CN113774082A - Method for expressing nucleic acid - Google Patents

Abstract

The invention provides a nucleic acid expression method, in particular to a nucleic acid construct, which adopts a nucleic acid construct driven by a specific promoter to successfully realize gRNA-guided efficient base site-directed mutagenesis in plants.

Description

A kind of method of nucleic acid expression

technical field

The present invention relates to the field of biotechnology, in particular, to a method for nucleic acid expression.

Background technique

At present, the single-base editing efficiency is low in dicotyledonous plants. Most dicotyledonous plants such as soybean cannot perform single-base editing at present, while Arabidopsis/tomato and other plants have low single-base editing efficiency, which seriously affects biological The application of technical breeding in agricultural production. Therefore, improving the editing efficiency of single base editing in dicotyledonous plants has great commercial value in agricultural production.

Therefore, there is an urgent need in the art to develop a method to improve the efficiency of single base editing in plants.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for improving the efficiency of single base editing in plants.

A first aspect of the present invention provides a nucleic acid construct having the structure of formula I of 5'-3' (5' to 3'):

P1-S1-L1-S2-S3 (I);

In the formula,

P1, S1, L1, S2, S3 are respectively the elements used to constitute the construct;

P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor;

S1 and S2 are each independently one or more (a) coding sequences for gene editing enzymes, (b) coding sequences for adenine deaminase and/or coding sequences for cytosine deaminase;

L1 is the coding sequence for no or linker peptide;

S3 is the coding sequence of no or uracil glucosidase inhibitor UGI;

Also, each "-" is independently a bond or a nucleotide linking sequence.

In another preferred example, the S1 is the coding sequence of adenine deaminase and/or the coding sequence of cytosine deaminase, and the S2 is the coding sequence of the gene editing enzyme.

In another preferred example, when S1 is the coding sequence of adenine deaminase, S3 is absent.

In another preferred example, when S1 is the coding sequence of cytosine deaminase, S3 is the coding sequence of the uracil glycosidase inhibitor UGI.

In another preferred embodiment, the elongation factor includes eukaryotic elongation factor or prokaryotic elongation factor.

In another preferred embodiment, the eukaryotic elongation factors include EF1α, EF1β, and EF2.

In another preferred embodiment, the prokaryotic elongation factor includes EF-Tu, EF-Ts, EF-G; preferably, includes EF1α; preferably, includes EF1α in plants.

In another preferred embodiment, the plant is selected from the group consisting of corn, rice, soybean, Arabidopsis, tobacco, tomato, or a combination thereof.

In another preferred embodiment, the first promoter is derived from one or more plants selected from the group consisting of corn, rice, soybean, Arabidopsis, tobacco, and tomato.

In another preferred embodiment, the first promoter is the promoter of tomato EF1a.

In another preferred embodiment, the sequence of the first promoter is shown in SEQ ID NO.:1.

In another preferred embodiment, the lengths of the L1 nucleotide sequences are each independently 3-120nt, preferably 3-96nt, and preferably a multiple of 3.

In another preferred example, the lengths of the amino acid sequences encoded by L1 are independently 3-40aa, preferably 6-32aa, preferably 18-32aa, and preferably 24-32aa.

In another preferred embodiment, the length of the nucleotide linker sequence is 1-300nt, preferably 1-100nt.

In another preferred embodiment, the nucleotide linker sequence does not affect the normal transcription and translation of each element.

In another preferred embodiment, the gene editing enzyme is an enzyme selected from the group of editing tools: CRISPR enzyme, TALEN enzyme, ZFN enzyme, or a combination thereof.

In another preferred embodiment, the gene editing enzyme is derived from microorganisms; preferably from bacteria.

In another preferred embodiment, the source of the gene editing enzyme is selected from the group consisting of Streptococcus _pyogenes , Staphylococcus aureus, Streptococcus canis, or a combination thereof.

In another preferred example, the gene editing enzyme has double-stranded or single-stranded DNA cleavage activity, or no cleavage activity.

In another preferred embodiment, the gene editing enzyme is a CRISPR enzyme with single-stranded DNA cleavage activity.

In another preferred embodiment, the gene editing enzyme includes wild type or mutant gene editing enzyme.

In another preferred embodiment, the identity of the gene editing enzyme and the mutated gene editing enzyme is ≥80%, preferably ≥90%; more preferably ≥95%, more preferably ≥98% or 99%.

In another preferred embodiment, the mutated gene editing enzyme is passed through one or more, preferably 1-15, preferably 1-10, preferably 1, wild-type gene editing enzymes. -7, more preferably 2-5, amino acid substitutions, deletions; and/or through 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids formed by adding.

In another preferred embodiment, the gene editing enzyme is selected from the group consisting of Cas9, Cas12, Cas13, Cms1, MAD7, or a combination thereof.

In another preferred embodiment, the gene editing enzyme is selected from the group consisting of nCas9, dCas9, nCas9NG, nCas9X, nCas12, nCas13, or a combination thereof.

In another preferred embodiment, the amino acid sequence of the gene editing enzyme is shown in SEQ ID NO.:2.

In another preferred embodiment, the coding sequence of the gene editing enzyme is selected from the following group:

(i) a polynucleotide whose sequence is shown in SEQ ID NO.: 3;

(ii) The homology between the nucleotide sequence and the sequence shown in SEQ ID NO.: 3 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99% ) polynucleotides;

(iii) truncation or addition of 1-60 (preferably 1-30, more preferably 1-10) nuclei at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.: 3 nucleotide polynucleotides;

(iv) a polynucleotide complementary to any of the polynucleotides of (i)-(iii).

In another preferred embodiment, the coding sequence of the gene editing enzyme is shown in SEQ ID NO.:3.

In another preferred embodiment, the adenine deaminase includes wild type and mutant type.

In another preferred embodiment, the adenine deaminase includes wild-type and/or mutant TadA.

In another preferred embodiment, the adenine deaminase includes TadA.

In another preferred embodiment, the mutant form of adenine deaminase includes TadA7-10.

In another preferred embodiment, the adenine deaminase is a fusion protein formed by TadA and TadA7-10.

In another preferred embodiment, the coding sequence of the adenine deaminase is selected from the following group:

(i) a polynucleotide whose sequence is shown in SEQ ID NO.: 5 or 19;

(ii) The homology between the nucleotide sequence and the sequence shown in SEQ ID NO.: 5 or 19 is ≥ 75% (preferably ≥ 85%, more preferably ≥ 90% or ≥ 95% or ≥ 98% or ≥ 99%) polynucleotides;

(iii) truncation or addition of 1-60 (preferably 1-30, more preferably 1-10) at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.: 5 or 19 ) polynucleotides of nucleotides;

(iv) a polynucleotide complementary to any of the polynucleotides of (i)-(iii).

In another preferred embodiment, the coding sequence of the adenine deaminase is shown in SEQ ID NO.5 or 19.

In another preferred embodiment, the amino acid sequence of the adenine deaminase is shown in SEQ ID NO.:4.

In another preferred embodiment, the cytosine deaminase includes wild type and mutant type.

In another preferred embodiment, the cytosine deaminase includes APOBEC.

In another preferred embodiment, the APOBEC is selected from the group consisting of: APOBEC1(A1), APOBEC2(A2), APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3H, APOBEC4(A4), activation-induced deaminase ( activation induced cytidine deaminase, AID), or a combination thereof.

In another preferred embodiment, the mutants of cytosine deaminase include CBE2.0, CBE2.1, CBE2.2, CBE2.3, and CBE2.4.

In another preferred embodiment, the amino acid sequence of the cytosine deaminase is shown in any of SEQ ID NO.: 6, 8-11.

In another preferred embodiment, the nucleic acid construct may also be operably linked to one or more localization signal sequences.

In another preferred embodiment, the localization signal is selected from the group consisting of a nuclear localization signal, a chloroplast localization signal, a mitochondrial localization signal, or a combination thereof.

In another preferred embodiment, the localization signals include nuclear localization signals, preferably 1-2 nuclear localization signals.

In another preferred embodiment, the nuclear localization signal includes bpNLS and SV40.

