CN116622758A - Method for improving genetic transformation and gene editing efficiency of plants - Google Patents

Abstract

The application belongs to the field of plant genetic engineering. In particular, the present application relates to a method for improving genetic transformation and gene editing efficiency of plants. More particularly, the present application relates to improving the regeneration efficiency of genetic transformation of plants and/or improving the efficiency of editing plant genes by expressing histone demethylase genes.

Description

A method to improve the efficiency of plant genetic transformation and gene editing

technical field

The invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to a method for improving the efficiency of plant genetic transformation and gene editing. More specifically, the present invention relates to increasing the regeneration efficiency of genetic transformation of plants and/or increasing the efficiency of gene editing in plants by expressing histone demethylase genes.

Background of the invention

Crop genetics and breeding have experienced artificial selection breeding, cross breeding, mutation breeding and molecular marker assisted breeding by means of molecular technology. As the genetic diversity of the varieties used gradually decreases, the bottleneck effect of traditional breeding becomes more and more obvious: it is difficult to breed breakthrough new varieties using conventional breeding techniques, which cannot meet the needs of human beings and the development of sustainable agriculture. The rapid development of life sciences has led to the post-genome era from the "reading" stage of biological genetic information, and the precise "rewriting" and even "new design" of genomes are gradually becoming a reality. This kind of biological technology aimed at designing and creating new traits or living organisms has shown great prospects in the fields of disease treatment, medicine, manufacturing, and especially agriculture.

Genome editing technology is a revolutionary technology emerging in the current life sciences. It can achieve precise, efficient and specific rewriting of the genome, and has a revolutionary role in promoting the research and exploration of the entire life sciences. Gene editing refers to the deletion, replacement, insertion and other operations of target genes to rewrite genetic information to obtain new functions or phenotypes, and even create new species. The development of efficient and precise breeding technology based on gene editing technology suitable for crops will break through the shortcomings of traditional breeding and realize molecular design breeding that is precisely transformed from the genome. It has important strategic significance for the development of future agriculture.

Current gene editing technologies mainly include ZFN, TALEN and CRISPR/Cas systems. Due to its high efficiency and flexibility, the CRISPR/Cas system is currently the simplest and most widely used gene editing technology system. In the CRISPR/Cas system, the Cas protein can be targeted to any position in the genome under the guidance of an artificially designed guide RNA (guide RNA). The base editing system is a new gene editing technology developed based on the CRISPR system. It is divided into a cytosine base editing system and an adenine base editing system. Fusion, under the targeting of the guide RNA, the Cas9 single-stranded nickase generates a single-stranded DNA region, so the deaminase can efficiently deaminate the C and A nucleotides on the single-stranded DNA at the targeted position , into U bases and I bases, and then repaired into T bases and G bases in the process of cell self-repair. Base editing technology overcomes the shortcomings of traditional DSB-mediated gene editing, and can efficiently achieve precise replacement of a single base. The powerful genome modification technology system mediated by the CRISPR/Cas system will provide strong technical support for plant genomics research and new plant molecular design and breeding, and will accelerate the cultivation of new crop varieties and realize the sustainable development of agriculture.

A key step in plant gene editing is to deliver the gene editing nuclease protein or encoding nucleic acid to plant cells to achieve the editing of the target gene. At present, the delivery technology of plant genome editing is mainly realized through genetic transformation and tissue culture technology, mainly including Agrobacterium-mediated method and gene gun method. Significant progress has been made in plant transformation and genetic modification in the past few years, but the transformation of major crops such as wheat is still limited by factors such as genotype and transformation material period.

Brief description of the drawings

Figure 1. Shows the map of vectors administered in the examples of the present application.

Figure 2 shows that histone demethylase JMJ706 can significantly improve the efficiency of wheat callus regeneration.

Detailed ways

1. Definition

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are described more fully in: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook"). Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "and/or" covers all combinations of the items connected by the term, and each combination should be deemed to have been individually listed herein. For example, "A and/or B" includes "A," "A and B," and "B." For example, "A, B, and/or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

When the term "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of said sequence, or may have additional amino acids or core elements at one or both ends of said protein or nucleic acid. Nucleotides, but still have the activity described in the present invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminal of the polypeptide may be retained in some practical situations (eg, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific amino acid sequence of a polypeptide in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, it also covers a methionine containing the methionine at this time. Sequence, correspondingly, its coding nucleotide sequence may also contain an initiation codon; and vice versa.

"Genome" as used herein encompasses not only chromosomal DNA present in the nucleus, but also organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

"Exogenous" with reference to a sequence means a sequence from a foreign species, or, if from the same species, a sequence that has undergone significant changes in composition and/or locus from its native form through deliberate human intervention.

"Nucleic acid sequence," "polynucleotide," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter designations as follows: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or Deoxyguanosine, "U" means uridine, "T" means deoxythymidine, "R" means purine (A or G), "Y" means pyrimidine (C or T), "K" means G or T," H" means A or C or T, "D" means A, T or G, "I" means inosine, and "N" means any nucleotide.

"Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of the corresponding naturally occurring amino acid, 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, gamma carboxylation of glutamic acid residues, hydroxylation ylation and ADP-ribosylation.

As used in the present 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, expression of a nucleic acid sequence can refer to transcription of the nucleic acid sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of the RNA into a precursor or mature protein.

The "expression construct" of the present invention may be a linear nucleic acid segment (including a DNA or RNA segment), a circular plasmid, or a viral vector.

An "expression construct" of the present invention may comprise a regulatory sequence and a nucleic acid sequence of interest operably linked thereto. The regulatory sequence and the nucleic acid sequence of interest may be of different origin, or of the same origin but arranged in a manner different from that normally found in nature.

"Regulatory sequence" and "regulatory element" are used interchangeably to refer to a sequence that is located upstream (5' non-coding sequences), midway or downstream (3' non-coding sequences) of a coding sequence and that affects the transcription, RNA processing or Stable or translated nucleotide sequences. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. "Promoter" refers to a nucleic acid segment capable of controlling the transcription of another nucleic acid segment. In some embodiments of the invention, the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from said cell. The promoter may be a constitutive promoter or a tissue specific promoter or a developmentally regulated promoter or an inducible promoter.

As used herein, the term "operably linked" means that a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) is linked to a nucleic acid sequence (such as a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequences is controlled and regulated by said transcriptional regulatory elements. Techniques for operably linking regions of regulatory elements to nucleic acid molecules are known in the art.

