CN114317596B - A method for mutating A to G in target sequence of plant genome - Google Patents

Abstract

The invention discloses a method for mutating A in a plant genome target sequence into G. The method comprises the following steps: introducing SpRYn, adenine deaminase and esgRNA into a plant body to realize the mutation of A in a target sequence of a plant genome into G; the esgRNA targets the target sequence; the esgRNA is shown in a formula I: the RNA-esgRNA backbone transcribed by the target sequence (formula I). Experiments prove that: the method can edit the base A in the target sequence of which the PAM sequence is NGC or NGA or NGG on the plant genome, realizes the replacement from the base A to the base G, expands the range of the editable A and improves the base replacement efficiency.

Description

A method for mutating A to G in target sequence of plant genome

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a method for mutating A to G in a plant genome target sequence.

Background technique

CRISPR-Cas9 technology has become a powerful genome editing method and is widely used in many tissues and cells. The CRISPR/Cas9 protein-RNA complex is positioned at the target site through guide RNA, and cuts to produce a DNA double-strand break (dsDNA break, DSB). Then the organism will instinctively start the DNA repair mechanism to repair the DSB. There are generally two types of repair mechanisms, one is non-homologous end joining (NHEJ) and the other is homologous recombination (homology-directed repair (HDR)). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base replacement, the application of using HDR to achieve precise base replacement is greatly limited due to the low efficiency of HDR and the need for DNA templates.

In 2017, David Liu's laboratory reported a new type of adenine base editors (ABE). Through seven rounds of evolution, the researchers fused the tRNA adenine deaminase (ecTadA) derived from Escherichia coli to the 5' end of Cas9 nickase (Cas9n), which can directly target a single base A (Adenine) in the cell. , the replacement of A) to G (Guanine, G), instead of generating DSB and initiating HDR repair, greatly improves the base editing efficiency of replacing A to G. The specific process is: when the sgRNA containing the genome targeting sequence is combined with ecTadA&ecTadA&Cas9n, the complex is positioned at the target site, and ecTadA catalyzes the adenine deamination reaction of A on the unpaired single-stranded DNA into inosine (I). , during the DNA repair process, I will be treated as G, and Cas9n will cut the phosphodiester bond of the paired DNA chain and introduce a cytosine C (Cytosine) to pair with I. Finally, a C-G pairing is generated during the subsequent repair process, thereby realizing the conversion from A to G.

At present, the ABE system has been widely used in plants to achieve A to G conversion, but the editing target is mainly limited to sequences whose PAM (Protospacer Adjacent Motif) is NGG, which greatly limits the range of editable A's. The variant of SpCas9, SpCas9-NG, can recognize NGN (N=A, T, C or G) PAM targets and was successfully developed into ABE (SpCas9-NG-ABE), greatly expanding the range of editable A in animals. , but compared to NGA, NGT and NGG PAM targets, SpCas9-NG-ABE has low editing ability for NGC PAM targets. There are very few target sequences reported in plants edited using SpCas9-NG-ABE.

Contents of the invention

The first object of the present invention is to provide a method for mutating A to G in a plant genome target sequence.

The method provided by the invention for mutating A in a plant genome target sequence to G is as follows 1) or 2):

Described 1) includes the following steps: introducing SpRYn, adenine deaminase and sgRNA into the plant to mutate A to G in the plant genome target sequence;

Described 2) includes the following steps: introducing the coding gene of SpRYn, the coding gene of adenine deaminase and the DNA molecule for transcribing sgRNA into the plant, so that the SpRYn, the adenine deaminase and the sgRNA are all obtained Expression to mutate A to G in the plant genome target sequence;

The sgRNA targeting target sequence;

The PAM sequence of the target sequence is NGC or NGA or NGG; N is A, T, C or G.

In the above method of mutating A in a plant genome target sequence to G, the sgRNA is esgRNA;

The esgRNA is shown in Formula I: the RNA-esgRNA skeleton transcribed by the target sequence (Formula I);

The esgRNA skeleton is n1) or n2) or n3):

n1) The RNA molecule obtained by replacing T in positions 617-702 of sequence 1 with U;

n2) An RNA molecule that has the same function by substituting and/or deleting and/or adding one or several nucleotides to the RNA molecule shown in n1);

n3) RNA molecules that are 75% or more identical to the nucleotide sequence defined by n1) or n2) and have the same function.