In another preferred embodiment, the nucleotide sequence of the nuclear localization signal is shown in any one of SEQ ID NO.: 12-14.

In another preferred embodiment, the amino acid sequence of the nuclear localization signal is shown in SEQ ID NO.:15.

In another preferred embodiment, the nucleotide sequence of the S3 element is shown in SEQ ID NO.:16.

In another preference, the nucleic acid construct is further operably linked to the second nucleic acid construct shown in one or more formula II:

P2-Y1(II)

In the formula,

P2 is the second promoter sequence;

Y1 is the coding sequence of gRNA;

Also, each "-" is independently a bond or a nucleotide linking sequence.

In another preferred example, when there are at least two nucleic acid constructs of formula II, their gRNA sequences may be different from each other.

In another preferred example, the nucleic acid construct of formula II is located at the 5' end or 3' end of the nucleic acid construct of formula I or distributed at both ends.

In another preferred embodiment, the gRNA includes crRNA, tracrRNA, and sgRNA.

In another preferred embodiment, the second promoter is derived from one or more plants selected from the group consisting of rice, corn, soybean, Arabidopsis, tobacco or tomato.

In another preferred embodiment, the second promoter includes an RNA polymerase III-dependent promoter.

In another preferred embodiment, the second promoter is an RNA polymerase III-dependent promoter.

In another preferred embodiment, the second promoter is selected from the group consisting of U6, U3, U6a, U6b, U6c, U6-1, U3b, U3d, U6-26, U6-29, H1, or a combination thereof.

In another preferred embodiment, the second promoter includes the U6 promoter.

In another preferred embodiment, the above-mentioned nucleotide elements of the present invention are linked in-frame, thereby expressing a fusion protein with the correct amino acid sequence.

In another preferred example, the nucleic acid construct of formula I and the nucleic acid construct of formula II also independently have a terminator.

In another preferred example, the nucleic acid construct of formula I and the nucleic acid construct of formula II share the same terminator.

In another preferred embodiment, the terminator includes a terminator suitable for plant gene editing.

In another preferred embodiment, the terminator is selected from the group consisting of NOS, Poly A, T-UBQ, rbcS, or a combination thereof.

In another preference, described construct has formula IIIa or formula IIIb structure:

P1-S1-L1-S2-S3-P2-Y1 (IIIa);

P2-Y1-P1-S1-L1-S2-S3 (IIIb);

In the formula, the definition of each element is as described above.

In another preferred embodiment, the nucleic acid construct can also be operably linked to the first integration element (I1) and the second integration element (I2).

In another preferred embodiment, the first integration element includes a 5' homology arm sequence. In another preferred embodiment, the second integration element includes a 3' homology arm sequence.

In another preferred embodiment, one or more additional expression cassettes are additionally inserted between the I1 and I2 elements.

In another preferred embodiment, the additional expression cassette is independent of the expression cassette containing the nucleic acid construct of formula I and the expression cassette containing the nucleic acid construct of formula II.

In another preferred embodiment, the additional expression cassette expresses a substance selected from the group consisting of marker genes.

In another preferred embodiment, the marker gene includes a resistance gene (eg, a hygromycin resistance gene, a herbicide resistance gene), a fluorescent gene, or a combination thereof.

The second aspect of the present invention provides a vector containing the nucleic acid construct described in the first aspect of the present invention.

In another preferred embodiment, the vector is a plant expression vector.

In another preferred embodiment, the vector is an expression vector that can transfect or transform plant cells.

In another preferred embodiment, the carrier is an Agrobacterium Ti carrier.

In another preferred embodiment, the construct is integrated into the T-DNA region of the vector.

In another preferred embodiment, the carrier is circular or linear.

The third aspect of the present invention provides a host cell, which contains the nucleic acid construct of the first aspect of the present invention, or has one or more nucleic acid constructs of the first aspect of the present invention integrated into its genome.

In another preferred embodiment, the cells are plant cells.

In another preferred example, the plant is selected from the group consisting of monocotyledonous plants, dicotyledonous plants, gymnosperms, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of grasses, legumes, crucifers, nightshade, umbelliferae, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of Arabidopsis, wheat, barley, oat, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, Cucumber, chrysanthemum, water spinach, celery, lettuce, or a combination thereof.

In another preferred example, the host cell is to introduce the nucleic acid construct of claim 1 into the cell by a method selected from the group consisting of: Agrobacterium transformation, biolistic method, microinjection method, electroshock method, Ultrasonic and polyethylene glycol (PEG) mediated methods.

A fourth aspect of the present invention provides a combination of reagents, comprising:

(i) the first nucleic acid construct, or the first carrier containing the first nucleic acid construct, the first nucleic acid construct having the structure of formula I from 5'-3':

P1-S1-L1-S2-S3 (I)

in,

P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor;

S1 and S2 are each independently one or more (a) coding sequences for gene editing enzymes, (b) coding sequences for adenine deaminase and/or coding sequences for cytosine deaminase;

L1 is the coding sequence for no or linker peptide;

S3 is the coding sequence of no or uracil glucosidase inhibitor UGI;

And, "-" is a bond or a nucleotide linking sequence;

(ii) the second nucleic acid construct, or the second carrier containing the second nucleic acid construct, the second nucleic acid construct having the structure shown in the formula (II) from 5'-3':

P2-Y1 (II);

Wherein, P2 is the second promoter;

Y1 is the coding sequence of gRNA;

Also, "-" is a bond or a nucleotide linking sequence.

In another preferred embodiment, the first carrier and the second carrier are different carriers.

In another preferred embodiment, the first nucleic acid construct and the second nucleic acid construct are located on different vectors.

In another preferred embodiment, the first carrier and the second carrier are the same carrier.

In another preferred embodiment, the first nucleic acid construct and the second nucleic acid construct are located on the same vector.

The fifth aspect of the present invention provides a kit containing the reagent combination described in the fourth aspect of the present invention.

In another preferred embodiment, the kit further contains labels or instructions.

A sixth aspect of the present invention provides a method for gene editing of plants, comprising the steps of:

(i) provide plants to be edited; and

(ii) introducing the nucleic acid construct described in the first aspect of the present invention, the vector described in the second aspect of the present invention, or the reagent combination described in the fourth aspect of the present invention into the plant cell of the plant to be edited, so that the Gene editing in plant cells.

In another preferred embodiment, the introduction is by Agrobacterium.

In another preferred embodiment, the introduction is by gene gun.

In another preferred embodiment, the gene editing is site-directed base substitution (or mutation).

In another preferred embodiment, the site-directed substitution (or mutation) includes mutating A to G.

In another preferred embodiment, the site-directed substitution (or mutation) comprises mutating C to T.

In another preferred embodiment, the plants include any higher plant types that can be transformed with technology, including monocotyledonous plants, dicotyledonous plants and gymnosperms.

In another preferred embodiment, the plant is a dicotyledonous plant.

In another preferred embodiment, the plant is selected from the group consisting of grasses, legumes, crucifers, nightshade, umbelliferae, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of Arabidopsis, wheat, barley, oat, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, Cucumber, chrysanthemum, water spinach, celery, lettuce, or a combination thereof.

A seventh aspect of the present invention provides a method for preparing gene-edited plant cells, comprising the steps of:

The nucleic acid construct described in the first aspect of the present invention, the vector described in the second aspect of the present invention, or the reagent combination described in the fourth aspect of the present invention are combined to transfect plant cells, so that the chromosomes in the plant cells are replaced by site-directed replacement ( or mutation) to produce the gene-edited plant cell.

In another preferred embodiment, the transfection adopts Agrobacterium transformation method or gene gun bombardment method.

The eighth aspect of the present invention provides the nucleic acid construct described in the first aspect of the present invention, the vector described in the second aspect of the present invention, the host cell described in the third aspect of the present invention, and the fourth aspect of the present invention. The reagent combination and the use of the kit according to the fifth aspect of the present invention are used for gene editing of plants.

A ninth aspect of the present invention provides a method for preparing a gene-edited plant, comprising the steps of:

The gene-edited plant cell prepared by the method of the seventh aspect of the present invention is regenerated into a plant body, thereby obtaining the gene-edited plant.