"Introducing" a nucleic acid molecule (eg, an expression construct) into a plant cell means presenting the nucleic acid molecule to the plant cell such that the nucleic acid molecule enters the interior of the plant cell.

"Regeneration" refers to the process of growing a whole plant from one or more plant cells (eg, plant protoplasts, callus, or explants).

2. Improved Plant Regeneration and Transformation

In one aspect, the present invention provides a method for improving regeneration efficiency of plant cells, the method comprising a) introducing into the plant cells an expression construct comprising a nucleic acid sequence encoding a histone demethylase. In some embodiments, the method further comprises b) regenerating whole plants from the plant cells obtained in a).

In one aspect, the present invention provides a method for improving the transformation efficiency of an exogenous nucleic acid sequence of interest in a plant or transforming an exogenous nucleic acid sequence of interest into a plant, the method comprising:

(a) introducing an expression construct comprising a nucleic acid sequence encoding a histone demethylase into a cell of the plant;

(b) introducing into said plant cell at least one expression construct comprising at least one exogenous nucleic acid sequence of interest

Build body;

(c) regenerating whole plants from said plant cells.

In another aspect, the present invention provides a method of increasing the efficiency of gene editing in plants or performing gene editing in plants, the method comprising:

(a) introducing into the cells of said plant an expression construct comprising a nucleic acid sequence encoding a histone demethylase,

(b) introducing into said plant cell at least one expression construct comprising at least one exogenous sequence of interest, wherein said at least one exogenous sequence of interest encodes a component of a gene editing system;

(c) regenerating whole plants from said plant cells.

The present invention also provides a kit for implementing the method of the present invention, which at least comprises an expression construct comprising a nucleic acid sequence encoding a histone demethylase.

The present invention also provides an expression construct comprising a nucleic acid sequence encoding a histone demethylase for improving plant cell regeneration efficiency in plant transformation, improving the transformation efficiency of exogenous nucleic acid sequences of interest in plants, or improving gene expression in plants. Uses for editorial efficiency.

In some embodiments, the histone demethylase is a H3K27me3 demethylase. In some embodiments, the histone demethylase is a plant-derived histone demethylase. In some embodiments, the histone demethylase is derived from rice histone demethylase JMJ706.

Histone demethylase JMJ706 is responsible for H3K27me3 demethylation in rice or wheat and is an important enzyme for epigenetic modification in plants. The present inventors surprisingly found that when rice-derived JMJ706 or wheat-derived JMJ30 is overexpressed in plants such as wheat cells, it can significantly improve the regeneration efficiency of plants such as wheat cells into complete plants such as wheat plants, and also significantly improve the efficiency of exogenous Efficiency of transformation of a nucleic acid sequence of interest into plants such as wheat plants. When the exogenous nucleic acid sequence of interest encodes the gene editing system, the efficiency of gene editing can be significantly improved.

In some embodiments, the histone demethylase suitable for the present invention comprises, for example, the amino acid sequence shown in SEQ ID NO: 1, or has at least 75%, at least 80%, at least 85%, at least 85%, Amino acid sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. In some embodiments, the coding nucleic acid sequence suitable for the histone demethylase of the present invention comprises, for example, the nucleotide sequence shown in SEQ ID NO: 2, or has at least 75%, at least 80% of SEQ ID NO: 2 %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical nucleotide sequences.

In some embodiments, the nucleic acid sequence encoding the histone demethylase and the at least one exogenous nucleic acid sequence of interest are placed in the same expression construct. In some embodiments, the nucleic acid sequence encoding the histone demethylase and the at least one exogenous nucleic acid sequence of interest are respectively placed in different expression constructs. In some embodiments, the nucleic acid sequence encoding the histone demethylase is placed in one expression construct, and the at least one exogenous nucleic acid sequence of interest is placed in another expression construct.

In some embodiments, the nucleic acid sequence encoding the histone demethylase and/or the at least one exogenous nucleic acid sequence of interest is operably linked to a transcriptional regulatory element.

Methods for expressing different proteins via the same expression construct are known in the art. For example, different proteins can be placed under the control of different transcriptional regulatory elements (eg, different promoters) in the same expression construct. Alternatively, different proteins can be fused by self-cleaving peptides (such as 2A peptides, including but not limited to P2A, E2A, F2A, and T2A, etc.), and then placed under the control of the same transcriptional regulatory elements (such as different promoters), so that in Separate distinct proteins are produced either during translation or post-translationally by self-cleavage of self-cleaving peptides. Alternatively, an internal ribosome entry site (IRES) can be inserted between the nucleic acid sequences encoding different proteins.

The "at least one exogenous nucleic acid sequence of interest" may be any nucleic acid sequence that needs to be transformed into a plant. For example, the exogenous nucleic acid sequence of interest may encode a nucleic acid sequence for a trait important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products. Nucleic acid sequences of interest may also include those involved in oil, starch, carbohydrate or nutrient metabolism, and those affecting fruit size, sucrose loading, and the like.

In some preferred embodiments, the "at least one exogenous nucleic acid sequence of interest" encodes a component of a gene editing system, so that gene editing can be performed on plants.

"Gene editing", also known as genome editing, uses sequence-specific nucleases or derivatives thereof to perform nucleotide insertions, deletions, or substitutions in the genome of an organism. Gene editing typically occurs by causing site-specific double-strand breaks (DSBs) at desired locations in the genome, followed by the introduction of desired DNA insertions, deletions, or substitutions in the process of repairing the DSBs. However, gene editing can also cover base editing techniques that do not involve DSBs, transcriptional activation or repression, epigenetic modification techniques, as long as they are sequence specific.

The present invention does not particularly limit the gene editing system used. For example, gene editing systems suitable for use in the present invention include, but are not limited to, zinc finger nuclease (ZFN), meganuclease (MGN), transcription activator-like effector nuclease (TALEN), and CRISPR (Clustered regularly interspaced short palindromic repeats, clustered regularly interspaced short palindromic repeats) system.

"Zinc finger nucleases" are artificial restriction enzymes made by fusing a zinc finger DNA binding domain to a DNA cleavage domain. A single zinc-finger DNA-binding domain of a ZFN typically contains 3-6 individual zinc-finger repeats, and each zinc-finger repeat can recognize, for example, a unique sequence of 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

Meganuclease generally refers to a homing endonuclease capable of recognizing nucleic acid sequences of 14-40 bases in length. The long recognition sequence makes the meganuclease highly specific, thereby reducing its off-target effects.

"Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences, usually by fusing the DNA-binding and DNA-cleavage domains of a transcription activator-like effector (TALE). preparation. TALEs can be engineered to bind virtually any desired DNA sequence.

A "CRISPR system" generally comprises two components that can form a complex with sequence specificity: a CRISPR nuclease or variant thereof, and a corresponding guide RNA. Therefore, for the CRISPR system, the "at least one exogenous nucleic acid sequence of interest" in the present invention may include the coding nucleic acid sequence of CRISPR nuclease or its variant, and the coding nucleic acid sequence of the corresponding guide RNA.

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, all of which can be applied to the present invention. For example, a suitable CRISPR gene editing system can be found at http://www.addgene.org/crispr/ . CRISPR gene editing systems cover systems that alter the genome sequence, as well as systems that are used for transcriptional regulation but do not alter the genome sequence.

As used herein, the term "CRISPR nuclease" generally refers to a nuclease found in naturally occurring CRISPR systems. "CRISPR nuclease variants" include modified forms of natural CRISPR nucleases, artificial mutants (including nickase mutants), catalytically active fragments, or fusions with other functional proteins/polypeptides, etc. Various artificial functional variants of CRISPR nucleases are known in the art, such as high-specificity variants or nickase variants, or fusion proteins thereof with cytidine deaminase or adenosine deaminase, etc. CRISPR nucleases or variants thereof can recognize, bind and/or cleave target nucleic acid structures by interacting with corresponding guide RNAs. Those skilled in the art know how to select a suitable CRISPR nuclease or variant thereof to achieve the purpose of the present invention.

The CRISPR nuclease or its variants used in the CRISPR gene editing system of the present invention can be selected from, for example, 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.

In some embodiments, the CRISPR nuclease or variant thereof comprises a Cas9 nuclease or variant thereof. A CRISPR gene editing system based on Cas9 nuclease or a variant thereof is also referred to herein as a CRISPR-Cas9 gene editing system. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S.pyogenes).

Cas9 nuclease variants can include Cas9 nickase (nCas9), in which one of the two subdomains (HNH nuclease subdomain and RuvC subdomain) in the DNA cleavage domain of the Cas9 nuclease is inactivated to form nickase. In some embodiments, Cas9 nickase can be used in combination 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.

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

In some embodiments, the CRISPR nuclease variant may also include a base editor. Base editors are usually fusion proteins comprising a deaminase and a CRISPR nuclease variant lacking DNA-cleavage activity.

As used herein, "CRISPR nuclease variants lacking DNA cleavage activity" include, but are not limited to, Cas9 nicking nuclease (nCas9), nuclease-dead Cas9 nuclease (dCas9) or nuclease-dead Cpf1 nuclease (dCpf1) . Nuclease-dead Cas9 nuclease (dCas9) or nuclease-dead Cpf1 nuclease (dCpf1) completely lack DNA cleavage activity. A variety of CRISPR nuclease variants lacking DNA cleavage activity are known in the art.

As used herein, "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments of the present invention, the deaminase refers to cytosine deaminase, which can accept single-stranded DNA as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuridine, respectively. pyrimidine. In some embodiments of the present invention, the deaminase refers to adenine deaminase, which can accept single-stranded DNA as a substrate and catalyze the formation of inosine (I) from adenosine or deoxyadenosine (A). A variety of 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, eg, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, 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 Nicloe M. Gaudelli et al. (doi: 10.1038/nature24644, 2017).

Base editing in target nucleotide sequences, such as C to T transitions or A Conversion to G. 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 purpose of the present invention. The base editor-based CRISPR gene editing system is also called base editing system.

As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to a guide RNA that is capable of forming a complex with a CRISPR nuclease or variant thereof and that is capable of targeting said complex due to a certain identity to a target sequence. The RNA molecule of the target sequence. For example, gRNAs employed by Cas9 nucleases or variants thereof typically consist of partially complementary crRNA and tracrRNA molecules that form a complex, where the crRNA contains sufficient identity to the target sequence to hybridize to the complementary strand of the target sequence and direct CRISPR complexation. A guide sequence that specifically binds to the target sequence (Cas9+crRNA+tracrRNA). However, it is known in the art that single-guide RNAs (sgRNAs) can be designed that contain features of both crRNAs and tracrRNAs. The gRNA used by Cpf1 nuclease or its variants is usually only composed of mature crRNA molecules, which can also be called sgRNA. It is within the purview of those skilled in the art to design an appropriate gRNA based on the CRISPR nuclease or variant thereof used and the target sequence to be edited.

The sequence-specific nucleases used for gene editing in the present 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 labels and other components. For example, base editors in CRISPR base editing systems usually contain one or more nuclear localization signals (NLS) to facilitate their entry into the nucleus to edit chromosomal DNA.

The expression constructs of the invention can be introduced into plant cells by one of a variety of methods known in the art, including but not limited to biolistic methods, PEG-mediated transformation of protoplasts, and Agrobacterium-mediated transformation.

In some embodiments, the plant cells of the invention are cells suitable for regeneration into whole plants by tissue culture.

Methods for regenerating whole plants by culturing transformed immature embryos are known in the art. During the regeneration process, transformants can also be selected based on the selectable marker carried on the introduced expression construct. In some embodiments, the regeneration is performed in the absence of selective pressure. In some embodiments, moderately stringent selection conditions can be used to select for transformants. The moderately stringent conditions refer to conditions that do not completely inhibit the growth of non-transformants. For example, moderately stringent selection conditions do not inhibit the growth of transformants but partially inhibit the growth of non-transformants. For example, under moderately stringent selection conditions, non-transformants can grow but at a slower or weaker rate than transformants. Moderately stringent selection conditions can be determined by those skilled in the art for a particular plant and a particular selectable marker.

In some embodiments, expression constructs of the invention are transiently transformed into plant cells. Transient transformation refers to the introduction of a construct into a cell, allowing it to function but not integrate into the cellular genome. This is particularly useful for gene editing, as non-transgenic modified plants can be produced.

Plants suitable for transformation or gene editing by the method of the present invention may be monocotyledonous or dicotyledonous. For example, examples of such plants include, but are not limited to, wheat, strawberry, rice, corn, soybean, sunflower, sorghum, canola, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava, and potato. The methods of the invention are particularly suitable for genetic transformation or gene editing in previously difficult-to-transform plant varieties or genotypes. In some embodiments, the plant is wheat, eg, the wheat is a different variety of wheat plant, eg KN199, Bobwhite, etc.

