Method for improving plant genetic transformation efficiency

Abstract

The invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for improving plant genetic transformation efficiency. More specifically, the invention relates to a VirG protein mutant or a functional fragment thereof, agrobacterium tumefaciens for expressing the VirG protein mutant or the functional fragment thereof, and application of the agrobacterium tumefaciens in plant genetic transformation.

Description

Technical Field

[0001] The present 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. More specifically, the present invention relates to a VirG protein mutant or a functional fragment thereof, Agrobacterium expressing the VirG protein mutant or a functional fragment thereof, and the use of the Agrobacterium in plant genetic transformation. Background of the Invention

[0003] At present, cutting-edge disruptive technologies such as gene editing and synthetic biology are continuously activating the potential explosive power of agriculture, and will lead to major adjustments and revolutionary breakthroughs in the agricultural industry structure. Against the backdrop of food crisis and tightening natural resources, precise design and intelligent creation of crops through cutting-edge technologies has become an inevitable choice for modern agricultural breeding. Crop gene delivery and transformation technology is a bridge between upstream cutting-edge technologies and downstream crop breeding applications, but its current development is relatively lagging, which limits the promotion of emerging biotechnology in crops and becomes a bottleneck restricting the development of precision design breeding. Therefore, it is imperative to develop efficient crop gene transformation and delivery technologies that meet the needs of modern agricultural development. Agrobacterium transformation is a gene delivery method that can introduce genes from any source into plants. This method has become the most important means of plant transformation due to its simple operation, low cost, and stable transformation efficiency, and has greatly promoted the development of basic plant research and the application of crop breeding. However, with the rapid development of science and technology and the gradual increase in human demand for food, the traditional Agrobacterium transformation method can no longer meet the current research and application needs, exposing a series of shortcomings that need to be solved urgently: First, species and genotype limitations: Due to the biological characteristics of Agrobacterium itself, the Agrobacterium transformation method is not applicable to many crops to be studied and developed. It is urgent to expand the host range to promote basic research and production applications of more species; Second, the delivery and transformation efficiency does not match the demand: The commonly used strains and vectors have been used for many years, and the transformation efficiency is relatively low, which cannot meet the needs of the development of emerging technologies.

[0004] Transforming Agrobacterium and improving its defects is an effective way to improve plant genetic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 , and functional tests of the obtained VirG mutants. (A) Functional test process of VirG variants to improve Agrobacterium transformation efficiency. (B) Vira without VirG expression, VirG (WT), VirG (N54D), VirG (LDD), VirG6 - virg -Genotype EHA105, VirG-free, VirG-expressing (WT), VirG (I23N), VirG (I77V), VirG (NV), VirG (NVN), VirG6 - Genotype EHA105, wild-type EHA105 not expressing VirG, expressing VirG (WT), VirG (NVN), VirG (LDD), and VirG6 were tested for transformation efficiency in tobacco leaves. The vector T-DNA transformed by all strain samples contains a firefly luciferase (F-LUC) expression frame. The transformation efficiency is positively correlated with the activity of F-LUC, and the transformation efficiency is characterized by the activity determination of F-LUC. RLU, relative luminescence unit. LDD represents F37L / N54D / E62D. VirG6 represents VirG (I23N / F37L / N54D / E62D / I77V / I182N). NV represents I23N / I77V. NVN represents I23N / I77V / I182N. (C) The upper part shows the expression construct used for the transcriptional activity test of VirG variants. Lower part, after using IPTG to induce the expression of VirG variants, the fluorescence intensity change curve of the fluorescent protein expressed by the Agrobacterium sample was detected. Staygold is a green fluorescent protein. (D) The effect of VirG variants on the mRNA levels of endogenous genes in different genotypes of EHA105. The data were normalized to the relative mRNA levels of each gene in WT EHA105. All values ​​and error bars are the mean ± sem of three independent experiments.

[0007] Figure 2 , shows the plant species and genotypes tested for transient transformation.

[0008] Figure 3 , shows the expression construct of the fluorescent protein-mediated visualization reporter system used in the transient transformation test method.

[0009] Figure 4 , transient expression tests in different plants and genotypes. (AI) Top, fluorescent protein-mediated visualization reporter system, where Figure I does not have this result. Middle, F-LUC reporter system. Bottom, the scientific name of the tested plant (genotype). The measurement time is slightly different in different plants, common tobacco 3Dpi (Day post injection), lettuce 2Dpi, sugar beet 3Dpi, Arabidopsis 3Dpi, Chinese cabbage 4Dpi, soybean HH43 5Dpi, soybean G2 3Dpi, tomato M82 3Dpi, tomato AC5Dpi. All values ​​and error bars are the mean ± sem of three independent experiments.

[0010] Figure 5 , shows the expression construct used for transient editing efficiency testing.

[0011] Figure 6 , transient editing efficiency test in tomato M82 and Arabidopsis Col-0. The editing sites are SlGGP1 and AtBRI1, respectively. The efficiency is defined as the ratio of indels (insertion and deletion) reads to the total number of reads. Blank is untreated plant tissue. Control is the Agrobacterium-treated group without VirG protein expression. All values ​​and error bars are mean ± sem of three independent experiments.