The tenth aspect of the present invention provides a gene-edited plant prepared by the method of the ninth aspect of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the structure of the ABE single base editor containing slEF1a.

Figure 2 shows the efficiency of different promoters in single base editing in tomato.

Figure 3 shows the single base editing efficiency in soybean using different promoters and different base editors.

Detailed ways

After extensive and in-depth research, the present inventor unexpectedly discovered a high-efficiency EF promoter (such as tomato EF promoter) for the first time. The promoter was constructed in the single-base editing system of ABE and CBE to drive ( a) Expression of a fusion protein composed of a gene editing enzyme and (b) adenine deaminase and/or cytosine deaminase, the promoter significantly improves the editing efficiency in plants. On this basis, the present inventors have completed the present invention.

the term

As used herein, the term "homology arms" refers to the flanking sequences on the targeting vector that are flanked by the exogenous sequence to be inserted that are completely identical to the genomic sequence, and are used to identify and recombine regions.

As used herein, the term "plant promoter" refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell. The plant promoter can be derived from plants, microorganisms (such as bacteria, viruses) or animals, or can be artificially synthesized or engineered.

As used herein, the term "gene editing" or "base mutation" or "base editing" refers to the substitution, insertion and/or deletion of a base at a position in a nucleotide sequence ). The "editing" or "mutation" in the present invention is preferably a single base mutation.

As used herein, the term "base substitution" refers to the mutation of a base at a position in a nucleotide sequence to a different base, such as A to G.

As used herein, the term "A.T to G.C" refers to the mutation or replacement of an A-T base pair with a G-C base pair at a position in a double-stranded nucleic acid sequence, especially a genomic sequence.

As used herein, the term "C.G to T.A" refers to a mutation or replacement of a C-G base pair with a T-A base pair at a position in a double-stranded nucleic acid sequence, especially a genomic sequence.

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

As used herein, the term "coding sequence for a Cas protein" refers to a nucleotide sequence encoding a Cas protein. Where the inserted polynucleotide sequence is transcribed and translated to produce a functional Cas protein, the skilled artisan will recognize that, because of the degeneracy of codons, a large number of polynucleotide sequences can encode the same polypeptide . In addition, skilled artisans will also recognize that different species have a certain preference for codons, and may optimize the codons of Cas proteins according to the needs of expression in different species. These variants are referred to by the term "Cas protein. Coding sequences" are specifically encompassed. In addition, the term specifically includes full-length sequences substantially identical to the Cas gene sequence, as well as sequences encoding proteins that retain Cas protein function.

As used herein, the "gRNA" is also referred to as guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art. In general, guide RNAs may comprise direct repeats and guidesequences, or consist essentially of or consist of direct repeats and guides (also referred to as spacers in the context of endogenous CRISPR systems ( spacer)) composition. In different CRISPR systems, gRNAs can include crRNA and tracrRNA, or only crRNA, depending on the Cas protein they depend on. crRNA and tracrRNA can be artificially fused to form single guide RNA (sgRNA). The gRNA of the present invention can be natural, or it can be artificially modified or designed and synthesized. In certain instances, a targeting sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence, typically having a 17- 23nt sequence length. In certain embodiments, when optimally aligned, the degree of complementarity between a targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

As used herein, the term "plant" includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds and plant cells and their progeny. The types of plants that can be used in the method of the present invention are not particularly limited, and generally include any plant type that can be subjected to gene editing techniques, including monocotyledonous, dicotyledonous and gymnosperm plants, angiosperms, mainly including woody plants.

As used herein, the term "expression cassette" refers to a polynucleotide sequence that contains the sequence components of the gene to be expressed and the elements required for expression. Components required for expression include a promoter and a polyadenylation signal sequence. In addition, the expression cassettes of the present invention optionally contain other sequences including, but not limited to, enhancers, secretion signal peptide sequences, and the like.

In the present invention, the description of the nucleotide sequence is from the 5' to 3' direction unless otherwise specified.

As used herein, a "uracil DNA glycosylase inhibitor (UGI)" is capable of inhibiting the re-catalysis of U back to C by intracellular uracil DNA glycosidase.

EF promoter

EF promoter refers to the promoter of elongation factors, and elongation factors (EF) refers to protein factors that promote polypeptide chain elongation during mRNA translation. Elongation factors in eukaryotes include: EF1α, EF1β and EF2. Elongation factors in prokaryotes include EF-Tu, EF-Ts, and EF-G. EF1a is eukaryotic elongation factor 1α, which is an essential component of protein biosynthesis. EF1A catalyzes the binding of aminoacyl tRNA to the ribosomal A site through a GTP-dependent mechanism. EF1A accounts for 3-10% of the total soluble protein and is considered to be one of the most abundant soluble proteins in the cytoplasm.

In a preferred embodiment, EF promoters include, but are not limited to: EF1a promoter, EF1β promoter, EF2 promoter, EF-Tu, EF-Ts, EF-G.

In a preferred embodiment, the promoter of the present invention refers to an EF1a promoter element derived from a plant of the Solanaceae family (preferably, from a tomato or similar plant).

A typical sequence of the promoter of the present invention is shown in SEQ ID NO.:1.

It should be understood that the term also includes promoters from other different Solanaceae plants that are homologous to the promoter shown in SEQ ID NO.: 1. In addition, the term also includes derived promoters or active fragments of the promoter shown in SEQ ID NO.: 1 or a homologous promoter thereof, mainly these derived promoters or active fragments retain the function of high gene editing efficiency, such as retaining At least 50% of the specific activation function of the promoter shown in SEQ ID NO.: 1 (indicated by the expression level of the exogenous gene that can be activated).

As used herein, the term "Solanaceae" includes tomato, potato, eggplant, pepper, wolfberry, tobacco.

As used herein, the term "promoter" or "promoter region (domain)" refers to a nucleic acid sequence that accurately and efficiently initiates the transcriptional function of a gene and directs the transcription of the gene nucleic acid sequence into mRNA, which is usually present in the coding sequence of the gene of interest. Upstream (5' end), typically, the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.

Herein, the promoter or promoter region (domain) includes promoter variants, which can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, and the like.

The present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more) of the preferred promoter sequence (SEQ ID NO.: 1) of the invention , most preferably more than 98%, such as 99%) homology nucleic acid, the nucleic acid also has the function of specifically improving the efficiency of plant gene editing. "Homology" refers to the level of similarity (ie, sequence similarity or identity) between two or more nucleic acids, in terms of percent identical positions.

It should be understood that although the examples of the present invention provide a promoter EF1a derived from Solanaceae, such as tomato, other similar plants (especially belonging to the same family as tomato) have certain homology to the promoter of the present invention Sexual (conservative) promoters are also included in the scope of the present invention, as long as those skilled in the art can easily isolate the promoters from other plants according to the information provided in this application after reading this application.

As used herein, "foreign" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

As used herein, a "cis-regulatory element" refers to a conserved base sequence that regulates transcription initiation and transcription efficiency of a gene.

The promoter of the present invention may be operably linked to a foreign gene, which may be foreign (heterologous) with respect to the promoter. The exogenous gene (also referred to as the target gene) in the present invention is not particularly limited, and can be a gene encoding a protein with a specific function, such as (a) gene editing enzymes and (b) adenine deaminase and/or cellular pyrimidine deaminase.

Representative examples of such exogenous genes include, but are not limited to, resistance genes, selectable marker genes, epitope tags, reporter gene sequences, nuclear localization signal sequences, transcriptional activation domains (eg, transcriptional activation domains (eg, transcriptional activation domains). , VP64), transcriptional repression domains (e.g., KRAB domains or SID domains), nuclease domains (e.g., Fok1), viral capsid protein genes, antibody genes; and domains with an activity selected from the group consisting of: Nucleotide deaminase, methylase activity, demethylase activity, transcriptional activation activity, transcriptional inhibitory activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity.