In the above-mentioned method of mutating A in a plant genome target sequence to G, the SpRYn is A1) or A2) or A3):

A1) The amino acid sequence is the protein shown in sequence 4;

A2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 4 in the sequence listing;

A3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of A1) or A2).

The adenine deaminase can be adenine deaminase from different sources, such as adenine deaminase derived from Escherichia coli (such as ecTadA, ecTadA* and other proteins), adenine deaminase derived from plants (such as OsTadA from rice, AtTadA from Arabidopsis thaliana and other proteins). In specific embodiments of the invention, the adenine deaminase is ecTadA and ecTadA*.

The ecTadA is C1) or C2) or C3):

C1) The amino acid sequence is the protein shown in sequence 2;

C2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing;

C3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of C1) or C2);

The ecTadA* is E1) or E2) or E3):

E1) The amino acid sequence is the protein shown in sequence 3;

E2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 3 in the sequence listing;

E3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of E1) or E2).

In order to facilitate the purification of the protein in A1), C1) or E1), the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence 4 or sequence 2 or sequence 3 in the sequence listing can be connected as shown in the following table Tag of.

Table, sequence of labels

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in A2), C2) or E2) is a protein that has 75% or more identity with the amino acid sequence of the protein shown in Sequence 4, Sequence 2 or Sequence 3 and has the same function. The said having 75% or more identity means having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% identity. .

The protein in the above A2), C2) or E2) can be synthesized artificially, or its encoding gene can be synthesized first and then biologically expressed.

The gene encoding the protein in A2), C2) or E2) above can be obtained by deleting one or several amino acids from the DNA sequence shown at positions 4132-8232, 2944-3441 or 3538-4035 of sequence 1. The codon of the residue, and/or a missense mutation of one or several base pairs, and/or the coding sequence of the tag shown in the table above is connected to its 5' end and/or 3' end. Positions 4132-8232, 2944-3441 and 3538-4035 of sequence 1 encode the proteins shown in sequence 4, sequence 2 and sequence 3 respectively.

The coding gene of SpRYn is b1) or b2) or b3):

b1) The cDNA molecule or DNA molecule shown at positions 4132-8232 of Sequence 1 in the sequence listing;

b2) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the above-mentioned SpRYn;

b3) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by b1) or b2) under stringent conditions and encodes the above-mentioned SpRYn;

The encoding gene of ecTadA is d1) or d2) or d3):

d1) The cDNA molecule or DNA molecule shown at positions 2944-3441 of Sequence 1 in the sequence listing;

d2) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) and encodes the above-mentioned ecTadA;

d3) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by d1) or d2) under stringent conditions and encodes the above-mentioned ecTadA;

The encoding gene of ecTadA* is f1) or f2) or f3):

f1) The cDNA molecule or DNA molecule shown at positions 3538-4035 of Sequence 1 in the sequence listing;

f2) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined by f1) and encodes the above-mentioned ecTadA*;

f3) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by f1) or f2) under stringent conditions and encodes the above-mentioned ecTadA*.

Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the SpRYn, the ecTadA or the ecTadA* of the present invention. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the SpRYn, the ecTadA or the ecTadA* of the present invention, as long as they encode the SpRYn, the ecTadA or The ecTadA* and having the same function are all derived from and equivalent to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher with the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2, 3 or 4 of the present invention, or a nucleotide sequence with 95% or greater identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The stringent conditions are to hybridize in a solution of 2×SSC and 0.1% SDS at 68°C and wash the membrane twice for 5 minutes each time, and then hybridize in a solution of 0.5×SSC and 0.1% SDS at 68°C. And wash the membrane twice, 15 minutes each time; or, hybridize and wash the membrane in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C.

The above-mentioned 75% or above identity may be 80%, 85%, 90% or 95% or above identity.

In the above method of mutating A to G in the plant genome target sequence, in 2), the coding gene of SpRYn, the DNA molecule for transcribing sgRNA, and the coding gene of adenine deaminase can be passed One or more recombinant expression vectors are introduced into the plant. In a specific embodiment of the present invention, the SpRYn coding gene, the DNA molecule transcribing esgRNA, the ecTadA coding gene and the ecTadA* coding gene are introduced into the plant through a recombinant expression vector.

