggtgcgcatg 1260 cactggacgg ggtgccccaa cagctgcgcg caggtgcagg tggccgacat cggcttcatg 1320 ggctgcctca ccaagaacag cagcggcaag atcgtcgagg cggcggacat cttcgtcggc 1380 ggccgcgtcg gcagcgactc gcacctcacc ggggtgtaca agaaggcggt gccgtgcgag 1440 gacctggtgc ccctcgtcgc cgacctcctg gtggagcggt tcggggccgt gcccagggag 1500 agggaggagg acgaggagta g 1521 <210> 5 <211> 587 <212> PRT <213> Solanum lycopersicum <400> 5 Put Here Sister Phe Sister Ile Lys Phe Leu Ala Pro Sister Leu Pro Donkey Pro 1 5 10 15 Thr Arg Phe Ser Lys Ser Ser Ile Val Lys Leu Asn Ala Thr Pro Pro 20 25 30 Gln Thr Val Ala Ala Ala Gly Pro Pro Glu Val Ala Ala Glu Arg Leu 35 40 45 Glu Pro Arg Val Glu Glu Lys Asp Gly Tyr Trp Ile Leu Lys Glu Gln 50 55 60 Phe Arg Gln Gly Ile Asn Pro Gln Glu Lys Val Lys Ile Glu Lys Glu 65 70 75 80 Pro Met Lys Leu Phe Met Glu Asn Gly Ile Glu Glu Leu Ala Lys Ile 85 90 95 Pro Ile Glu Glu Ile Asp Gln Ser Lys Leu Thr Lys Asp Asp Ile Asp 100 105 110 Val Arg Leu Lys Trp Leu Gly Leu Phe His Arg Arg Lys Asn Gln Tyr 115 120 125 Gly Arg Phe Met Met Arg Leu Lys Leu Pro Asn Gly Val Thr Thr Ser 130 135 140 Ala Gln Thr Arg Tyr Leu Ala Ser Val Ile Arg Lys Tyr Gly Glu Glu 145 150 155 160 Gly Cys Ala Asp Ile Thr Thr Arg Gln Asn Trp Gln Ile Arg Gly Val 165 170 175 Val Leu Pro Asp Val Pro Glu Ile Leu Lys Gly Leu Glu Glu Val Gly 180 185 190 Leu Thr Ser Leu Gln Ser Gly Met Asp Asn Val Arg Asn Pro Val Gly 195 200 205 Asn Pro Leu Ala Gly Ile Asp Pro Glu Glu Ile Val Asp Thr Arg Pro 210 215 220 Tyr Thr Asn Leu Leu Ser Gln Phe Ile Thr Gly Asn Ser Arg Gly Asn 225 230 235 240 Pro Ala Val Ser Asn Leu Pro Arg Lys Trp Asn Pro Cys Val Val Gly 245 250 255 Ser His Asp Leu Tyr Glu His Pro His Ile Asn Asp Leu Ala Tyr Met 260 265 270 Pro Ala Ile Lys Asp Gly Arg Phe Gly Phe Asn Leu Leu Val Gly Gly 275 280 285 Phe Phe Ser Ala Lys Arg Cys Asp Glu Ala Ile Pro Leu Asp Ala Trp 290 295 300 Val Pro Ala Asp Asp Val Val Pro Val Cys Lys Ala Ile Leu Glu Ala 305 310 315 320 Phe Arg Asp Leu Gly Phe Arg Gly Asn Arg Gln Lys Cys Arg Met Met 325 330 335 Trp Leu Ile Asp Glu Leu Gly Val Glu Gly Phe Arg Ala Glu Val Val 340 345 350 Lys Arg Met Pro Gln Gln Glu Leu Glu Arg Ala Ser Pro Glu Asp Leu 355 360 365 Val Gln Lys Gln Trp Glu Arg Arg Asp Tyr Leu Gly Val His Pro Gln 370 375 380 Lys Gln Glu Gly Tyr Ser Phe Ile Gly Leu His Ile Pro Val Gly Arg 385 390 395 400 Val Gln Ala Asp Asp Met Asp Asp Leu Ala Arg Leu Ala Asp Glu Tyr 405 410 415 Gly Ser Gly Glu Leu Arg Leu Thr Val Glu Gln Asn Ile Ile Ile Pro 420 425 430 Asn Ile Glu Asn Ser Lys Ile Asp Ala Leu Leu Lys Glu Pro Ile Leu 435 440 445 Ser Lys Phe Ser Pro Asp Pro Pro Ile Leu Met Lys Gly Leu Val Ala 450 455 460 Cys Thr Gly Asn Gln Phe Cys Gly Gln Ala Ile Ile Glu Thr Lys Ala 465 470 475 480 Arg Ser Leu Lys Ile Thr Glu Glu Val Gln Arg Gln Val Ser Leu Thr 485 490 495 Arg Pro Val Arg Met His Trp Thr Gly Cys Pro Asn Thr Cys Ala Gln 500 505 510 Val Gln Val Ala Asp Ile Gly Phe Met Gly Cys Leu Thr Arg Asp Lys 515 520 525 Asp Lys Lys Thr Val Glu Gly Ala Asp Val Phe Leu Gly Gly Arg Ile 530 535 540 Gly Ser Asp Ser His Leu Gly Glu Val Tyr Lys Lys Ala Val Pro Cys 545 550 555 560 Asp Glu Leu Val Pro Leu Ile Val Asp Leu Leu Ile Lys Asn Phe Gly 565,570,575 Ala Val Pro Arg Glu Arg Glu Glu Thr Glu Asp 580,585 <210> 6 <211> 