The resistance genes are selected from the group consisting of herbicide resistance genes, virus resistance genes, cold resistance genes, high temperature resistance genes, drought resistance genes, waterlogging resistance genes, or insect resistance genes. The selectable marker gene is selected from the group consisting of gus (β-glucuronidase) gene, hyg (hygromycin) gene, neo (neomycin) gene, or gfp (green fluorescent protein) gene.

The present invention also provides a gene expression cassette, the expression cassette has the following elements in order from 5' to 3': a promoter, a gene ORF sequence, and a terminator. Preferably, the promoter sequence is shown in SEQ ID NO.: 1 or the homology with the sequence shown in SEQ ID NO.: 1 is ≥90%, preferably ≥95%, more preferably ≥98%.

The present invention also provides a recombinant vector comprising the promoter and/or gene expression cassette of the present invention. As a preferred manner, the downstream of the promoter of the recombinant vector contains multiple cloning sites or at least one restriction enzyme cleavage site. When the target gene needs to be expressed, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operably linking the target gene to the promoter. As another preferred mode, the recombinant vector includes (from 5' to 3' direction): a promoter, a target gene, and a terminator. If desired, the recombinant vector may also include elements selected from the group consisting of: 3' polynucleotideization signal; untranslated nucleic acid sequences; transport and targeting nucleic acid sequences; resistance selectable markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancers; or operons.

Those of ordinary skill in the art can use well-known methods to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.

The promoters, expression cassettes or vectors of the present invention can be used to transform suitable host cells to allow the host to express proteins. Host cells can be prokaryotic cells, such as E. coli, Streptomyces, Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to those of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism (such as E. coli), it can be treated with CaCl ₂ or electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Agrobacterium transformation or biolistic transformation and other methods can also be used to transform plants, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants.

As a preferred mode of the present invention, the method for preparing a transgenic plant is as follows: a vector carrying a promoter and a gene of interest (the two are operably linked) is transferred into Agrobacterium, and then the vector containing the promoter and the gene of interest is transferred to Agrobacterium. The fragment integrates into the chromosome of the plant. The transgenic recipient plants involved are, for example, Arabidopsis, wheat, barley, oats, maize, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, Lettuce, etc. In the example of the present invention, the recombinant vector is pCAMBIA1300 vector, and the promoter of the present invention is constructed into this vector to transform plants.

In a preferred embodiment, the present invention clones an EF promoter (such as tomato SlEF1a promoter), and uses the promoter to drive the expression of the fusion protein coding sequence of Cas enzyme and deaminase, and finally obtains a pair of dicotyledonous leaves. System for high-efficiency single-base substitution and gene knockout in plants.

adenine deaminase

As used herein, the term "adenine deaminase" is an enzyme that catalyzes the hydrolytic deamination of adenine to produce hypoxanthine and ammonia. Adenine A is converted into hypoxanthine I, which can pair with cytosine and be read and replicated as guanine (G) at the DNA level, resulting in the conversion of A·T pairing into G·C pairing. TadA adenine deaminase is derived from Escherichia coli, and the ecTadA mutant that has been obtained so far has been artificially modified. The dimer of TadA and ecTadA is currently commonly used adenine deaminase.

In the present invention, suitable TadA includes both the wild-type form and its specific mutant form, TadA7-10, and may also include a combination of wild-type and mutant forms. TadA7-10 can carry out deamination reaction with DNA as substrate.

In the present invention, the coding sequence of adenine deaminase in the nucleic acid construct can be codon-optimized in a host-preferred manner according to different applicable hosts.

cytosine deaminase

As used herein, the term "cytosine deaminase (APOBEC)" is an enzyme capable of catalyzing the deamination of intracellular cytosine to form uracil, converting cytosine C to uracil U, and damaging DNA by polymerase during re-replication In the process of DNA replication, uracil will be recognized as T, resulting in the conversion of C·G pairing to T·A pairing. 11 APOBECs family members have been found, including APOBEC1 (A1), APOBEC2 (A2), APOBEC3A~H (3A, 3B, 3C, 3D, 3E, 3F, 3H), APOBEC4 (A4) and activation-induced deaminase (activation induced cytidine deaminase, AID).

In the present invention, suitable cytosine deaminase includes both the wild-type form and its specific mutant form (such as CBE2.0, CBE2.1, CBE2.2, CBE2.3, CBE2.4), or Contains a combination of wild-type and mutant forms. Mutant forms of cytosine deaminase are capable of deamination reactions using DNA as a substrate.

In the present invention, the coding sequence of cytosine deaminase in the nucleic acid construct can be codon-optimized in a host-preferred manner according to different applicable hosts.

In a preferred embodiment of the present invention, the preferred cytosine deaminase is CBE2.0, CBE2.1, CBE2.2, CBE2.3, CBE2.4.

The amino acid sequence of CBE2.0 is shown in SEQ ID NO.:6, and its nucleotide sequence is shown in SEQ ID NO.:7.

The amino acid sequence of CBE2.1 is shown in SEQ ID NO.:8.

The amino acid sequence of CBE2.2 is shown in SEQ ID NO.:9.

The amino acid sequence of CBE2.3 is shown in SEQ ID NO.:10.

The amino acid sequence of CBE2.4 is shown in SEQ ID NO.:11.

Constructs of the present invention

The present invention provides a nucleic acid construct for gene editing of plants, and the nucleic acid construct has the structure of formula I of 5'-3':

P1-S1-L1-S2-S3 (I);

In the formula,

P1, S1, L1, S2, S3 are the elements used to constitute the construct, respectively

, which is defined as described in the first aspect of the present invention;

Also, each "-" is a bond or a nucleotide linking sequence.

In a preferred embodiment, the nucleic acid construct is further operably linked to the second nucleic acid construct shown in one or more formula II:

P2-Y1(II);

In the formula, the definitions of P2 and Y1 are as described in the first aspect of the present invention.

In a preferred embodiment, the nucleic acid construct is further operably linked to a first integration element (I1) and a second integration element (I2).

Among them, the I1 element (or the left integration element) and the I2 element (or the right integration element) can cooperate to integrate the elements located therebetween (ie, the nucleotide sequence from P1 to Y1) into the genome of the plant cell .

Representative I1 and I2 are Ti elements from Agrobacterium. Of course, other elements that can serve a similar integration function can also be used in the present invention.

The various elements used in the constructs of the present invention are either known in the art or can be prepared by methods known to those skilled in the art. For example, the corresponding elements can be obtained by conventional methods, such as PCR method, total artificial chemical synthesis method, and enzyme cleavage method, and then connected together by well-known DNA ligation techniques to form the construct of the present invention.

Inserting the construct of the present invention into an exogenous vector (especially a vector suitable for manipulation of transgenic plants) constitutes the vector of the present invention.

The vector of the present invention is transformed into a plant cell so as to mediate the vector of the present invention to integrate into the plant cell chromosome, and express in the plant to obtain a gene-edited plant cell.

The gene-edited plant cell of the present invention is regenerated into a plant body, thereby obtaining a gene-edited plant.

The above-mentioned nucleic acid construct constructed by the present invention can be introduced into plant cells by conventional plant recombination technology (such as Agrobacterium transfer technology), thereby obtaining the carrier carrying the nucleic acid construct (or carrying the nucleic acid construct) plant cells, or obtain plant cells with the nucleic acid construct integrated into the genome.

In the present invention, the plant individual incorporating the nucleic acid construct can be isolated or removed from its progeny by routine screening or by other means known in the art, so as to obtain a gene-edited plant that does not contain the nucleic acid construct body.

Specifically, the present invention uses a specific EF promoter, such as tomato EF1a, to drive the expression of a gene editing enzyme (such as Cas9) and a deaminase fusion protein coding sequence, thereby improving gene editing efficiency.

Vector construction

The main feature of the vector is that the specific EF promoter (such as tomato EF1a), the coding sequences of deaminase and Cas fusion protein, optionally also nuclear localization signal, UGI coding sequences are linked together, thereby forming the present invention. specific nucleic acid constructs. When the nucleic acid construct is expressed in the cytoplasm, the fusion protein encoded by the nucleic acid construct can be transferred into the nucleus very efficiently, and guided to the target position in the genome by the guide RNA encoded by the formula II construct, As a result, base substitutions from A.T to G.C or C.G to T.A are performed at the target position, and the risk of insertion/deletion is basically avoided or eliminated, and the efficiency of gene editing can be significantly improved.