[0012] Figure 7 , Functional testing of VirG variants derived from EHA105 and VirG variants derived from LBA4404 in Agrobacterium LBA4404. RLU, relative luminescence units, relative light units. DETAILED DESCRIPTION

[0013] I. Definition

[0014] 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. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: 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"). At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0015] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "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."

[0016] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0017] As used herein, "amino acid position references SEQ ID NO: x" (SEQ ID NO: x is a specific sequence listed herein) means that the position number of the specific amino acid being described is the position number of the amino acid corresponding to the amino acid on SEQ ID NO: x. The correspondence of amino acids in different sequences can be determined according to sequence alignment methods known in the art. For example, amino acid correspondence can be determined by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ), where two sequences can be aligned using the Needleman-Wunsch algorithm using default parameters. For example, if the alanine at position 360 from the N-terminus of a polypeptide is aligned with the amino acid at position 365 of SEQ ID NO: x in a sequence alignment, then the alanine in the polypeptide may also be described herein as "the alanine at position 365 of the polypeptide, the amino acid position references SEQ ID NO: x".

[0018] The amino acid abbreviations herein are well known in the art and are shown in the following table:

[0019] name Three letter symbols Single letter symbols name Three letter symbols Single letter symbols Alanine Ala A Proline Pro P Arginine Arg R Serine Ser S Aspartic acid Asp D Methionine Met M Cysteine Cys C Lysine Lys K Glutamine Gln Q Valine Val V Glutamate Glu E Isoleucine Ile I Histidine His H Phenylalanine Phe F Glycine Gly G Leucine Leu L Asparagine Asn N Tryptophan Trp W Tyrosine Tyr Y Threonine Thr T

[0020] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0021] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, a sequence that has been significantly altered in composition and / or locus from its native form through deliberate human intervention.

[0022] "Nucleic acid sequence", "polynucleotide", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally may contain synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "D" represents A, T or G, "I" represents inosine, and "N" represents any nucleotide.

[0023] "Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical 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 attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0024] As used herein, "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 the transcription of the nucleic acid sequence (such as transcription to generate mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0025] The "expression construct" of the present invention can be a linear nucleic acid fragment (including a DNA or RNA fragment), a circular plasmid, or a viral vector.

[0026] The "expression construct" of the present invention may comprise a regulatory sequence and an interesting nucleic acid sequence operably linked thereto. The regulatory sequence and the interesting nucleic acid sequence may be of different origins, or of the same origin but arranged in a manner different from that normally found in nature.

[0027] "Regulatory sequence" and "regulatory element" are used interchangeably and refer to nucleotide sequences that are located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affect the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation recognition sequences. "Promoter" refers to a nucleic acid fragment that can control the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that can control the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0028] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0029] To "introduce" a nucleic acid molecule (eg, an expression construct) into a plant cell means to present the nucleic acid molecule to the plant cell such that the nucleic acid molecule enters the interior of the plant cell.

[0030] "Regeneration" refers to the process of growing a whole plant from one or more plant cells (eg, plant protoplasts, callus tissue, or explants).

[0031] 2. Agrobacterium VirG protein mutants

[0032] In one aspect, the present invention provides a VirG protein mutant or a functional fragment thereof, wherein the VirG protein mutant has an amino acid mutation at at least one or more positions selected from position 23, 37, 54, 62, 77 or 182 relative to the wild-type VirG protein, and the amino acid positions refer to SEQ ID NO:1.

[0033] VirG protein is a transcription factor in Agrobacterium, which can activate the expression of downstream target genes such as endogenous virulence genes virB4, virC1, virD2, and virE2. An exemplary wild-type VirG protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 9.

[0034] In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 54 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 37, 54, and 62 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 23 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 77 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 23 and 77 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has an amino acid mutation at least at position 23, 77, and 182 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some preferred embodiments, the VirG protein mutant has amino acid mutations at least at positions 23, 37, 54, 62, 77 and 182 relative to the wild-type VirG protein, and the amino acid positions refer to SEQ ID NO:1.

[0035] In some embodiments, the VirG protein mutant has at least the amino acid at position 23 replaced by N, the amino acid at position 37 replaced by L, the amino acid at position 54 replaced by D, the amino acid at position 62 replaced by D, the amino acid at position 77 replaced by V, and / or the amino acid at position 182 replaced by N relative to the wild-type VirG protein, and the amino acid positions refer to SEQ ID NO:1.

[0036] In some embodiments, the VirG protein mutant is substituted with D at least at amino acid position 54 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant is substituted with L at least at amino acid position 37, D at amino acid position 54 and D at amino acid position 62 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant is substituted with N at least at amino acid position 23 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant is substituted with V at least at amino acid position 77 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant is substituted with N at least at amino acid position 23 and V at amino acid position 77 relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant has at least an amino acid substituted by N at position 23, an amino acid substituted by V at position 77, and an amino acid substituted by N at position 182 relative to the wild-type VirG protein, and the amino acid positions are referenced to SEQ ID NO: 1. In some preferred embodiments, the VirG protein mutant has at least an amino acid substituted by N at position 23, an amino acid substituted by L at position 37, an amino acid substituted by D at position 54, an amino acid substituted by D at position 62, an amino acid substituted by V at position 77, and an amino acid substituted by N at position 182 relative to the wild-type VirG protein, and the amino acid positions are referenced to SEQ ID NO: 1.