Furthermore, the recombinant vector also includes a gene encoding a screening agent resistance protein.

Furthermore, the recombinant vector includes an expression cassette containing a DNA molecule for transcribing esgRNA and a sequence containing the coding gene of ecTadA, the coding gene of ecTadA*, the coding gene of SpRYn, and the self-cleaving oligopeptide. and an expression cassette of the gene encoding the screening agent resistance protein.

The number of expression cassettes containing DNA molecules transcribing esgRNA can be one, two or more. Specifically, it can be one or two.

The self-cleaving oligopeptide may be a 2A self-cleaving oligopeptide derived from a viral genome, such as foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Erythropus lucidum beta tetra Thosea asigna virus (T2A) peptide, porcine asigna virus-1 (PTV-1) (P2A) peptide, Theile virus 2A peptide and encephalomyocarditis virus 2A peptide. Specifically, it may be P2A peptide.

The screening agent resistance protein may specifically be hygromycin phosphotransferase.

In specific embodiments of the present invention, the recombinant expression vector is specifically SpRYn-ABE-1 recombinant expression vector, SpRYn-ABE-2 recombinant expression vector, SpRYn-ABE-5 recombinant expression vector, and SpRYn-ABE-6 recombinant expression vector. Vector, SpRYn-ABE-7 recombinant expression vector.

Another object of the present invention is to provide a new use of the above-mentioned method of mutating A to G in a plant genome target sequence.

The present invention provides the application of the above method of mutating A to G in a plant genome target sequence in plant genome base replacement or plant genome base editing.

The present invention also provides the application of the above method of mutating A to G in the plant genome target sequence in improving the plant genome base replacement efficiency or the plant genome base editing efficiency.

The present invention also provides the application of the above method of mutating A to G in the plant genome target sequence in preparing plant mutants.

Another object of the present invention is to provide a new use of a set of reagents; the set of reagents includes the above-mentioned SpRYn, the above-mentioned adenine deaminase and the above-mentioned sgRNA;

The present invention provides the application of a complete set of reagents in any of the following T1)-T11):

T1) Mute A to G in the plant genome target sequence;

T2) Prepare a product in which A in the plant genome target sequence is mutated to G;

T3) Plant genome base replacement;

T4) Prepare plant genome base replacement products;

T5) Plant genome base editing;

T6) Preparation of plant genome base editing products;

T7) Improve the base substitution efficiency of plant genome;

T8) Prepare products that improve the base substitution efficiency of plant genomes;

T9) Improve plant genome base editing efficiency;

T10) Prepare products that improve the efficiency of base editing in plant genomes;

T11) Preparation of plant mutants;

The PAM sequence of the target sequence is NGC or NGA or NGG; N is A, T, C or G.

Further, the set of reagents also includes the above-mentioned self-cleaving oligopeptide and the above-mentioned screening agent resistance protein.

Furthermore, the set of reagents is composed of the above-mentioned SpRYn, the above-mentioned adenine deaminase, the above-mentioned sgRNA, the above-mentioned self-cleaving oligopeptide and the above-mentioned screening agent resistance protein.

In any of the above methods or applications, the PAM sequence is a DNA sequence connected to the 3' end of the target sequence. The first N from the 5' end of the PAM sequence is connected to the 3' end of the target sequence. The size of the target sequence may be 15-25 bp, further may be 18-22 bp, and further may be 20 bp.

Further, the NGC may be AGC or GGC.

The NGA may be TGA.

The NGG may be AGG or GGG or CGG.

In any of the above methods or applications, the target sequence may be one, two or more.

In any of the above methods or applications, the base editing or base replacement is to mutate A to G in the plant genome target sequence. The A can be a base A located at any position in the target sequence.

In any of the above methods or applications, the plant is S1) or S2) or S3):

S1) Monocots or dicots;

S2) Gramineae;

S3) Rice (such as Nipponbare).

The present invention provides a method for mutating A to G in a plant genome target sequence. The method includes the steps of introducing SpRYn, adenine deaminase and esgRNA into the plant. It has been proved through experiments that the method of the present invention can edit base A in the target sequence whose PAM sequence is NGC, NGA or NGG on the plant genome, realize the replacement of base A to base G, and expand the editable While increasing the range of A, it also improves the base substitution efficiency.