Since adenine deaminase mutates A to G and cytosine deaminase mutates C to T, the DNA double-strand cleavage activity of the Cas protein is not required. Therefore, in the present invention, the Cas protein is a mutant Cas protein without cleavage activity or with single-strand cleavage activity. In a preferred embodiment, the Cas protein of the present invention can be nCas9, the amino acid sequence of which is shown in SEQ ID NO.:2. Generally, in order to increase the activity of the fusion protein, the proteins are usually connected by some flexible short peptides, namely Linker (linker peptide sequence). Preferably, XTEN can be selected for this Linker, and its coding sequence is shown in SEQ ID NO.: 17, and its amino acid sequence is shown in SEQ ID NO.: 18.

Select the expression cassette of the guide RNA suitable for plant cells, and construct it in the same vector as the open expression cassette (ORF) of the above fusion protein.

In the present invention, the vector may be of the type such as plasmid, virus, cosmid, bacteriophage, etc., which are well known to those skilled in the art and are numerously described in the art. Preferably, the expression vector in the present invention is a plasmid. Expression vectors may contain promoters, ribosome binding sites for translation initiation, polyadenylation sites, transcription terminators, enhancers, and the like. The expression vector may also contain one or more selectable marker genes for use in selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase, or genes conferring neomycin resistance, genes conferring resistance to tetracycline or ampicillin, and the like.

The nucleic acid constructs of the present invention can be inserted into a vector by a variety of methods, for example, by digestion of the insert and vector with appropriate restriction endonucleases followed by ligation. Various cloning techniques are known in the art and are within the knowledge of those skilled in the art.

Vectors suitable for use in the present invention include commercially available plasmids such as but not limited to: pBR322 (ATCC37017), pCAMBIA1300, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) ) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), among others.

genetic transformation

In the present invention, there is no particular limitation on the method of introducing the construct of formula I of the present invention into cells or integrating into the genome. This can be done by conventional methods, for example by introducing the construct of formula I or the corresponding vector into plant cells by a suitable method. Representative introduction methods include, but are not limited to: Agrobacterium transfection, biolistic, microinjection, electroshock, ultrasonic, and polyethylene glycol (PEG) mediated methods, and the like.

In the present invention, the recipient plants are not particularly limited, including various crop plants (such as grasses), forestry plants, horticultural plants (such as flower plants) and the like. Representative examples include, but are not limited to, rice, soybean, tomato, corn, tobacco, wheat, sorghum, potato, and the like.

After the above-mentioned DNA vector or fragment is introduced into plant cells, the fusion protein and gRNA are expressed in DNA in the transformed plant cells. A gene editing enzyme (such as Cas9 nuclease) fused with adenine deaminase and/or cytosine deaminase, under the guidance of the corresponding gRNA, mutates the A at the target position to G (and then mutates the T of the complementary strand to C). ) or mutate the C at the target position to a T (which in turn mutates the G of the complementary strand to A).

For the plant cells, tissues or organs subjected to site-directed replacement of the plant genome by the method of the present invention, conventional methods can be used to regenerate and obtain corresponding gene-edited plants. For example, by tissue culture, the plant after the base substitution is obtained by regeneration.

application

The present invention can be used in the field of plant genetic engineering, for plant research and breeding, especially the genetic improvement of crops, forestry crops or horticultural plants with economic value.

The main advantages of the present invention include:

(1) For the first time, the present invention combines a specific promoter (such as Ef1a promoter) with a gene editing enzyme (such as Cas9 nuclease), adenine deaminase and/or cytosine deaminase, and optionally also includes a nuclear localization signal , the coding sequences of UGI are connected together, thereby forming the specific nucleic acid construct of the present invention, the nucleic acid construct of the present invention has successfully realized the base site-directed mutation (such as A is mutated to G) guided by gRNA in plants, and the mutation efficiency Very high (up to ≥70% or higher).

(2) The specific nucleic acid construct of the present invention can edit some gene loci where other promoters do not work, breaking the obstacle of gene editing limited by genotype.

(3) The specific nucleic acid construct of the present invention can edit some plants for which other promoters do not work, such as soybean, effectively expanding the scope of use of the gene editing system and breaking the species barrier.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989), or according to manufacturer's recommended conditions. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

Example 1 Single base editing efficiency of different promoters in tomato

1. Target selection

Solyc05g012020, which affects fruit development in tomato, was selected as the target gene, and 6 target sites were selected to design sgRNAs. The sequences of the designed 6 sgRNAs were as follows: sgRNA1: TACTGGAGTTGTACCTGGA (SEQ ID NO.: 20), sgRNA2: GGAACAGCTTTGAACGTCAAT (SEQ ID NO. .:21), sgRNA3: GAACAGCCTTCTCATCATGA (SEQ ID NO.:22), sgRNA4: GGTGAGGATTTGGGACAATT (SEQ ID NO.:23), sgRNA5: CTGTGAATCTGATGAAGTTT (SEQ ID NO.:24), sgRNA6: GAAAAGTAATAACAAAGGGC (SEQ ID NO.:25) ).

2. Vector construction

The expression cassette of ABE single base editor was obtained by homologous recombination technology (see Figure 1), and the nucleotide sequence of the adenine deaminase ABE7.10 is shown in SEQ ID NO.: 5 or 19, and the The nucleotide sequence of the SlEF1a promoter is shown in SEQID NO.:1, and the specific operations are as follows:

A) Using tomato genomic DNA as a template, the target fragment was amplified with forward/reverse primers pSlEF1a-F/pSlEF1a-R to obtain a PCR product (length about 1583bp, primer annealing temperature 58).

PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45s, 35 cycles, and extension at 72°C for 5 minutes

B) Recover the vector backbone by digestion with restriction enzymes Sbf1 and SalI

proAtU6-gRNA-pro35S-ABE7.10-nspCas9

C) Connect the PCR product obtained from A to the backbone vector obtained from B by homologous recombination to obtain a single base editing vector proAtU6-gRNA-proSlEF1a-ABE7.10-nspCas9

PCR reaction conditions: 50°C for 30min

D) Transform E. coli, pick a single clone and sequence to verify that the fragment is successfully ligated into the vector.

A single base editing vector containing 35S, UBI, AtRPS5A, SlRPS5A1, SlRPS5A2, and SlTCTP promoters was constructed in the same way.

3. Genetic transformation

(A) The above-mentioned construction plasmid is directly transformed into Agrobacterium EHA105:

(1) Plasmid DNA was added to Agrobacterium competent cells, then ice-bathed for 30min, placed in liquid nitrogen for 5min, then immediately placed in a 37°C water bath for 5min, and placed on ice for 5min

(2) Take out the centrifuge tube, add 700ul of YEP medium, and shake for 2-4hrs.

(3) Take out the bacterial liquid and spread it on the YEP medium plate containing the corresponding antibiotics, and invert the culture in the incubator, and the colonies can be seen in about 2 days.

(B) Tomato transgenic

(1) Take tomato sterile seedlings of 7-10d age (the cotyledons are fully expanded, the first true leaves are slightly exposed), and the cotyledons are cut into 5mm square leaves (the tip of the leaf and a small part of the base are cut off, and the middle part is reserved) , placed face up in the pre-culture medium, and incubated in the dark at 25°C for 2 days.

(2) The bacterial liquid stored at -80°C was streaked on solid YEB medium, and cultured in the dark at 28°C for 2 days. A single colony was picked and added to 5 ml of liquid YEB medium at 28° C., 200 rpm, and cultured for 1 d. Take 2ml of bacterial solution and add 50ml of fresh YEB medium, 28℃, 200rpm. Centrifuge at 5000 rpm for 10 min at 4°C, resuspend the cells with infection buffer, and adjust the OD600 to about 0.6-0.8.