[0037] In some embodiments, the VirG protein mutant comprises at least one or more amino acid substitutions selected from I23N, F37L, N54D, E62D, I77V, and I182N relative to the wild-type VirG protein, and the amino acid positions are referenced to SEQ ID NO: 1.

[0038] In some embodiments, the VirG protein mutant comprises at least the amino acid substitution N54D relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant comprises at least the amino acid substitution F37L, N54D and E62D relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant comprises at least the amino acid substitution I23N relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant comprises at least the amino acid substitution I77V relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant comprises at least the amino acid substitution I23N and I77V relative to the wild-type VirG protein, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the VirG protein mutant comprises at least amino acid substitutions I23N, I77V and I182N relative to the wild-type VirG protein, and the amino acid positions are referenced to SEQ ID NO: 1. In some preferred embodiments, the VirG protein mutant comprises at least amino acid substitutions I23N, F37L, N54D, E62D, I77V and I182N relative to the wild-type VirG protein, and the amino acid positions are referenced to SEQ ID NO: 1.

[0039] As used herein, "functional fragment" refers to a fragment that at least partially or completely retains the function (eg, transcriptional activation function) of the full-length VirG protein mutant from which it is derived.

[0040] In some embodiments, the transcriptional activation activity of the VirG protein mutant of the present invention or its functional fragment is increased, for example, by at least 50-at least 500 times or more, relative to the wild-type VirG protein. For example, the expression of downstream target genes such as endogenous virulence genes virB4, virC1, virD2, virE2 in Agrobacterium containing the VirG protein mutant of the present invention or its functional fragment can be increased by at least 50-at least 500 times or more. In some embodiments, the transcriptional activation activity of the VirG protein mutant of the present invention or its functional fragment is independent of the activity of VirA.

[0041] In some embodiments, the VirG protein mutant or its functional fragment may further comprise one or more additional amino acid mutations, such as conservative amino acid substitutions, relative to the wild-type VirG protein. These additional amino acid mutations, such as conservative amino acid substitutions, do not substantially or significantly change the function of the protein.

[0042] In some embodiments, the VirG protein mutant comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity with any one of SEQ ID NOs: 2-8, 10 and 11. In some specific embodiments, the VirG protein mutant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-8, 10 and 11, preferably an amino acid sequence as shown in SEQ ID NOs: 8 or 11.

[0043] In another aspect, the present invention also provides a nucleic acid comprising a nucleotide sequence encoding the VirG protein mutant or a functional fragment thereof of the present invention. In some embodiments, the nucleic acid is an isolated nucleic acid or a recombinant nucleic acid.

[0044] In another aspect, the present invention also provides an expression cassette, which comprises a nucleotide sequence encoding the VirG protein mutant of the present invention or a functional fragment thereof operably linked to a regulatory sequence. In some embodiments, the regulatory sequence is a natural promoter of the virG gene or a functional variant thereof.

[0045] In another aspect, the present invention also provides an expression construct comprising the expression cassette of the present invention.

[0046] 3. Improved Agrobacterium

[0047] In another aspect, the present invention provides an Agrobacterium comprising and / or expressing the VirG protein mutant or a functional fragment thereof according to the present invention.

[0048] In some embodiments, the Agrobacterium is Agrobacterium tumefaciens. In some embodiments, the Agrobacterium can be different strains of Agrobacterium tumefaciens, including but not limited to EHA105 strain, GV3101 strain, LBA4404 strain, AGL-1 strain, etc. In some embodiments, the Agrobacterium can be Agrobacterium strains of different genotypes, such as vira-virg- or virg- strains.

[0049] In some embodiments, the Agrobacterium comprises the nucleic acid, expression cassette and / or expression construct of the present invention, or is transformed with the nucleic acid, expression cassette and / or expression construct of the present invention, thereby expressing the VirG protein mutant or a functional fragment thereof of the present invention.

[0050] In some embodiments, the Agrobacterium further comprises at least one exogenous nucleic acid sequence of interest to be transformed into a plant. In some embodiments, the Agrobacterium comprises an expression construct containing at least one exogenous nucleic acid sequence of interest to be transformed into a plant.

[0051] The expression construct containing at least one exogenous nucleic acid sequence of interest to be transformed into the plant can be derived from a vector suitable for transforming the exogenous nucleic acid sequence into the plant / plant cell by Agrobacterium transfection, such as a binary expression vector well known in the art. The backbones of these vectors are easily available to those skilled in the art, such as pCambia, pRK2, pSa, pRi, pBRR1 series vectors.

[0052] In some embodiments, the nucleic acid sequence encoding the VirG protein mutant or a functional fragment thereof and the at least one exogenous nucleic acid sequence of interest to be transformed into the plant are located in the same expression construct.

[0053] 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 encode a nucleic acid sequence that is important for agronomy, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Nucleic acid sequences of interest can also include those nucleic acid sequences involved in oil, starch, carbohydrate or nutrient metabolism, as well as those nucleic acid sequences that affect grain size, sucrose loading, etc.