Description of the drawings

Figure 1 is a schematic diagram of the structure of each component of the SpRYn-ABE base editing system vector. Among them, n is the number of targets, which can be 1 or 2. OsU6 can be OsU6a or OsU6b. When one target is used, OsU6a is used, and when two targets are used, OsU6a and OsU6b are used respectively.

Figure 2 is a schematic diagram of the structure of each component of the SpCas9n-NG-ABE base editing system vector. Among them, n is the number of targets, which can be 1 or 2. OsU6 can be OsU6a or OsU6b. When one target is used, OsU6a is used, and when two targets are used, OsU6a and OsU6b are used respectively.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5' terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal core of the corresponding DNA/RNA. glycosides.

Primer pair NGC-A1 consists of primer NGC-A1-F: 5’-GGAGCTGGATGAGGTGCT-3’ and primer NGC-A1-R: 5’-GGAAGAAGAAAAGTAGGGAGA-3’, which is used to amplify the target NGC-A1.

Primer pair NGC-A2 consists of primer NGC-A2-F: 5’-AGTAACCCCTCTGGGTAAGT-3’ and primer NGC-A2-R: 5’-GCAAGATATCTGCTTATCTTC-3’, which is used to amplify the target NGC-A2.

Primer pair NGC-A3 consists of primer NGC-A3-F: 5’-TCAATTAGTTGTACCCGGTGA-3’ and primer NGC-A3-R: 5’-CGCCCACCACTGATCGATCG-3’, which is used to amplify the target NGC-A3.

Primer pair NGT-A1 consists of primer NGT-A1-F: 5’-CAGAGTACTGCATTGTTGCACCT-3’ and primer NGT-A1-R: 5’-TAATCAGCCATAAAGATTATCATG-3’, which is used to amplify the target NGT-A1.

Primer pair NGT-A2 consists of primer NGT-A2-F: 5’-GTGTCGCATCACGATTGCGA-3’ and primer NGT-A2-R: 5’-ACAACCAAGTGCAGAGCCT-3’, which is used to amplify the target NGT-A2.

Primer pair NGT-A3 consists of primer NGT-A3-F: 5’-TCAATTAGTTGTACCCGGTGA-3’ and primer NGT-A3-R: 5’-CGCCCACCACTGATCGATCG-3’, which is used to amplify the target NGT-A3.

Primer pair NGT-A4 consists of primer NGT-A4-F: 5’-TCAATTAGTTGTACCCGGTGA-3’ and primer NGT-A4-R: 5’-CGCCCACCACTGATCGATCG-3’, which is used to amplify the target NGT-A4.

Primer pair NGA-A1 consists of primer NGA-A1-F: 5’-CAGAGTACTGCATTGTTGCACCT-3’ and primer NGA-A1-R: 5’-TAATCAGCCATAAAGATTATCATG-3’, which is used to amplify the target NGA-A1.

Primer pair NGG-A1 consists of primer NGG-A1-F: 5’-CAGGGAGTTCATGATTGGAG-3’ and primer NGG-A1-R: 5’-GCGTCGCATGTGATATTTGTCA-3’, which is used to amplify the target NGG-A1.

Primer pair NGG-A2 consists of primer NGG-A2-F: 5’-CGCAGGCATTGCTGGACTTC-3’ and primer NGG-A2-R: 5’-CATTCTTGATCAGCTCGAT-3’, which is used to amplify the target NGG-A2.

Primer pair NGG-A3 consists of primer NGG-A3-F: 5’-ATCCAGAAGAGCATCGCAGGT-3’ and primer NGG-A3-R: 5’-AGCAATTAATTGAGTTCAAG-3’, which is used to amplify the target NGG-A3.

Primer pair NGG-A4 consists of primer NGG-A4-F: 5’-ATGAAGCGCGAGTACCAAGA-3’ and primer NGG-A4-R: 5’-CGGATCCCAGCCTCCTGCGT-3’, and is used to amplify the target NGG-A4.

In the following examples, A·G base substitution refers to the mutation of A to G at any position in the target sequence.

A·G base substitution efficiency = number of positive T0 seedlings with A·G base substitution/total number of positive T0 seedlings analyzed × 100%.

Nipponbare rice: Reference: Liang Weihong, Wang Gaohua, Du Jingyao, et al. Effects of sodium nitroprusside and its photolysis products on Nipponbare rice seedling growth and expression of five hormone marker genes [J]. Journal of Henan Normal University (Nature Edition), 2017 (2):48-52.; Available to the public from the Beijing Academy of Agricultural and Forestry Sciences.