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

Codon optimization refers to the replacement of at least one codon of a native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence and modifying the nucleic acid sequence in order to enhance expression in the host cell of interest. Different species display certain codons for specific amino acids Codon bias (differences in codon usage between organisms) is often associated with messenger RNA (mRNA) translation efficiency, which is thought to depend on the nature and nature of the codons being translated Availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell generally reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored to be most optimal in a given organism based on codon optimization. Optimal gene expression. Codon usage 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 adjusted in different ways Applicable. 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).

In one aspect, the invention provides plants obtained by the methods of the invention and progeny thereof.

Example

A further understanding of the invention may be obtained by reference to certain specific examples which are given herein for illustration only and are not intended to limit the scope of the invention in any way. Apparently, various modifications and changes can be made to the present invention without departing from the essence of the present invention. Therefore, these modifications and changes are also within the protection scope of the present application.

Example 1. Overexpression of histone demethylases improves the efficiency of genetic transformation and gene editing in wheat.

1. Control group selection

In this study, pUBI-GFP and mGRF4-GIF1 were selected as negative and positive controls. Among them, mGRF4-GIF1 (WO2021185358A1) is a protein complex established by the inventor's research group. Studies have found that transient expression of mGRF4-GIF1 can effectively promote wheat regeneration and editing efficiency (Qiu, F., Xing, S., Xue, C. et al., Sci. China LifeSci., 2022).

2. Experimental carrier construction

In this study, diploid rice histone demethylases JMJ703, JMJ704, and JMJ706 were selected for research, and whether they could improve the genetic transformation and gene editing of wheat through different constructions.

First NCBI found rice JMJ703 gene (amino acid sequence is shown in SEQ ID NO:3, nucleotide sequence is shown in SEQ ID NO:4), JMJ704 gene (amino acid sequence is shown in SEQ ID NO:5, nucleotide sequence is shown in SEQ ID NO:5) ID NO:6), rice JMJ706 gene (the amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2). The above coding sequence was placed under the control of the ZmUBI promoter and cloned into the gene editing vector p163, and the obtained vectors were named pOsJMJ703, pOsJMJ704, and pOsJMJ706, respectively. And construct the vector of the control group in the same way. The carrier map is shown in Figure 1.

3. Transformation and regeneration of wheat plants

The vector constructed above was transformed into wheat callus by gene gun. The results of wheat callus regeneration are shown in Fig. 2 . It can be seen that compared with the negative control group, the overexpression of rice JMJ706 can significantly improve the efficiency of wheat callus regeneration.

sequence listing

SEQ ID NO: 1 Amino acid sequence of rice JMJ706

MQQVEGRNCLPAAEVRIGLETLKRRRLERMRLTAQNNAGDGPPVPARSGGDALRTPANCGVRLHANNGTALPSRTTQNKDPFAKRRVDKFDMSSLEWIDKIEECPVYYPTKEEFEDPIGYIQKIAPVASKYGICKIVSPVSASVPAGVVLMKEQPGFKFMTRVQPLRLAKWAEDDTVTFMSERK YTFRDYEKMANKVFAKKYSSASCLPAKYVEEEFWREIAFGKMDFVEYACDVDGSAFSSSPHDQLGKSNWNLKNFSRLSNSVLRLLQTPIPGVTDPMLYIGMLFSMFAWHVEDHYLYSINYHHCGAFKTWYGIPGDAAPGFEKVASQFVYNKDILVGEGEDAAFDVLLGKTTMFPPNVLLDH NVPVYKAVQKPGEFVITFPRSYHAGFSHGFNCGEAVNFAISDWFPLGSVASRRYALLNRTPLLAHEELLCRSAVLLSHKLLNSDPKSLNKSEHPHSQRCLKSCFVQLMRFQRNTRGLLAKMGSQIHYKPKTYPNLSSCSMCRRDCYITHVLCGCNFDPVCLHHEQELRSCPCKSNQVVYVREDIQELEAL SRKFEKDICLDKEISGFDSYKQAEKNEPFFEITRNLRNTEVNLIEDAFSGATAADAAKSSPATSTLTSFAQHDVPVLAEAIVCANQADQLYSTTEQTISSPLVKGTDAVGANSSMADANNGTGSCNASAVEYSGNSDSESEIFRVKRRSGVSVKPASDAKTSNLSDQQVLRRLKKVRPEIQQHNKRPED YGHCSVPSGRMSMKNLNSSSSCGEEHWRMKRRQLETQQDESSYSAKQKSYSYPSTSYSFRGEFVEMSRDAAAEVRPKRLKIRLPSSSTNRVVEQGSSGQRFTRDDKSLGC