[0054] In some preferred embodiments, the "at least one exogenous nucleic acid sequence of interest" encodes components of a gene editing system, thereby enabling gene editing of plants.

[0055] "Gene editing", also known as genome editing, uses sequence-specific nucleases or their derivatives to insert, delete or substitute nucleotides in the genome of an organism. Gene editing usually causes site-specific double-strand breaks (DSBs) at the desired location in the genome, and then introduces the desired DNA insertion, deletion or substitution in the process of repairing the DSBs. However, gene editing can also cover base editing techniques, transcriptional activation or inhibition, and epigenetic modification techniques that do not involve DSBs, as long as they are sequence-specific.

[0056] 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 nucleases (ZFNs), meganucleases (MGNs), transcription activator-like effector nucleases (TALENs), and CRISPR (Clustered regularly interspaced short palindromic repeats) systems.

[0057] "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 separate zinc finger repeats, each of which can recognize a unique sequence of, for example, 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

[0058] Meganucleases are generally homing endonucleases that can recognize nucleic acid sequences of 14-40 bases in length. The long recognition sequence gives meganucleases strong specificity, thereby reducing their off-target effects.

[0059] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences, typically made by fusing the DNA binding domain of a transcription activator-like effector (TALE) to a DNA cleavage domain. TALEs can be engineered to bind to almost any desired DNA sequence.

[0060] The "CRISPR system" generally comprises two components that can form a complex with sequence specificity: a CRISPR nuclease or a variant thereof, and a corresponding guide RNA. Therefore, for the CRISPR system, the "at least one exogenous nucleic acid sequence of interest" described in the present invention may include a nucleic acid sequence encoding a CRISPR nuclease or a variant thereof, and a nucleic acid sequence encoding a corresponding guide RNA.

[0061] 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, suitable CRISPR gene editing systems can be found in http: / / www.addgene.org / crispr / CRISPR gene editing systems include systems that change the genome sequence as well as systems that are used for transcriptional regulation but do not change the genome sequence.

[0062] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in a naturally occurring CRISPR system. "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. A variety of 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 cut target nucleic acid structures by interacting with corresponding guide RNAs. Those skilled in the art know how to select suitable CRISPR nucleases or variants thereof to achieve the purposes of the present invention.

[0063] The CRISPR nuclease or its variant used in the CRISPR gene editing system of the present invention can be selected 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.

[0064] In some embodiments, the CRISPR nuclease or variant thereof includes a Cas9 nuclease or variant thereof. A CRISPR gene editing system based on a Cas9 nuclease or variant thereof is also referred to herein as a CRISPR-Cas9 gene editing system. The Cas9 nuclease can be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S. pyogenes) (having an amino acid sequence shown in SEQ ID NO: 15).

[0065] Cas9 nuclease variants may 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 a nickase. In some embodiments, the Cas9 nickase can be combined with two gRNAs targeting the upstream and downstream of the sequence to be edited to achieve the deletion of the sequence to be edited, or to achieve the replacement of the sequence to be edited in the presence of a donor sequence.

[0066] In some embodiments, the CRISPR nuclease or variant thereof may also include a Cpf1 (Cas12a) nuclease or variant thereof, such as a highly specific variant. The Cpf1 nuclease may be a Cpf1 nuclease from a different species, such as a Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceaebacterium ND2006. The CRISPR gene editing system based on the Cpf1 nuclease or variant thereof is also referred to herein as a CRISPR-Cpf1 system.

[0067] In some embodiments, the CRISPR nuclease variant may also include a base editor. A base editor is typically a fusion protein comprising a deaminase and a CRISPR nuclease variant lacking DNA cleavage activity.

[0068] 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.

[0069] As used in the present invention, "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments of the present invention, the deaminase refers to a cytosine deaminase, which can accept single-stranded DNA as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments of the present invention, the deaminase refers to an adenine deaminase, which can accept single-stranded DNA as a substrate and can catalyze adenosine or deoxyadenosine (A) to form inosine (I). 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, 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 Nicloe M. Gaudelli et al. (doi: 10.1038 / nature24644, 2017).

[0070] By using a CRISPR nuclease variant lacking DNA cleavage activity and fused to a deaminase (forming a so-called "base editor"), base editing in the target nucleotide sequence, such as conversion of C to T or conversion of A to G, can be achieved. A variety of 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 CRISPR gene editing system based on the base editor is also referred to as a base editing system.

[0071] As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to an RNA molecule that can form a complex with a CRISPR nuclease or its variants and can target the complex to a target sequence due to a certain homology with the target sequence. For example, the gRNA used by the Cas9 nuclease or its variants is generally composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein the crRNA contains a guide sequence that has sufficient homology to the target sequence so as 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 sequence. However, it is known in the art that a single guide RNA (sgRNA) can be designed, which contains the features of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or its variants is generally composed of only mature crRNA molecules, which may also be referred to as sgRNA. It is within the capabilities of those skilled in the art to design a suitable gRNA based on the CRISPR nuclease or its variants used and the target sequence to be edited.

[0072] In some embodiments, the gene editing system also encompasses the so-called Prime editing system.