(3) Infect the cotyledons of the pre-cultured 2d in the bacterial liquid for 5-10 min, blot the excess bacterial liquid on the filter paper dish, and place the co-cultivation medium face-up on the co-cultivation medium (also can be soaked with non-bacterial infection liquid) filter paper) and incubated at 25 °C for 2 d.

(4) The cotyledons co-cultured for 2 days were transferred to sterilized medium, cultured at 25°C for 7 days, the first 2-3 days of dark culture, and the latter 4-5 days of light culture. After 7 days of co-cultivation, the cotyledons were transferred to the selection medium and cultured for 30-45 days. Subculture every 15d.

(5) Detection of marker genes after sterilization (taking GUS as an example), take several pieces of cotyledons after sterilization for 7 days for GUS staining, and adjust the infection time according to the size of the stained area. (Not every batch, but periodically to check bacterial activity).

(6) Detect the damage of Agrobacterium to the cotyledons, take a number of cotyledons after sterilization for 7 days, make them continue to grow in the sterilization medium for about 30d, subculture once every 15 days, observe the cotyledon differentiation rate, and judge the effect of bacterial liquid on the cotyledons. the damage level, adjust the infection time.

(7) When the young bacteria to be differentiated grow to about 2cm, the seedling is cut off, transferred to the rooting medium, and cultivated until the root grows

(8) Transfer the differentiated robust seedlings to a rooting medium containing antibiotics for rooting culture for one week, and cultivate the seedlings at room temperature for 2-3 days, then cultivate in a greenhouse medium.

(9) Gene editing detection. The leaves of each plant were taken, genomic DNA was extracted, and primers were designed on both sides of the target site of the gRNA. The amplified fragments were subjected to Sanger sequencing to determine the genotype of each plant.

4. Experimental results

The slEF1a promoter reached up to 70% editing efficiency in single base editing, which was 2-20 times higher than other promoters (see Figure 2).

5. Experimental conclusion

The slEF1a promoter can efficiently drive the expression of the fusion protein of deaminase and Cas9, effectively expand the scope of application of single-base editing tools, and is of great significance for plant trait improvement and variety cultivation.

Example 2 Single base editing efficiency of different promoters in soybean

The GmELF3a and GmALS1 genes in soybean were selected, and different promoters and base editors were selected to investigate the single-base editing efficiency of different promoters in soybean. The gRNAs used are shown in the following table:

First, according to the method of Example 1, the SlEF1a promoter (pSlEF1a), the CaMV35S promoter (35S) and the AA6 promoter (pAA6, reference: CN101370939A) and ABE7.10 (SEQ ID NO.: 5 or 19) were investigated. And the editing efficiency when Cas9 is used in conjunction; as shown in Figure 3, the "A to G gRNA1" is the result when using different promoters in conjunction with the above-mentioned adenine deaminase, in soybean, using the SlEF1a promoter The resulting editing efficiency is much higher than the CaMV35S promoter and the AA6 promoter.

In addition, using the above method, the above-mentioned adenine deaminase is replaced with cytosine deaminase, and the amino acid sequence of the cytosine deaminase is shown in SEQ ID NO.: 6, 8-11, the preferred SEQ ID NO. For the cytosine deaminase shown in ID NO.:6, the editing efficiency of different promoters when used in conjunction with cytosine deaminase and Cas9 was investigated; as shown in Figure 3, the "C to T gRNA2" is In order to use the results of using different promoters in combination with the above cytosine deaminase, in soybean, the editing efficiency produced by the SlEF1a promoter is much higher than that of the CaMV35S promoter and the AA6 promoter.

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

sequence listing

<110> Shandong Shunfeng Biotechnology Co., Ltd.

<120> A kind of method of nucleic acid expression

<130> P2020-0390

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<170> SIPOSequenceListing 1.0

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gattagtttg tcaaatagta gagttcattt aaaattcttc agccatatag ttctattttt 60

aagctagtcg acttttttttt tcttactgaa aattaatatt ttttttctttt tgaaatacta 120

atacatctaa atttaacaat tgccaaagtg atttttaatt agcttgctgg ctaatcacaa 180

taaaaattac tctcctttac tatataagta aatttttatt gctatatttg ttattattat 240

tattattatt aatatttatt ttctacaaat ttaataatat tttattttat atcattttaa 300

aaagataagt aatgaaatat taagaattcg tttataattc ttttgcaggt gggtttctat 360

ttgtaagcta atctttttca gttatccttt ttttaaaatc tttattatta ttatagctat 420

atcttttatc ttttaaaatt aacattatct attaaagata atttcaataa aagagtaaaa 480

attaatttag agttctactg tcttcaaatt tctattttaa aaaatacttt taaaacttga 540

tgtatttttt acgtggtttt tcactatgac ttaatttctg ttttattata atatgtataa 600

atataaaaat agattttcca taacatatta taaaaaatgt aaggggcatt tacgtaaata 660

gatagactta aaagaggcac cgagtgaacc ctaattctca tcgttgagac tataaaatgc 720

ccattatccc attcgcacag tctcttcatt acttttgctg ttatttctcc tcagctgtgc 780

cgcatatcgc ctaatttttc ttctctaagg tttcatcatc ttcaccaatt tctttaatct 840

cgattcaatt ttttatgttt gatctgttat tgttctgtca ctacatgtgt ttttcagttg 900

ttttactaga tgattttcac tgtcttcttg ttagatcata catatattga aaatgttttg 960

gattgacttt tttgtattgt gaatatctgt tattgtttga ttgttgttca gtatttacac 1020

acccgatctg tgttatgagc ttggtcataa ctatttctct gtatgtaaat acagatctgt 1080

taatgtttgt aatcaatttt tcatatgcac tgttgatatt gttctctctc ctgtcctgtt 1140

atatgttgat atgattcggt ttttgtataa cttgaactaa acactagtcc taaatgtttt 1200

ttttactatt taagatttat ataatatgga tagatttttt gagttcctag tctctgaaga 1260

ggttaagctt gctgtagttg tttaccagtt gaggtgcaat actaaaaatc aattcaatta 1320

ctgatatttt ttgctgttta ggttttttgac aaagtacttt aatttgcttt attgaactaa 1380

aaacgtagtc ctgaattcat tgcaagtgtg aaagctatag ttcattgttt ttgttgcaat 1440

tcttgaaaaa ttaattggtc aagctataat ggattttact ttttctgttt taatattgaa 1500

tttgctgaat ttatgaatgg gttgcatggt ttttgaaata tgttgttgtg tgttgtgtaa 1560

atgcagtttc ttagtgtctc aag 1583

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Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

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Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

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Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

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Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

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Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

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Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

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Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

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Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 3

<211> 4101

<212> DNA

<213> Streptococcus pyogenes

<400> 3

gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga ttttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

<210> 4

<211> 364

<212> PRT

<213> Artificial sequence

<400> 4

Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr

1 5 10 15

Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val

20 25 30

Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile

35 40 45

Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln

50 55 60

Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr

65 70 75 80

Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser

85 90 95

Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala

100 105 110

Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg

115 120 125

Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu

130 135 140

Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys

145 150 155 160

Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly

165 170 175

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly

180 185 190

Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp

195 200 205

Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu

210 215 220

Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu

225 230 235 240

Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu

245 250 255

Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu

260 265 270

Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala

275 280 285

Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg

290 295 300

Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr

305 310 315 320

Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp

325 330 335

Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val

340 345 350

Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp

355 360

<210> 5

<211> 1092

<212> DNA

<213> Artificial sequence

<400> 5

tctgaagtcg agtttagcca cgagtattgg atgaggcacg cactgaccct ggcaaagcga 60

gcatgggatg aaagagaagt ccccgtgggc gccgtgctgg tgcacaacaa tagagtgatc 120

ggagagggat ggaacaggcc aatcggccgc cacgacccta ccgcacacgc agagatcatg 180

gcactgaggc agggaggcct ggtcatgcag aattaccgcc tgatcgatgc caccctgtat 240

gtgacactgg agccatgcgt gatgtgcgca ggagcaatga tccacagcag gatcggaaga 300

gtggtgttcg gagcacggga cgccaagacc ggcgcagcag gctccctgat ggatgtgctg 360

caccaccccg gcatgaacca ccgggtggag atcacagagg gaatcctggc agacgagtgc 420

gccgccctgc tgagcgattt ctttagaatg cggagacagg agatcaaggc ccagaagaag 480

gcacagagct ccaccgactc tggaggatct agcggaggtt cctctggaag cgagacacca 540

ggcacaagcg agtccgccac accagagagc tccggcggct cctccggagg ctcctctgag 600

gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa gagggcacgc 660

gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acaatagagt gatcggcgag 720

ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat tatggccctg 780

agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct gtacgtgaca 840

ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatcgg ccgcgtggtg 900

tttggcgtga ggaacgcaaa aaccggcgcc gcaggctccc tgatggacgt gctgcactac 960

cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga atgtgccgcc 1020

ctgctgtgct atttctttcg gatgcctaga caggtgttca atgctcagaa gaaggcccag 1080

agctccaccg ac 1092

<210> 6

<211> 228

<212> PRT

<213> Artificial sequence

<400> 6

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Thr Cys Leu Leu Tyr Glu Ile Lys Trp Gly Thr Ser His Lys