WPAI*

SEQ ID NO:2 Rice JMJ706 coding sequence

ATGCAACAGGTGGAGGGCAGGAACTGTCTTCCTGCGGAGGTCAGGATTGGCCTCGAGACGCTCAAGAGGCGCCGGCTTGAGAGGATGCGTTTGACTGCTCAGAACAATGCCGGCGACGGTCCTCCGGTGCCCGCAAGGAGCGGTGGGGATGCGCTAAGGACTCCCGCAAACTGCGGGGTCAGGTTGCATGCTAACAATGGCACAG CTCTACCTAGCAGAACCACCCAGAACAAGGACCCTTTTGCAAAGCGCAGGGTGGACAAGTTTGATATGTCTAGCCTAGAATGGATTGACAAGATCGAAGAATGCCCTGTGTACTATCCTACCCAAGGAGGAGTTCGAGGATCCCATTGGTTATATACAGAAGATTGCACCTGTGGCTTCGAAATACGGAATTTGCAAAATCGTATCTCCAAGTAAGCGCTTCTGTTC CTGCTGGTGTCGTGTTGATGAAGGAACAGCCTGGTTTCAAGTTCATGACCAGGGTTCAGCCGCTTCGCCTCGCCAAATGGGCTGAAGATGACACGGTCACTTTCTTCATGAGCGAAAGAAAGTACACTTTCCGGGATTATGAGAAAATGGCCAACAAGGTGTTCGCCAAGAAATACTCAAGTGCTAGTTGTCTCCAGCTAAGTACGTGGAGGAGGAAT TCTGGCGCGAAATTGCTTTTGGTAAAATGGATTTTGTTGAATATGCCTGTGATGTTGATGGTAGTGCTTTCTCTCTTCTCCTCATGATCAACTTGGGAAAAGCAACTGGAACTTGAAGAATTTTTCACGGCTTTCCAATTCTGTGCTTAGACTTCTGCAGACACCAATTCCAGGAGTAACAGATCCAATGCTTTATATCGGGATGCTCTTCAGCATGTTTGC TTGGCATGTGGAAGATCATTATTTGTACAGCATCAATTACCATCATTGTGGGGCATTTAAGACATGGTATGGCATACCGGGTGATGCTGCTCCTGGGTTTGAAAAGGTGGCTAGCCAGTTTGTATACAACAAGGATATTTTGGTTGGTGAAGGAGAGGATGCAGCATTTGATGTTCTCTTGGGGAAGACAACAATGTTCCCCCAAATGTCTTGTTA GACCACAACGTTCCTGTTTATAAAGCTGTGCAAAAACCTGGGGAGTTTGTCATTACTTTTCCCTCGTTCCTACCACGCGGGTTTCAGCCACGGCTTCAATTGTGGCGAGGCTGTCAACTTTGCTATCAGTGACTGGTTTCCTCTGGGTTCTGTGGCCAGCAGACGCTACGCGCTTCTGAACAGAACACCCTTGCTTGCACACGAGGAGTTACTTTGCCGTT CTGCAGTGCTTCTGTCCCAAACTGTTAAACAGCGACCCAAAATCCCTCAATAAATCTGAGCATCCACATTCACAGCGTTGTTTGAAGTCTTGCTTTGTGCAGTTGATGCGATTCCAGAGAAACACACGTGGCCTACTTGCTAAAATGGGCTCTCAGATACATTATAAGCCAAAAACATACCCGAATCTCTCATGTAGCATGTGTCGGCGTGATTGCTACATTACACATGTGT TGTGTGGATGCAACTTTGACCCAGTCTGTCTTCATCACGAACAAGAACTCCGGAGCTGCCCTTGTAAATCTAACCAGGTTGTCTAACGTTAGGGAGGACATACAGGAGCTAGAAGCTCTATCAAGAAAATTTGAGAAGGATATTTGCTTGGATAAGGAAATAAGTGTTTTGACTCATACAAGCAGGCCGAAAAGAATGAGCCATTTTTTGAGATAACTCGGAAC CTCAGGAACACTGAAGTAAATTTGATAGAGGATGCCTTCTCAGGAGCAACTGCTGCTGATGCTGCAAAGAGTTCTCCTGCAACGTCAACACTGACATCTTTTGCACAACATGATGTGCCTGTTCTTGCTGAAGCAATTGTCTGTGCTAATCAAGCCGACCAATTATACTCCACCACCGAGCAAACCATCAGCTCACCTTTAGTCAAAGGAACTGATGCTGTGG GTGCAAATTCATCCAGCATGGCTGATGCTAATAACGGAACTGGTTCTTGTAATGCTTCAGCTGTGGAATACAGTGGAAATTCAGATTCTGAATCTGAAATATTTCGAGTCAAGCGCAGGTCTGGCGTATCAGTAAAGCCTGCATCTGATGCCAAGACATCAAACTTGTCTGATCAACAGGTTCTCAGGCGGTTGAAGAAGGTGCGCCCTGAAATACAACAGCA CAATAAGCGACCAGAAGACTATGGTCACTGTTCAGTTCCCCAGGTCGTATGAGTATGAAGAATTTGAATTCATCCTCTCATGTGGTGAAGAACACTGGAGGATGAAGCGGCGGCAGTTGGAGACTCAGCAGGATGAGAGCAGTTATTCTGCAAAGCAGAAGTCGTATCCGTACCACCAGCTTCTTTCCGAGGAGAGTTTGTGGAAATGAGTAGAGATG CTGCTGCAGAAGTCCGACCAAAGCGACTGAAAATCCGGCTACCTTCTTCTAGCACGAACAGAGTGGTTGAGCAGGGCAGTTCAGGGCAAAAGATTTACAAGGGATGACAAGTCGCTTGGTTGTTGGCCTGCAATTTAG