[0073] 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 tags and other elements. For example, the base editors in the CRISPR base editing system usually contain one or more nuclear localization signals (NLS) to facilitate their entry into the cell nucleus to achieve editing of chromosomal DNA.

[0074] IV. Improved Plant Transformation

[0075] In one aspect, the present invention provides use of the VirG protein mutant of the present invention or a functional fragment thereof or the Agrobacterium of the present invention, such as Agrobacterium tumefaciens, in transforming an exogenous nucleic acid sequence of interest into a plant.

[0076] In one aspect, the present invention provides a method for transforming an exogenous nucleic acid sequence of interest into a plant, the method comprising infecting a plant with an Agrobacterium of the present invention, such as Agrobacterium tumefaciens, wherein the Agrobacterium, such as Agrobacterium tumefaciens, comprises the exogenous nucleic acid sequence of interest. In some embodiments, the Agrobacterium comprises an expression construct containing the exogenous nucleic acid sequence of interest to be transformed into the plant. The exogenous nucleic acid sequence of interest or an expression construct comprising the same is as defined above.

[0077] In some embodiments, the infecting plants with Agrobacterium of the present invention, such as Agrobacterium tumefaciens, comprises treating the plants, plant parts, plant organs, plant tissues or plant cells under suitable conditions. For example, soaking the plants, plant parts, plant organs, plant tissues or plant cells with a bacterial solution containing Agrobacterium of the present invention, such as Agrobacterium tumefaciens.

[0078] In some embodiments, the Agrobacterium of the present invention, such as Agrobacterium tumefaciens, does not need to be treated with acetosyringone.

[0079] The plant part or plant organ of the present invention may be an inflorescence or a flower. The plant tissue may be a callus.

[0080] The plant cell of the present invention may be a cell suitable for regenerating into a complete plant by tissue culture. Examples of suitable plant cells include, but are not limited to, protoplast cells, callus cells, immature embryo cells, and explant cells.

[0081] Methods for regenerating a transformed complete plant by culturing a transformed protoplast, callus, immature embryo or explant are known in the art. In the regeneration process, the transformant can also be screened based on the selective marker carried on the introduced expression construct. In some embodiments, the regeneration is carried out in the absence of selection pressure. In some embodiments, the transformant can be screened with medium stringent screening conditions. The medium stringent conditions refer to conditions that do not completely suppress the growth of non-transformants. For example, medium stringent screening conditions do not suppress the growth of transformants but partially suppress the growth of non-transformants. For example, under medium stringent screening conditions, non-transformants can grow but are slower or weaker than the growth of transformants. Medium stringent screening conditions are those that can be determined by those skilled in the art for specific plants and specific selective markers.

[0082] Plants suitable for transformation or gene editing by the method of the present invention can be monocots or dicots. For example: cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beets (sugar beets and fodder beets), pear trees, stone fruits and soft fruits (apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries and blackberries), legumes (beans, lentils, peas, soybeans), oil plants (rapeseed, mustard, olives, sunflowers, coconuts, castor beans, cocoa beans, peanuts), cucumber plants (cucumbers, melons), fiber plants (cotton, flax, jute), citrus fruit trees (orange, lemon, grapefruit, tangerine), vegetables (spinach, lettuce, asparagus, cabbage cane, carrot, onion, tomato, potato, red pepper), Lauraceae (avocado, cinnamon, camphor tree), or corn, tobacco, nuts, coffee, sugar cane, tea, grapevine, hops, banana and natural rubber plants and decorative plants (various flowers, shrubs, deciduous trees and coniferous trees); the plants are not limited to these.

[0083] For example, examples of the plant include, but are not limited to, wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, potato, lettuce, beet, Arabidopsis, Chinese cabbage, radish, cucumber, apple, pepper, peanut, sesame, and rubber tree.

[0084] The method of the present invention is particularly suitable for genetic transformation or gene editing in plant varieties or genotypes that were previously difficult to transform. In some specific embodiments, the plant can be soybean cultivar HH43, soybean cultivar G2, tomato cultivar M82, sugar beet, Arabidopsis thaliana, or Chinese cabbage.

[0085] Compared with Agrobacterium containing wild-type VirG protein, the efficiency of plant transformation using the Agrobacterium of the present invention is increased by at least 2 times to at least 400 times or more.

[0086] In order to obtain efficient expression in plants, in some embodiments of the present invention, the exogenous nucleic acid sequence of interest is codon-optimized for the plant species whose genome is to be modified.

[0087] Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted for use in different ways. See, Nakamura et al., 2001. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0088] In one aspect, the present invention provides plants and progeny thereof obtained by the method of the present invention.

[0089] Example

[0090] A further understanding of the present invention can be obtained by referring to some specific embodiments given herein, which are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Obviously, various modifications and changes can be made to the present invention without departing from the essence of the present invention, and therefore, these modifications and changes are also within the scope of the present application.