Recovery medium: N6 solid medium containing 200 mg/L timentin.

Screening medium: N6 solid medium containing 50 mg/L hygromycin.

Differentiation medium: N6 solid medium containing 2 mg/L KT, 0.2 mg/L NAA, 0.5 g/L glutamic acid, and 0.5 g/L proline.

Rooting medium: N6 solid medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, and 0.5g/L proline.

Example 1. The SpRYn-ABE base editing system can realize base editing of target sites whose PAM sequence is NGC in the rice genome.

1. Construction of recombinant expression vector

The following recombinant expression vectors were artificially synthesized, SpRYn-ABE-1 recombinant expression vector, SpRYn-ABE-2 recombinant expression vector, SpCas9n-NG-ABE-1 recombinant expression vector and SpCas9n-NG-ABE-2 recombinant expression vector. The schematic structural diagram of each element of the SpRYn-ABE-1 recombinant expression vector and the SpRYn-ABE-2 recombinant expression vector is shown in Figure 1. The schematic structural diagram of each element of the SpCas9n-NG-ABE-1 recombinant expression vector and SpCas9n-NG-ABE-2 recombinant expression vector is shown in Figure 2. Each vector is a circular plasmid, and the specific structural descriptions are as follows:

The sequence of the SpRYn-ABE-1 recombinant expression vector is sequence 1 in the sequence list. Positions 131-596 of sequence 1 are the nucleotide sequence of the OsU6a promoter, positions 597-616 are the nucleotide sequence of the target NGC-A1, positions 617-702 are the nucleotide sequence of the esgRNA backbone, and Positions 703-709 are the PolyT sequence; positions 710-1042 of sequence 1 are the nucleotide sequence of the OsU6b promoter, positions 1043-1062 are the nucleotide sequence of the target NGC-A2, and positions 1063-1148 are the esgRNA The nucleotide sequence of the backbone, positions 1149-1160 are the PolyT sequence; positions 1167-2880 of sequence 1 are the nucleotide sequence of the OsUbq3 promoter, and positions 2944-3441 are the coding sequence of the ecTadA protein (does not contain the stop codon) codon), encoding the ecTadA protein shown in sequence 2; positions 3538-4035 of sequence 1 are the coding sequence of ecTadA* protein (not containing a stop codon), encoding the ecTadA* protein shown in sequence 3; position 4132 of sequence 1 Position -8232 is the coding sequence of SpRYn protein (without start codon and stop codon), encoding the SpRYn protein shown in sequence 4; positions 8389-8445 of sequence 1 are the coding sequence of P2A, positions 8446-9471 It is the coding sequence of hygromycin phosphotransferase, and positions 9811-10063 are the nucleotide sequence of the Nos terminator. The SpRYn-ABE-1 recombinant expression vector contains two target sites, NGC-A1 and NGC-A2. The sequences are shown in Table 1.

The sequence of the SpRYn-ABE-2 recombinant expression vector is the sequence obtained by replacing the 131-1160th sequence of Sequence 1 in the Sequence Listing with Sequence 5 in the Sequence Listing, while keeping other sequences unchanged. Positions 1-466 of sequence 5 are the nucleotide sequence of the OsU6a promoter, positions 467-486 are the nucleotide sequence of the target NGC-A3, positions 487-572 are the nucleotide sequence of the esgRNA backbone, and positions 487-572 are the nucleotide sequence of the esgRNA backbone. Bits 573-584 are PolyT sequences. The NGC-A3 target sequence is shown in Table 1.

The sequence of the SpCas9n-NG-ABE-1 recombinant expression vector is the sequence obtained by replacing the 4132-8232nd sequence of Sequence 1 in the Sequence Listing with Sequence 6 in the Sequence Listing, while keeping other sequences unchanged. Sequence 6 is the coding sequence of SpCas9n-NG protein (does not contain start codon and stop codon).

The sequence of the SpCas9n-NG-ABE-2 recombinant expression vector is to replace the 4132-8232nd sequence of sequence 1 contained in the SpRYn-ABE-2 recombinant expression vector with the sequence 6 in the sequence list, and keep other sequences unchanged. The sequence obtained after the change.