SEQ ID NO: 3 rice JMJ703 coding sequence

ATGATGGGGGTTACCACCACGCTCAACGAGGACACTGAACCCTCTATTCCACCTGGATTTGGACCTTTTGCTACCCTTCCGTTATGGGGAATCCACAATGATGCCAAACCTGCTGTTACTCATTCTACTCCTGTTCAAGCATTGCAAAGCATTAGAAAAGACAGCGAAGAATGCCAACCCAGTGCGGCTGTGTCTCGGAGTGATACACCTTGCA GCACTTCCGGAACCCAGACATGCAGAAAATCACTGCGTAACAGACCCCAATAGACTATAGCCGCTTTGAACATATATCGGATGAAGATTCTGATGTCGAAATAGTGGAAAAGGATGTAAGTTCAACGAGACGCAGACAACAGCTACCGAAAGGAGTACTTCGAGGATGTGCAGAATGCAGTGACTGTCAAAAAGGTTATCGCAAAATGGAATCCAGCTGGTGC ACGCAGGCCTGTTCTTGATGAGGCTCCTGTTTTTCTATCCAACAGAGGAGGAATTTGAAGACACTCTAAAATACATTGAGAGTATACGGCCAATGGCGGAACCATATGGTATTTGCCGTATTGTCCCACCATCTTCTTGGAAGCCCTCCATGCCTTCTTAAAGATAAAAGCATATGGGAAGGATCAAAATTCTCTACTCGGGTACAAAAGGTTGACAAGCTCAAAACC GTAAATCATCAAAAAAGGGCAGAAGAGGTGGAATGATGAAGAGGAGAAAGCTTGCAGAGTCAGAGGAGAACAGTGCCACTGCTCACACTCAGAGAGGGATGCAGCAAAGTCCAGAGAGAGATTTGGATTTGAACCTGGGCCAGAGTTCACGTTACAGACATTTCAGAAATATGCAGATGATTTCAGTAAGCAGTACTTTAGGAAAGATACATCGATGGATTCAGTACCATCA GTGGAAGATATTGAAGGTGAGTACTGGCGCATCGTTGAGGTTCCCACAGAAGAGATAGAGGTGATATATGGTGCTGATCTGGAGACTGGAACTTTCGGCAGTGGTTTTCCAAAAATTATCTCCTGAGACAAAATCTGATGCTGAGGATAAATATGCACAATCTGGTTGGAATCTAAATAACTTGCCTAGACTACAAGGTTCAGTTCTTCTTTCGAGG GCGGTGACATTTCTGGTGTTCTAGTGCCTTGGGTGTATGTTGGCATGTGTTTTTCATCATTCTGCTGGCATGTTGAAGACCATCATTTATACTCACTAAACTACATGCATTGGGGTGCCCCAAAGTTGTGGTATGGAGTTCCAGGAAAGGATGCTGTGAATTTGGAATCTGCAATGAGGAAAACATCTACCTGAATTATTTGAGGAGCAACCTGAT TTGCTACACAACCTTGTTACCCAGTTTTTCACCATCGCTGCTGAAATCTGAAGGAGTACATGTATACCGTTGTGTTCAGCATGAGGGCGAGTTTGTCTTGACATTCCCAAGGGCGTACCATGCTGGTTTCAATTGTGGCTTCAATTGTGCCGAAGCTGTTAATGTGGCTCCTATTGATTGGTTACCGATTGGACATAATGCTGTAGAGCTTTATCGTGAGCA AGCTAGGAAAATAACCATTTCTCATGATAAGTTGTTGTTGGGGGCTGCAAGAGAAGCAATAAGGCTCAGTGGGATATCCTATTCCTCAAGAGGAATACTGCTGATAATATGAGGTGGAAGAGTATATGCGGAGCTGATAGCACTATATTCAAGGCTCTTAAGGCACGAATTGAGACAGAGTTGGTGCAAAGGAAAACTCTAGGTGTTCCAGCTCAATCAAG GAAAATGGATGCTGAATTCGATTCCATTGATAGGGAATGTGCCTTGTGCTACTATGATTTACATCTTTCTGCTTCTGGCTGTCCATGCTGCCCAGAGAAATATGCTTGCCTTGTACATGCAAAGCAACTTTGCTCATGTGACTGGGACAAAAGGTTTTTCTATTCCGCTATGATGTCAATGAGCTAAATATCTTAGCTGATGCTTTAGGGGGGAAATTAAGT GCCATTCATAGATGGGGCGTCCTCTGATCTTGGATTAAGTTTGAGTTCATGTGTCAAACGAGAAAAGGTCCAAGATTCCAAGACTGTTCGCAGATTAACTGATGGTCCAAGAAGGTCTTACATGTCACAGGCATCAGCAGTATCCTTGGTTTCTTTCTTACTTCCAATGAACAGAAAGATGAAGGAAATAAGATCATGAAGATAGCTAGCCCACAGACAAATAATGTGTGC CCTTCTGTCGAGCAAAGGAAATCAGAGAATATTTCACCATTGAAGGAGCCATGTGTAAGGAATGAGTTGTCATGTACAACAAATTCTGATAGTAACGGATTGCAATATAATGGAGGACTTGGAGGCCATAAAGGATCTGCACCAGGCTTGCCAGTTTCTTCTAGCCCATCATTTTCTTCCAACGTTGCAACAAGGCCCATTAGTACTTCAAGTGTATCCATGAAAATT GTGCAAGGCTTGGTGGCATCTAAAAGTTGTATACAAGCTTCTCTCGAACTGGAGACAGTAGATCATTGCTTGGTGAGCATCATAACAGATCACCGGCAATGATTCATGATGGAACCAACATGAAGTCCAGTTTGGAAAGCTCAAAACAATTCTTGCAGGTTGATTGCATCTGACTATAATGCAACTCCGTGTCATTCATCCAAAGGATCAGGTATTAGTAACCACCAGGG ACTAATGCCTCAGTAGTGACTCTGAAAGATAGCAGCCAGGTCCATAGTGCGTCAAGTCAGCAGTTTGTCAGAACTGGCCCATGGACACAAAGTGCTTCTCATGAAGCATCATCACCTAGTACCCTCTGCTTTGAAGCCTTCTTTAGATCCCCCTGCCATGAAAAATCTGTATGGGGGTTTTACTCAAGGCAGTGCCCATCCTGGACCTCCAAGTTTCAGTAATCAGCAACC AAATGATGGGCGTCTTCAAAGAACATCTGAATCTCTACCAGGTGTGGAAGCTAGAGCTAGGGGACATCCAACTGTCACGGCACAGCCTGCACTAGAATTCACAGCAGGAATGGAGGTGCACAGAAGGGTCCTCGCATAGCCAATGTTGTGTGCATCGTTTCAAGTGCTCTGTTGAACCTCTCGAAATTGGTGTTGTGCTATCAGGGAGGCTGTGGTCT TCAAGCCAAGCAATCTTCCCGAAAGGGTTTAGAAGCAGAGTGAAATACTTCAGCATTGTGGATCCAATCCAAATGGCATACTACATATCGGAAATACTGGATGCTGGGATGCAGGGGCCTCTGTTTATGGTAAAATTAGAACTGTCCAGGTGAAGTTTTTCATTAACTTATCTCCAACCAAGTGTTGGAACATGGTCCGTGAAAGGCTGAACATGGAAATAAG GAGGCAACTTAATATGGGAAAATCAAATCTTCCTACATTGCAGCCTCCAGGATCAGTTGATGGTCTTGAAATGTTTGGTTTATTACCCACCAATAGTTCAGGCAATTTGGGCGCGGGACAGAGATCACATCTGTACAGAGTACTGGAGATCAAGGCCCCATGTTTCCATTGAGGATCCAAACAATCGGCATATGTTATCTCAGGGTCCACCTCTCTTGCC CTGAGGGGTCTCATCCAAAGGGCTAACCGGGATGAATTGCAAGTCCTGCGGAGTTTGATGACGAACAGCAACAATTTGGATGATAGCTCCAGGCAACAGGCCGCGCACATTATCGAAGAGGAGATTGCGAAGCAATTGTGCTGA