35 40 45

Ile Trp Arg His Ser Ser Lys Asn Thr Thr Lys His Val Glu Val Asn

50 55 60

Phe Ile Glu Lys Phe Thr Ser Glu Arg His Phe Cys Pro Ser Thr Ser

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser

85 90 95

Lys Ala Ile Thr Glu Phe Leu Ser Gln His Pro Asn Val Thr Leu Val

100 105 110

Ile Tyr Val Ala Arg Leu Tyr His His Met Asp Gln Gln Asn Arg Gln

115 120 125

Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Thr

130 135 140

Ala Pro Glu Tyr Asp Tyr Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro

145 150 155 160

Gly Lys Glu Ala His Trp Pro Arg Tyr Pro Pro Leu Trp Met Lys Leu

165 170 175

Tyr Ala Leu Glu Leu His Ala Gly Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 7

<211> 684

<212> DNA

<213> Artificial sequence

<400> 7

agcagtgaaa ccggaccagt ggcagtggac ccaaccctga ggagacggat tgagccccat 60

gaatttgaag tgttctttga cccaagggag ctgaggaagg agacatgcct gctgtacgag 120

atcaagtggg gcacaagcca caagatctgg cgccacagct ccaagaacac cacaaagcac 180

gtggaagtga atttcatcga gaagtttacc tccgagcggc acttctgccc ctctaccagc 240

tgttccatca catggtttct gtcttggagc ccttgcggcg agtgttccaa ggccatcacc 300

gagttcctgt ctcagcaccc taacgtgacc ctggtcatct acgtggcccg gctgtatcac 360

cacatggacc agcagaacag gcagggcctg cgcgatctgg tgaattctgg cgtgaccatc 420

cagatcatga cagccccaga gtacgactat tgctggcgga acttcgtgaa ttatccacct 480

ggcaaggagg cacactggcc aagataccca cccctgtgga tgaagctgta tgcactggag 540

ctgcacgcag gaatcctggg cctgcctcca tgtctgaata tcctgcggag aaagcagccc 600

cagctgacat ttttcaccat tgctctgcag tcttgtcact atcagcggct gcctcctcat 660

attctgtggg ctacaggcct taaa 684

<210> 8

<211> 228

<212> PRT

<213> Artificial sequence

<400> 8

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Ala Cys Leu Leu Tyr Glu Ile Lys Trp Gly Thr Ser His Lys

35 40 45

Ile Trp Arg Asn Ser Gly Lys Asn Thr Thr Lys His Val Glu Val Asn

50 55 60

Phe Ile Glu Lys Phe Thr Ser Glu Arg His Phe Cys Pro Ser Ile Ser

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser

85 90 95

Lys Ala Ile Arg Glu Phe Leu Ser Gln His Pro Asn Val Thr Leu Val

100 105 110

Ile Tyr Val Ala Arg Leu Phe Gln His Met Asp Gln Gln Asn Arg Gln

115 120 125

Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Thr

130 135 140

Ala Ser Glu Tyr Asp His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro

145 150 155 160

Gly Lys Glu Ala His Trp Pro Arg Tyr Pro Pro Leu Trp Met Lys Leu

165 170 175

Tyr Ala Leu Glu Leu His Ala Gly Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 9

<211> 150

<212> PRT

<213> Artificial sequence

<400> 9

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro Glu Phe Phe Asn Arg Asn Tyr Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Thr Tyr Leu Leu Tyr Glu Ile Lys Trp Gly Lys Glu Ser Lys

35 40 45

Ile Trp Arg His Thr Ser Asn Asn Arg Thr Gln His Ala Glu Val Asn

50 55 60

Phe Leu Glu Asn Phe Phe Asn Glu Leu Tyr Phe Asn Pro Ser Thr His

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser

85 90 95

Lys Ala Ile Val Glu Phe Leu Lys Glu His Pro Asn Val Asn Leu Glu

100 105 110

Ile Tyr Val Ala Arg Leu Tyr Leu Cys Glu Asp Glu Arg Asn Arg Gln

115 120 125

Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile Arg Ile Met Asn

130 135 140

Leu Pro Asp Tyr Asn Tyr

145 150

<210> 10

<211> 228

<212> PRT

<213> Artificial sequence

<400> 10

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro Phe Tyr Phe Gln Phe Asn Asn Asp Pro Arg Ala Cys Arg

20 25 30

Arg Lys Thr Tyr Leu Cys Tyr Glu Leu Lys Gln Asp Gly Ser Thr Trp

35 40 45

Val Trp Lys Arg Thr Leu His Asn Lys Gly Arg His Ala Glu Ile Cys

50 55 60

Phe Leu Glu Lys Ile Ser Ser Leu Glu Lys Leu Asp Pro Ala Gln His

65 70 75 80

Tyr Arg Ile Thr Trp Tyr Met Ser Trp Ser Pro Cys Ser Asn Cys Ala

85 90 95

Gln Lys Ile Val Asp Phe Leu Lys Glu His Pro His Val Asn Leu Arg

100 105 110

Ile Tyr Val Ala Arg Leu Tyr Tyr His Glu Glu Glu Arg Tyr Gln Glu

115 120 125

Gly Leu Arg Asn Leu Arg Arg Ser Gly Val Ser Ile Arg Val Met Asp

130 135 140

Leu Pro Asp Phe Glu His Cys Trp Glu Thr Phe Val Asp Asn Gly Gly

145 150 155 160

Gly Pro Phe Gln Pro Trp Pro Gly Leu Glu Glu Leu Asn Ser Lys Gln

165 170 175

Leu Ser Arg Arg Leu Gln Ala Gly Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 11

<211> 228

<212> PRT

<213> Artificial sequence

<400> 11

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro Phe His Phe Gln Phe Asn Asn Asp Pro Arg Ala Tyr Arg

20 25 30

Arg Lys Thr Tyr Leu Cys Tyr Glu Leu Lys Gln Asp Gly Ser Thr Trp

35 40 45

Val Leu Asp Arg Thr Leu Arg Asn Lys Gly Arg His Ala Glu Ile Cys

50 55 60

Phe Leu Asp Lys Ile Asn Ser Trp Glu Arg Leu Asp Pro Ala Gln His

65 70 75 80

Tyr Arg Val Thr Trp Tyr Met Ser Trp Ser Pro Cys Ser Asn Cys Ala

85 90 95

Gln Gln Val Val Asp Phe Leu Lys Glu His Pro His Val Asn Leu Arg

100 105 110

Ile Phe Ala Ala Arg Leu Tyr Tyr His Glu Gln Arg Arg Tyr Gln Glu

115 120 125

Gly Leu Arg Ser Leu Arg Gly Ser Gly Val Pro Val Ala Val Met Thr

130 135 140

Leu Pro Asp Phe Glu His Cys Trp Glu Thr Phe Val Asp His Gly Gly

145 150 155 160

Arg Pro Phe Gln Pro Trp Asp Gly Leu Glu Glu Leu Asn Ser Arg Ser

165 170 175

Leu Ser Arg Arg Leu Gln Ala Gly Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 12