[0091] Example 1: Effects of different VirG mutants on transformation efficiency of Agrobacterium tumefaciens with different genotypes

[0092] Several mutants of VirG protein of Agrobacterium strain EHA105 were obtained through directed evolution of Agrobacterium protein, including the following mutations: N54D, F37L / N54D / E62D (LDD), I23N, I77V, I23N / I77V (NV), I23N / I77V / I182N (NVN). In addition, through the combination of mutation sites, a mutant VirG6 containing mutations I23N / F37L / N54D / E62D / I77V / I182N was obtained. Figure 1 As shown in A, the functional strength of various variants was determined using a tobacco transient expression system.

[0093] 1.1 Vector construction

[0094] 1. Control plasmid: pCambia-LUC-control

[0095] 2. Plasmid containing VirG (WT): pCambia-LUC-VirG-WT

[0096] 3. Plasmid containing VirG (N54D): pCambia-LUC-VirG-N54D

[0097] 4. Plasmid containing VirG (F37L / N54D / E62D): pCambia-LUC-VirG-LDD

[0098] 5. Plasmid containing VirG (I23N): pCambia-LUC-VirG-I23N

[0099] 6. Plasmid containing VirG (I77V): pCambia-LUC-VirG-I77V

[0100] 7. Plasmid containing VirG (I23N / I77V): pCambia-LUC-VirG-NV

[0101] 8. Plasmid containing VirG (I23N / I77V / I182N): pCambia-LUC-VirG-NVN

[0102] 9. Plasmid containing VirG (I23N / F37L / N54D / E62D / I77V / I182N): pCambia-LUC-VirG6 The above expression vector was constructed as follows: Figure 1 As shown in A.

[0103] In this example, the wild-type VirG sequence of EHA105 is from pTiBo542. The VirG sequences on other Ti plasmids may be different, but similar effects can be achieved. According to previous studies, the main translation of this protein starts at the 27th TTG codon, and the amino acid residue encoded by this codon is recorded as the first amino acid. The wild-type VirG sequence of EHA105 is shown in SEQ ID NO: 1.

[0104] 1.2 Transfer the vectors in 1.1 into each Agrobacterium tumefaciens genotype

[0105] pCambia-LUC-control, pCambia-LUC-VirG-WT, pCambia-LUC-VirG-N54D, pCambia-LUC-VirG-LDD, and pCambia-LUC-VirG6 were transformed into vira-virg-EHA105;

[0106] pCambia-LUC-control, pCambia-LUC-VirG-WT, pCambia-LUC-VirG-I23N, pCambia-LUC-VirG-I77V, pCambia-LUC-VirG-NV, pCambia-LUC-VirG-NVN, pCambia-LUC-VirG6 were transferred into virg-EHA105;

[0107] pCambia-LUC-control, pCambia-LUC-VirG-WT, pCambia-LUC-VirG-NVN, pCambia-LUC-VirG-LDD, and pCambia-LUC-VirG6 were transformed into WT EHA105.

[0108] 1.3 Determination of transformation efficiency of each Agrobacterium obtained in 1.2

[0109] like Figure 1 As shown in B, each mutant obtained by evolution has a higher efficiency than the control group (control) and the wild-type VirG group in different Agrobacterium genotype backgrounds. This shows that the VirG protein mutants obtained by evolution can promote the transformation efficiency of Agrobacterium in Nicotiana benthamiana. The VirG6 variant has the best effect in promoting transformation efficiency, and it is further tested.

[0110] Example 2: VirG6 mutant protein has higher transcriptional activity

[0111] VirG protein is a transcriptional activator in Agrobacterium, which can activate the expression of downstream target genes after being phosphorylated by VirA protein. Based on the fact that VirG6 can improve the transformation efficiency of Agrobacterium in Nicotiana benthamiana, we further explored the reason why VirG6 has this activity and detected its transcriptional function through transcriptional activation experiments and RT-qPCR experiments.

[0112] Constructed as Figure 1 C, and transfer them into vira - virg - In Agrobacterium, it was found that after IPTG induction, Agrobacterium expressing VirG6 gradually showed enhanced green fluorescence signals within 11 hours, while the control group and the group expressing wild-type VirG did not. This indicates that VirG6 can directly activate the transcription of downstream genes independently of VirA.

[0113] qPCR results showed that overexpression of VirG6 in EHA105 could increase the mRNA levels of endogenous virulence genes virB4, virC1, virD2, and virE2 to 531.8 times, 250.0 times, 192.6 times, and 84.6 times, respectively; while the related genes in EHA105 expressing VirG (WT) did not increase significantly. Previous research results show that acetosyringone treatment can enhance the infection activity of Agrobacterium (Sheikholeslam and Weeks, 1987; Owens and Smigocki, 1988). The mRNA levels of each virulence gene were detected two hours after acetosyringone treatment. Compared with the hundreds of times increase in the expression level of downstream genes by VirG6, acetosyringone treatment only increased by 1.4 times, 2.7 times, 2.8 times, and 1.8 times, respectively. The expression of VirG6 protein helps the transcription level of Agrobacterium virulence genes much more than acetosyringone treatment. This result suggests that the improvement of VirG6 protein on Agrobacterium-mediated plant transformation ability may also be much higher than acetosyringone treatment.

[0114] The above results indicate that the evolved VirG6 protein has higher transcriptional activation activity, which may be the reason for its improved efficiency of Agrobacterium-mediated plant transformation.