SEQ ID NO: 4 Amino acid sequence of rice JMJ703

MMGVTTTLNEDTEPSIPPGFGPFATLPLWGIHNDAKPAVTHSTPVQALQSIRKDSEECQPSAAVSRSDTPCSTSGTQTCRKSLRNRPPIDYSRFEHISDEDSDVEIVEKDVSSTRRRQQLPKGVLRGCAECSDCQKVIAKWNPAGARRPVLDEAPVFYPTEEEFEDTLKYIESIRPMAEPYGICRIVPPSSWKPPC LLKDKSIWEGSKFSTRVQKVDKLQNRKSSKKGRRGGMMKRRKLAESEENSATAHTQTGMQQSPERFGFEPGPEFTLQTFQKYADDFSKQYFRKDTSMDSVPSVEDIEGEYWRIVEVPTEEIEVIYGADLETGTFGSGFPKLSPETKSDAEDKYAQSGWNLNNLPRLQGSVLSFEGG DISGVLVPWVYVGMCFSSFCWHVEDHHLYSLNYMHWGAPKLWYGVPGKDAVNLESAMRKHLPELFEEQPDLLHNLVTQFSPSLLKSEGVHVYRCVQHEGEFVLTFPRAYHAGFNCGFNCAEAVNVAPIDWLPIGHNAVELYREQARKITISHDKLLLGAAREAIRAQWDILFLKRNTADN MRWKSICGADSTIFKALKARIETELVQRKTLGVPAQSRKMDAEFDSIDRECALCYYDLHLSASGCPCCPEKYACLVHAKQLCSCDWDKRFFLFRYDVNELNILADALGGKLSAIHRWGVSDLGLLSLSSCVKREKVQDSKTVRRLTDGPRRSYMSQASAVSLVSSSTSNEQKDEGNKIMKIASPQTNNVCPS VEQRKSENISPLKEPCVRNELSCTTTNSDSNGLQYNGGLGGHKGSAPGLPVSSPSFSSNVATRPISTSSVSMKIVQGLVASKSCIQASSRTGDSRSLLGEHHNRSPAMIHDGTNMKSSLESSNNNSCRLIASDYNATPCHSSKDQVLVTPGTNASVVTLKDSSQVHSASSQQFVRTGPWTQSASHEASSPSTSALKPSLDPP AMKNLYGGFTQGSAHPGPPSFSNQQPNDGRLQRTSESLPGVEARARGHPTVTAQPALEIHSRNGGAQKGPRIANVVHRFKCSVEPLEIGVVLSGRLWSSSQAIFPKGFRSRVKYFSIVDPIQMAYYISEILDAGMQGPLFMVKLENCPGEVFINLSPTKCWNMVRERLNMEIRRQLNMGKSNLP TLQPPGSVDGLEMFGLLSPPIVQAIWARDRDHICTEYWRSRPHVLIEDPNNRHMLSQGPPLLALRGLIQRANRDELQVLRSLMTNSNNLDDSSRQQAAHIIEEEIAKQLC*

SEQ ID NO:5 Rice JMJ704 coding sequence

ATGGTTTCCTCCCGCGACCCCGGCGAGGAGGCCAGCGCGCCGCCGCCCCCGCCCCCGCGCCGCGGCGAGAAGCGGCGAATGCGCGGCCGCACCCCGTCGCCGGAGCCGGCCTCCGCGCCGCAGGATCTCTGCCCATCAGGAGCTTGCGGGGACAATGTTGCTGGAGCTACAACTACAAATGGAAAGTGGCATCCACATGAATCGTACAGAC CTGAAATTGATGATGCCCCTGTTTCACTCCAACGGAAGAGGAGTTTAAAGATCCAATTAGATATATTACGAGCATTCGTCCCCAAGCAGAAAAGTGGAATTTGTCGTATTGTTCCCACCATCTTCTTGGCGACCGCCTTGTTCTCTGAAGGAGAAGAACTTCTGGGAATGTACAGAGTTCAATACCCGTGTTCAACAAGTTGACAAGCTTCAAAACCGGGAACCCACAA AGAAAAAAATCACAACCTCGAGTTCAGAAAGAAGAGGAAGAGGAGAAAGAGACTGAGATTTGGGATGACTCACAGGCGTCCTAGTGCAAATACATCAGAAGACTGCGCAGATGCAGACGAGAAGTTTGGCTTTCAATCTGGCTCAGATTTCACACTAGATGAGTTTCACGAAATATGCAGATGAGTTTAAGCAGCAGTATTTTGGAATAAAGGGAAGTGACGAAATCCCT TTCTGAAATTAAAAAGAAGAAAAAAAATTGGCAACCATCGGTCGATGAAATAGAGGGAGAATATTGGCGGATAGTTGTATGCCCCACTGACGAAGTTGAGGTGGATTATGGTGCTGATTTGGACACTTCAATGTTCAGTAGTGGATTCTCTAAATTATCTTCAGATTCAAATAGACGAGATCCATATGGTTTATCTTGTTGGAATTTGAACAATCTTCC ACGTATTCCTGGGTCTGTACTGTCATTTGAAACTGAGGATATATCTGGCGTCGTAGTCCCTTGGCTTTATGTAGGGATGTGCTTCTCATCATTCTGTTGGCACGTGGAAGATCATTTCCTTTATTCTATGAATTACATGCATTTTGGTGAACCAAAAGTATGGTATGGTGTTCCTGGTGCTGATGCAGTGAAGCTGGAAGAAGCTATGAGAAAGAAC TTACCAAGATTGTTTGAAGAACAGCCTGATCTCCTACATGAGCTGGTTACGCAATTATCTCCTTCTGTTTCTAAATCAGAAGGAGTTCCTGTTTATCGTGTTGTTCAGAATCCAGGCGAGTTTGTTCTAACGCTACCGCGAGCTTACCATTCTGGGTTCAACTGTGGCTTCAACTGTGCGGAGGCAGTAAATGTCGCACCTGTGGATTGGCTGCCTCACGGACAAT GTGCTGTTGAGCTCTACAGGGAGCAGCGGCGCAAGACATCCATATCACATGACAAATTATTACTAAAAACTGCAAATGAAGCTGTCAGACAGCTTTGGATGAACCTTAGCGACTGCAAAAGTGAACAAGGAGTATACAGATGGCAGGATACTTGCGGAAAGGACGGAATGCTGACAAGTGCAATTAAGACAAGGGTTAAAATGGAGAAGGCAGCACGGGGA GGGAATATGGCACTGCGATATAAGAAAATGGATGGGGATTATGATTCAGCTGACCGGGAATGCTTTTCATGTTTTTTATGATCTCCATTTGTCAGCTGTCAGCTGCCAATGCTCCCCAAATCGTTTTGCTTGCTTAAACCATGCAAACATTCTATGTTCATGTGAAATGGACAGAAAAACCGCGTTGTTGCGGTATACCATAGAGGAGCTCCATACTCTTT GTTGCAGCTCTAGAGGGTGATCCAACTGCGGTCTACCAGTGGGGACAGAATGATTTAGGTTTAGTCTGCCCATCTGGTTCTACTCAGTACAAGAAGATGGACTTGGGTGAAAACACGGAATTTCCGGATTCAGCAACCAACGTCAATCATGGCTGCAGCTTAGGAAGTCAAGATCAATATCACTATGACCCCGCAAAGCCAGCAGGATACCAGCAAGAGAAG GGAATCCAGATTGCTTCAGAAAAACATGATAAGAACAAGATGGTTGTCAATCTTGAGTCTCCAGCAACAGCTAGTAATCCAAGCAGGTCAAAGTCTGACTGCAGTGGCTCACTGTCCTTGAATCATTCATCTGAGTTACCATCTTCAAGAATTCAAACAGGAAATTCTACGCTAGCTTCATTACCCAGAGAAACTGTTTGGTGTTGACATTAAATCCAATTTAGCACAGTC TTCTGATGGCCAAGTTAGTCAATTGGCCAAGCCTTCCTCGAGCCAAACTGATGAAGTCTCTAAGCCAGCAATAGCTAAGTATACGGTTGAGCTGCTAGACAGTGGAACAATGATGATTGGTAAAAAGTGGTGCAATCAGCAAGCTATATTCCCCAAAGGATTTAAGAGTCGAGTTCATTTCATAGTGTACTAGATCCAACAAGGACATGCTGCTACATCTCCGAA GTTCTTGATGCTGGGCTTCTTGGACCATTGTTTAGGGTGACTGTCGAAGGTCTTCCAGAAGTTTCGTTTACTCACACACACCAATGCAATGTTGGGACAGTGTAAGAGACAGAGTAAATGAAGAAATAGCAAAACAAATAAGTTTTGGAAAATCTGGCCTTCCTGATTTTCTATCCTGCAATTCTTTGAATGGACTTGAAATGTTTGGGTTCTTATCCTCC CCTATAATTAAGGAAATCGAGGCTCTAGATCCCTGTCACCAATGCTTGGACTATTGGTTGTCAAGGGTTTCTTCTGTTGGAACTGAACTCCCCTCGGAATCTGTGATGGCAGCAATGGTTAATGACTCCACTAACCCCCCAATAAAGTTGCTCGGGATTGAGATTAACCGGAGGGAATCAGAACAATCAAGTAGCTTCAATAATTCCTGTGTGAGGAGGTC ACACTTGGCAGGTTGCTGA