1761 <212> DNA <213> Solanum lycopersicum <400> 6 atggcatctt tttctcatcaa atttttggca ccttcattgc caatccaac tagattttcc 60 aagagtagta ttgtcaagct caatgcaact ccgccgcaga cagtggctgc ggcggggcct 120 ccagaggttg ctgctgagag actagaacca agagttgagg aaaagatgg atattggata 180 ctaaaagagc agtttaggca aggattaat cctcaagaga agtgagat tgagagaa 240 cctatgaagt tgttcatgga aaatgtatt gaggagttag ctaagattcc attgaagg 300 atagatcaat caaagcttac taaggatgac attgatgtta ggctcaagtg gcttggccctc 360 ttccatagga gaaagaatca atatgggaga ttcatgatga ggttgaact tccaatgga 420 gtaacaaa gtgctcagac tcgatatttg gcgagtgtga taaggaata tggaggaaa 480 ggatgtgctg atattacgac aaggcaaat tggcagattc gtggagtagt gctgcctgat 540 gtgcctgaga ttctaaaggg acttgaagaa gttggcttga ctagtttgca gagtggcatg 600 gataatgtca ggaatccagt tggaatcct ctggctggaa ttgatctga agaatagtt 660 gabacagac cttacacta tttgctcc caatttatca ctggtaattc acgaggcaat 720 ccggctgttt ctaacttgcc aaggaagtgg aatccgtgtg tagtaggtc tcatgatctt 780 tatgagcacc ctcatatcaa tgatcttgca tacatgcctg ccataaaga tggacgattt 840 ggattcacc tgcttgtggg agggttctc agtgccaaaa gatgtgatga ggcaatttcct 900 cttgatgcat gggttccagc cgatgatgtt gttccggttt gcaagcaat actggaagct 960 tttagagacc ttggttcag agggaacagg cagaagtgta gatgatgtg gttgatcgat 1020 gaactgggtg tagaggatt caggggcagag gtcgtaaga gatgcctca gcaagagcta 1080 gagagagcat ctccggaaga cttggttcag aaacaatggg aaagagaga ttatctttggt 1140 gtacatccac agaaacagga aggctatagc tttattggtc ttcacattcc agtgggtcgt 1200 gtccaagcag acgacatgga tgatctagct cgttttggctg atgagtacgg ctcaggagag 1260 ctacggctga ctgtggaaca gaacattat attcccaaca ttgagaactc aaagattgac 1320 gcactgctaa aagagcctat tttgagcaaa ttttcacctg atccacctat tctcatgaaa 1380 ggtttagtgg cttgtactgg taaccagttt tgtggacaag ccattattga aacgaaagct 1440 cgttccctga agatcaccga agaggttcaa aggcaagtat ctctaacgag gccagtaagg 1500 atgcactgga caggctgcc aaatacgtgt gcacaagttc aagttgcaga cattggattc 1560 atgggatgcc tgactagaga taaagacaag aagactgtgg aaggcgccga tgttttctta 1620 ggaggcagaa tagggagtga ctcacatttg ggtgaagtat acaagaaggc agttccttgt 1680 gatgaattag taccacttat tgtggactta cttattaaga actttggtgc agttccacga 1740 gaaagagaag aaacagaaga t 1761

Claims

1. A method for improving the efficiency of plant gene editing, the method comprising: (a) Overexpressing NiR in the cells of the plant, specifically by introducing an expression construct containing a nucleic acid sequence encoding the plant's endogenous NiR into the cells of the plant; (b) Introducing into the plant cells at least one expression construct containing at least one exogenous nucleic acid sequence of interest, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system; and (c) Regenerating a complete plant from the plant cells. The gene editing system is a CRISPR system; the plant is selected from wheat and tomato; and the NiR is the amino acid sequence shown in SEQ ID NO: 3 or 5.

2. The method of claim 1, wherein the coding nucleic acid sequence of NiR and the at least one exogenous nucleic acid sequence of interest are placed in the same expression construct.

3. The method of claim 1, wherein the CRISPR system is a base editing system.

4. The method of claim 1, wherein the cells are selected from protoplast cells, callus cells, cells of immature embryos, and explant cells.

Images