[0115] Example 3: VirG6 mutant improves transformation efficiency of Agrobacterium in different species and genotypes

[0116] To test whether the improvement of Agrobacterium transformation efficiency by VirG6 mutants is widely applicable to various plants and genotypes, seven plants from five families were tested, including two genotypes each of pakchoi and soybean, and four genotypes of tomato ( Figure 2 ).

[0117] Fluorescent protein-mediated visualization reporter system and luciferase reporter system were used to characterize the transformation efficiency. In order to detect the fluorescent protein signal more easily, the BeYDV replicon system was introduced to increase the expression of fluorescent protein and amplify the fluorescent protein signal. Figure 3 As shown. Figure 4 As shown, the results of the fluorescent protein reporter system showed that among all the plants tested, the parts infected with Agrobacterium containing VirG6 protein showed the strongest fluorescent signal; while in the control group, except for common tobacco and lettuce, there was no visible fluorescent signal in other plants. Compared with the control, the transformation efficiency of Agrobacterium expressing wild-type VirG protein in common tobacco, lettuce, beet, and chicken feather vegetable was significantly improved, but there was no visible signal in the remaining other species. The luciferase-mediated quantitative reporter system provides basically consistent and more accurate information ( Figure 4 ). The transformation efficiency of Agrobacterium expressing VirG6 protein in tobacco, lettuce, sugar beet, Arabidopsis, Chinese cabbage, soybean HH43, soybean G2, tomato M82, and tomato AC was increased by 13.9 times, 3.7 times, 19.2 times, 38.2 times, 28.4 times, 70.3 times, 386.3 times, 279.5 times, and 2.1 times respectively compared with the control group.

[0118] The above results indicate that the improvement of Agrobacterium-mediated transformation efficiency by VirG6 protein can be applied to a variety of plants of different species and genotypes, greatly removing the species and genotype restrictions of Agrobacterium transformation method.

[0119] Example 4. VirG6 mutant improves gene editing efficiency in different species mediated by Agrobacterium transformation

[0120] In order to test whether the VirG6 variant can also improve the gene editing efficiency in different species, Arabidopsis thaliana (Col-0) and tomato (M82) were selected for editing tests. The editing vector was constructed as follows Figure 5 . The AtBRI1 and SlGGP1 gene loci were edited respectively. Figure 6 As shown, the next-generation sequencing results showed that at different editing sites of the two plants, Agrobacterium containing VirG6 could create the highest editing efficiency (insertion and / or deletion ratio), which was about 6.8% (AtBRI1) and 1.1% (SlGGP1), respectively, which were much higher than the control group using conventional editing vectors (0.2% (AtBRI1) and 0.02% (SlGGP1)) and the experimental group using wild-type VirG (0.15% (AtBRI1) and 0.03% (SlGGP1)). The above results suggest that the VirG6 mutant can improve the gene editing efficiency mediated by Agrobacterium transformation in different plants.

[0121] Example 5: VirG6 mutant and its mutation site improve the transformation efficiency of multiple Agrobacterium strains

[0122] In order to detect whether the obtained Agrobacterium variants (derived from EHA105) can improve the ability of other Agrobacterium strains to transform plants and whether the mutation sites can also be applied in VirG derived from other Agrobacterium strains. The vectors containing the VirG variants derived from EHA105 and the VirG variants derived from LBA4404 were transformed into Agrobacterium LBA4404, and these Agrobacteria were used to infect Nicotiana benthamiana, and the LUC activity at 2Dpi was detected. The results showed that ( Figure 7 ), the efficiency of transforming tobacco by the LBA4404 strain containing VirG6 of EHA105 was higher than that of the control group, and the efficiency of transforming tobacco by the LBA4404 strain containing the VirG6 variant of LBA4404 was also higher than that of the control group. This indicates that the VirG6 variant and its mutation site derived from EHA105 can also be applied to other strains.

[0123] Sequence information

[0124] SEQ ID NO:1EHA105 VirG (WT)

[0125] LKHVLLIDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRP NVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0126] SEQ ID NO:2EHA105 VirG (N54D)

[0127] LKHVLLIDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLDLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0128] SEQ ID NO:3EHA105 VirG(F37L / N54D / E62D)

[0129] LKHVLLIDDDVAMRHLIIEYLTIHAFKVTAVADSTQLTRVLSSATVDVVVVDLDLGREDGLDIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0130] SEQ ID NO:4EHA105 VirG(I23N)

[0131] LKHVLLIDDDVAMRHLIIEYLTNHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0132] SEQ ID NO:5EHA105 VirG(I77V)

[0133] LKHVLLIDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIVISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0134] SEQ ID NO:6EHA105 VirG(I23N / I77V)

[0135] LKHVLLIDDDVAMRHLIIEYLTNHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIVISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0136] SEQ ID NO:7EHA105 VirG(I23N / I77V / I182N)

[0137] LKHVLLIDDDVAMRHLIIEYLTNHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIVISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLNASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0138] SEQ ID NO:8EHA105 VirG(I23N / F37L / N54D / E62D / I77V / I182N)