SEQ ID NO:6 Amino acid sequence of rice JMJ704

MVSSRDPGEEASAPPPPPPRRGEKRRMRGRTPSPEPASAPQDLCPSGACGDNVAGATTTNGKWHPHESYRPEIDDAVFTPTEEEFKDPIRYITSIRPQAEKYGICRIVPPSSWRPPCSLKEKNFWECTEFNTRVQQVDKLQNREPTKKKSQPRVQKKRKRRKRLRFGMTHRRPSANTSEDCADADEKFGF QSGSDFTLDEFQKYADEFKQQYFGIKGSDEIPLSEIKKKKKNWQPSVDEIEGEYWRIVVCPTDEVEVDYGADLDTSMFSSGFSKLSSSNRRDPYGLSCWNLNNLPRIPGSVLSFETEDISGVVVPWLYVGMCFSSFCWHVEDHFLYSMNYMHFGEPKVWYGVPGADAVKLEEAMRKN LPRLFEEQPDLLHELVTQLSPSVLKSEGVPVYRVVQNPGEFVLTLPRAYHSGFNCGFNCAEAVNVAPVDWLPHGQCAVELYREQRRKTSISHDKLLLKTANEAVRQLWMNLSDCKSEQGVYRWQDTCGKDGMLTSAIKTRVKMEKAARGGNMALRYKKMDGDYDSADRECFSCF YDLHLSAVSCQCSPNRFACLNHANILCSCEMDRKTALLRYTIEELHTLVAALEGDPTAVYQWGQNDLGLVCPSGSTQYKKMDLGENTEFPDSATNVNHGCSLGSQDQYHYDPAKPAGYQQEKGIQIASEKHDKNKMVVNLESPATASNPSRSKSDCSGSLSLNHSSELPSSRIQTGNSTLASITTEKLFGVDI KSNLAQSSDGQVSQLAKPSSSQTDEVSKPAIAKYTVELLDSGTMMIGKKWCNQQAIFPKGFKSRVTFHSVLDPTRTCCYISEVLDAGLLGPLFRVTVEGLPEVSFTHTSPMQCWDSVRDRVNEEIAKQISFGKSGLDFLSCNSLNGLEMFGFLSSPIIKEIEALDPCHQCLDAYWLSRVSSVGTELPSESVMAAMVNDSTNPPIKLL GIEINRRESEQSSSFNNSCVRRSHLAGC*

Claims (10)

1. A method for improving plant cell regeneration efficiency, said method comprising: (a) introducing into said plant cell an expression construct comprising a nucleic acid sequence encoding a histone demethylase, (b) regenerating whole plants from said plant cells. 2. A method of transforming at least one exogenous nucleic acid sequence of interest into a plant, said method comprising: (a) introducing into the cells of said plant an expression construct comprising a nucleic acid sequence encoding a histone demethylase, (b) introducing into said plant cell at least one expression construct comprising at least one exogenous nucleic acid sequence of interest; and (c) regenerating whole plants from said plant cells. 3. A method of gene editing in a plant, said method comprising: (a) introducing into the cells of said plant an expression construct comprising a nucleic acid sequence encoding a histone demethylase, (b) introducing into the plant cell at least one expression construct comprising at least one exogenous nucleic acid sequence of interest, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of the gene editing system; (c) regenerating whole plants from said plant cells. 4. The method of claim 2 or 3, wherein the nucleic acid sequence encoding the histone demethylase and the at least one exogenous nucleic acid sequence of interest are placed in the same expression construct. 5. The method of any one of claims 1-4, wherein the histone demethylase comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1. 6. The method of claim 5, wherein the histone demethylase is from rice. 7. The method of any one of claims 3-6, wherein the gene editing system is selected from the group consisting of CRISPR systems, TALENs, meganucleases and zinc finger nucleases. 8. The method of claim 7, wherein the gene editing system is a CRISPR system, such as a base editing system. 9. The method of any one of claims 1-8, wherein the cells are selected from the group consisting of protoplast cells, callus cells, immature embryo cells, and explant cells. 10. The method of any one of claims 1-9, wherein the plant is selected from the group consisting of wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, Cassava and potatoes.