<211> 57

<212> DNA

<213> Artificial sequence

<400> 12

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtc 57

<210> 13

<211> 51

<212> DNA

<213> Artificial sequence

<400> 13

aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt c 51

<210> 14

<211> 66

<212> DNA

<213> Artificial sequence

<400> 14

gcttctccaa agcgtccgcg tgaccgtcac gatggagaat tgggtggacg caaacgtgca 60

agaggt 66

<210> 15

<211> 22

<212> PRT

<213> Artificial sequence

<400> 15

Ala Ser Pro Lys Arg Pro Arg Asp Arg His Asp Gly Glu Leu Gly Gly

1 5 10 15

Arg Lys Arg Ala Arg Gly

20

<210> 16

<211> 579

<212> DNA

<213> Artificial sequence

<400> 16

agcggcggga gcggcgggag cggcgggagc ggggggagca ctaatctgag cgacatcatt 60

gagaaggaga ctgggaaaca gctggtcatt caggagtcca tcctgatgct gcctgaggag 120

gtggaggaag tgatcggcaa caagccagag tctgacatcc tggtgcacac cgcctacgac 180

gagtccacag atgagaatgt gatgctgctg acctctgacg cccccgagta taagccttgg 240

gccctggtca tccaggattc taacggcgag aataagatca agatgctgag cggaggctcc 300

ggaggatctg gaggcagcac caacctgtct gacatcatcg agaaggagac aggcaagcag 360

ctggtcatcc aggagagcat cctgatgctg cccgaagaag tcgaagaagt gatcggaaac 420

aagcctgaga gcgatatcct ggtccatacc gcctacgacg agagtaccga cgaaaatgtg 480

atgctgctga catccgacgc cccagagtat aagccctggg ctctggtcat ccaggattcc 540

aacggagaga acaaaatcaa aatgctgtct ggcggctca 579

<210> 17

<211> 96

<212> DNA

<213> Artificial sequence

<400> 17

tctggagggt cctccggcgg atcgtccggc agcgagacgc caggcacctc cgagagcgct 60

acgcctgaat cctccggggg atcttcagga ggatca 96

<210> 18

<211> 32

<212> PRT

<213> Artificial sequence

<400> 18

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1 5 10 15

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

<210> 19

<211> 1092

<212> DNA

<213> Artificial sequence

<400> 19

tccgaagtcg agttttccca tgagtactgg atgagacacg cattgactct cgcaaagagg 60

gcttgggatg aacgcgaggt gcccgtgggg gcagtactcg tgcataacaa tcgcgtaatc 120

ggcgaaggtt ggaataggcc gatcggacgc cacgacccca ctgcacatgc ggaaatcatg 180

gcccttcgac agggagggct tgtgatgcag aattatcgac ttatcgatgc gacgctgtac 240

gtcacgcttg aaccttgcgt aatgtgcgcg ggagctatga ttcactcccg cattggacga 300

gttgtattcg gtgcccgcga cgccaagacg ggtgccgcag gttcactgat ggacgtgctg 360

catcacccag gcatgaacca ccgggtagaa atcacagaag gcatattggc ggacgaatgt 420

gcggcgctgt tgtccgactt ttttcgcatg cggaggcagg agatcaaggc ccagaaaaaa 480

gcacaatcct ctactgactc tggagggtcc tccggcggat cgtccggcag cgagacgcca 540

ggcacctccg agagcgctac gcctgaatcc tccgggggat cttcaggagg atcatccgaa 600

gtcgagtttt cccatgagta ctggatgaga cacgcattga ctctcgcaaa gagggctcgg 660

gatgaacgcg aggtgcccgt gggggcagta ctcgtgctta acaatcgcgt aatcggcgaa 720

ggttggaata gggcgatcgg actccacgac cccactgcac atgcggaaat catggccctt 780

cgacagggag ggcttgtgat gcagaattat cgacttatcg atgcgacgct gtacgtcacg 840

tttgaacctt gcgtaatgtg cgcgggagct atgattcact cccgcattgg acgagttgta 900

ttcggtgtcc gcaacgccaa gacgggtgcc gcaggttcac tgatggacgt gctgcattac 960

ccaggcatga accaccgggt agaaatcaca gaaggcatat tggcggacga atgtgcggcg 1020

ctgttgtgct acttttttcg catgccgagg caggtgttca atgcccagaa aaaagcacaa 1080

tcctctactg ac 1092

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<400> 20

tactggagtt gtacctgga 19

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<400> 21

ggaacagctt gaacgtcaat 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

gaacagcctt ctcatcatga 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

ggtgaggatt tgggacaatt 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

ctgtgaatct gatgaagttt 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

gaaaagtaat aacaaagggc 20

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence

<400> 26

aaatatccac accttactaa gg 22

<210> 27

<211> 22

<212> DNA

<213> Artificial sequence

<400> 27

aggtcccccg ccggatgatc gg 22

Claims (10)

1. a nucleic acid construct is characterized in that, described nucleic acid construct has the formula I structure of 5 '-3 ' (5 ' to 3 '): P1-S1-L1-S2-S3(I); In the formula, P1, S1, L1, S2, S3 are respectively the elements used to constitute the construct; P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor; S1 and S2 are each independently one or more (a) coding sequences for gene editing enzymes, (b) coding sequences for adenine deaminase and/or coding sequences for cytosine deaminase; L1 is the coding sequence for no or linker peptide; S3 is the coding sequence of no or uracil glucosidase inhibitor UGI; Also, each "-" is independently a bond or a nucleotide linking sequence. 2. nucleic acid construct as claimed in claim 1 is characterized in that, described S1 is the coding sequence of adenine deaminase and/or the coding sequence of cytosine deaminase, and described S2 is the coding sequence of gene editing enzyme. coding sequence. 3. A carrier, wherein the carrier contains the nucleic acid construct of claim 1. 4. A host cell, characterized in that the cell contains the nucleic acid construct of claim 1, or its genome is integrated with one or more of the nucleic acid constructs of claim 1. 5. a reagent combination, is characterized in that, comprises: (i) the first nucleic acid construct, or the first carrier containing the first nucleic acid construct, the first nucleic acid construct having the structure of formula I from 5'-3': P1-S1-L1-S2-S3 (I) in, P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor; S1 and S2 are each independently one or more (a) coding sequences for gene editing enzymes, (b) coding sequences for adenine deaminase and/or coding sequences for cytosine deaminase; L1 is the coding sequence for no or linker peptide; S3 is the coding sequence of no or uracil glucosidase inhibitor UGI; And, "-" is a bond or a nucleotide linking sequence; (ii) the second nucleic acid construct, or the second carrier containing the second nucleic acid construct, the second nucleic acid construct having the structure shown in the formula (II) from 5'-3': P2-Y1 (II); Wherein, P2 is the second promoter; Y1 is the coding sequence of gRNA; Also, "-" is a bond or a nucleotide linking sequence. 6 . A kit, characterized in that, the kit contains the reagent combination of claim 5 . 7 . 7. A method for gene editing of plants, comprising the steps of: (i) provide plants to be edited; and (ii) introducing the nucleic acid construct according to claim 1, the vector according to claim 3 or the reagent combination according to claim 5 into the plant cell of the plant to be edited, thereby performing gene editing in the plant cell . 8. A method for preparing gene-edited plant cells, comprising the steps of: The nucleic acid construct described in claim 1, the carrier described in claim 3 or the reagent combination described in claim 5 are transfected into plant cells, so that the chromosome in the plant cell is replaced (or mutated) at a site, thereby making The gene-edited plant cells are obtained. 9. the purposes of the described nucleic acid construct of claim 1, the described carrier of claim 3, the described host cell of claim 4, the described reagent combination of claim 5, the described test kit of claim 6 , characterized in that it is used for gene editing of plants. 10. A method for preparing a gene-edited plant, comprising the steps of: The gene-edited plant cell prepared by the method of claim 8 is regenerated into a plant body, thereby obtaining the gene-edited plant.

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