[0139] LKHVLLIDDDVAMRHLIIEYLTNHAFKVTAVADSTQLTRVLSSATVDVVVVDLDLGREDGLDIVRNLAAKSDIPIIVISGDRLEETDKVVALELGASDFIAKPFSTREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLNASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0140] SEQ ID NO:9LBA4404 VirG(WT)

[0141] LKHVLLVDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSIREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0142] SEQ ID NO:10LBA4404 VirG(N54D)

[0143] LKHVLLVDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLDLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDFIAKPFSIREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0144] SEQ ID NO:11LBA4404 VirG(I23N / F37L / N54D / E62D / I77V / I182N)

[0145] LKHVLLVDDDVAMRHLIIEYLTNHAFKVTAVADSTQLTRVLSSATVDVVVVDLDLGREDGLDIVRNLAAKSDIPIIVISGDRLEETDKVVALELGASDFIAKPFSIREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDVLSREQLLNASRVRDEEVYDRSIDVLILRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA*

[0146] References

[0147] Owens LD, Smigocki AC(1988)Transformation of Soybean Cells Using Mixed Strains of Agrobacterium tumefaciens and Phenolic Compounds.Plant Physiol 88:570 - 573

[0148] Sheikholeslam SN, Weeks DP(1987)Acetosyringone promotes high - efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens.Plant Molecular Biology 8:291 - 298。

Claims

1. A VirG protein mutant or a functional fragment thereof, wherein the VirG protein mutant has an amino acid mutation at least at one or more positions selected from position 23, 37, 54, 62, 77 or 182 relative to the wild-type VirG protein, wherein the amino acid position refers to SEQ ID NO:

1.

2. The VirG protein mutant or a functional fragment thereof according to claim 1, wherein the VirG protein mutant has at least the amino acid at position 23 replaced by N, the amino acid at position 37 replaced by L, the amino acid at position 54 replaced by D, the amino acid at position 62 replaced by D, the amino acid at position 77 replaced by V, and / or the amino acid at position 182 replaced by N relative to the wild-type VirG protein, and the amino acid positions refer to SEQ ID NO:

1.

3. The VirG protein mutant or a functional fragment thereof according to claim 1 or 2, wherein the VirG protein mutant comprises at least one or more amino acid substitutions selected from I23N, F37L, N54D, E62D, I77V, I182N relative to the wild-type VirG protein, and the amino acid positions refer to SEQ ID NO:

1.

4. The VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 3, wherein the VirG protein mutant comprises at least i) amino acid substitution N54D; ii) amino acid substitutions F37L, N54D and E62D; iii) amino acid substitution I23N; iv) amino acid substitution I77V; v) amino acid substitutions I23N and I77V; vi) amino acid substitutions I23N, I77V and I182N; or vii) amino acid substitutions I23N, F37L, N54D, E62D, I77V and I182N, The amino acid positions are referenced to SEQ ID NO:

1.

5. The VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 4, wherein the transcriptional activation activity of the VirG protein mutant or the functional fragment thereof is improved relative to the wild-type VirG protein, or the transcriptional activation activity of the VirG protein mutant or the functional fragment thereof is independent of the activity of VirA.

6. The VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 5, wherein the VirG protein mutant comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with any one of SEQ ID NOs: 2-8, 10 and 11. Preferably, the VirG protein mutant comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-8, 10 and 11, and more preferably comprises the amino acid sequence shown in SEQ ID NO: 8 or 11.

7. A nucleic acid comprising a nucleotide sequence encoding the VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 6.

8. An expression cassette comprising a nucleotide sequence encoding the VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 6, operably linked to a regulatory sequence.

9. An expression construct comprising the expression cassette of claim 8.

10. An Agrobacterium comprising and / or expressing the VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 6.

11. The Agrobacterium of claim 10, wherein the Agrobacterium is Agrobacterium tumefaciens.

12. The Agrobacterium of claim 11, wherein the Agrobacterium is selected from the group consisting of Agrobacterium tumefaciens EHA105 strain, LBA4404 strain, GV3101 strain, and AGL-1 strain.

13. The Agrobacterium of any one of claims 10 to 12, wherein the Agrobacterium comprises the nucleic acid of claim 7, the expression cassette of claim 8 and / or the expression construct of claim 9, or is transformed with the nucleic acid of claim 7, the expression cassette of claim 8 and / or the expression construct of claim 9, thereby expressing the VirG protein mutant or a functional fragment thereof.

14. The Agrobacterium of any one of claims 10 to 13, wherein the Agrobacterium further comprises at least one exogenous nucleic acid sequence of interest to be transformed into a plant.

15. Use of the VirG protein mutant or a functional fragment thereof according to any one of claims 1 to 6 or the Agrobacterium according to any one of claims 10 to 13 in transforming an exogenous nucleic acid sequence of interest into a plant.

16. A method for transforming an exogenous nucleic acid sequence of interest into a plant, the method comprising infecting the plant with the Agrobacterium of any one of claims 10 to 13, wherein the Agrobacterium comprises the exogenous nucleic acid sequence of interest.

17. The use of claim 15 or the method of claim 16, wherein the plant is selected from the group consisting of wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, potato, lettuce, beet, Arabidopsis, Chinese cabbage, Chinese cabbage, radish, cucumber, apple, pepper, peanut, sesame, and rubber tree.

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