The target nucleotide sequence of esgRNA of each vector and the corresponding PAM sequence are shown in Table 1.

Table 1. Target nucleotide sequence of esgRNA of each vector and corresponding PAM sequence

2. Base editing of target sites in rice plants

Follow the following steps 1- 11. Do the following:

1. Introduce the vector into Agrobacterium EHA105 (product of Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1010) to obtain recombinant Agrobacterium.

2. Use culture medium (YEP medium containing 50 μg/ml kanamycin and 25 μg/ml rifampicin) to culture the recombinant Agrobacterium at 28°C and 150 rpm with shaking until the OD ₆₀₀ is 1.0-2.0, at room temperature, 10000 rpm Centrifuge for 1 minute, resuspend the bacteria in infection solution (replace the sugar in the N6 liquid medium with glucose and sucrose, the concentrations of glucose and sucrose in the infection solution are 10g/L and 20g/L respectively) and dilute to OD ₆₀₀ is 0.2, and the Agrobacterium infection solution is obtained.

3. Peel and thresh the mature seeds of rice variety Nipponbare, place them in a 100 mL Erlenmeyer flask, add 70% (v/v) ethanol aqueous solution and soak for 30 seconds, then place them in 25% (v/v) sodium hypochlorite aqueous solution, and shake and sterilize at 120 rpm for 30 minutes. , rinse 3 times with sterile water, absorb the water with filter paper, then place the seed embryo face down on N6 solid medium, and cultivate it in the dark at 28°C for 4-6 weeks to obtain rice callus.

4. After completing step 3, soak the rice callus in Agrobacterium infection solution A (Agrobacterium infection solution A is a liquid obtained by adding acetosyringone to the Agrobacterium infection solution. The added amount of acetosyringone satisfies Soak in acetosyringone and Agrobacterium infection solution for 10 minutes (the volume ratio of acetosyringone and Agrobacterium infection solution is 25μl:50ml), and then place it on a petri dish covered with two layers of sterilized filter paper (containing about 200ml of infection solution without Agrobacterium) , cultured in the dark at 21°C for 1 day.

5. Place the rice callus obtained in step 4 on the recovery medium and cultivate it in the dark at 25-28°C for 3 days.

6. Take the rice callus obtained in step 5, place it on the screening medium, and cultivate it in the dark at 28°C for 2 weeks.

7. Take the rice callus obtained in step 6, place it on the screening medium again, and cultivate it in the dark at 28°C for 2 weeks to obtain rice resistant callus.

8. Take the rice resistant callus obtained in step 7 and place it on the differentiation medium. Cultivate it in the light at 25°C for about 1 month. Move the differentiated seedlings to the rooting medium and cultivate it in the light at 25°C for 2 weeks to obtain rice T0. Seedling.

9. Extract the genomic DNA of rice T0 seedlings and use it as a template to perform PCR amplification using a primer pair consisting of primer F (5'-ttattgccactagttcattctacttat-3') and primer R (5'-ggggtacttctcgtggtagg-3') to obtain PCR Amplification product; subject the PCR amplification product to agarose gel electrophoresis, and then make the following judgment: if the PCR amplification product contains a DNA fragment of approximately 1854 bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product contains If the amplified product does not contain a DNA fragment of approximately 1854 bp, the corresponding rice T0 seedling is not a positive rice T0 seedling.

10. For each vector, take the genomic DNA of the rice positive T0 seedlings obtained in step 9 as a template. For the NGC-A1 target, use primers to perform PCR amplification of NGC-A1 to obtain a PCR amplification product; for the NGC-A2 target For the NGC-A3 target point, use primers to perform PCR amplification of NGC-A2 to obtain a PCR amplification product; for the NGC-A3 target point, use primers to perform PCR amplification of NGC-A3 to obtain a PCR amplification product.

11. Perform Sanger sequencing and analysis on the PCR amplification product obtained in step 10. Sequencing results are only analyzed for each target region. The number of positive T0 seedlings with A·G base substitution at each target site was counted respectively, and the A·G base substitution efficiency was calculated. The results are shown in Table 2.

The results show that the SpRYn-ABE base editing system realizes A·G base substitution for all three targets, while the SpCas9n-NG-ABE base editing system only realizes the editing of the NGC-A1 target, and the A·G base The replacement efficiency is lower at 9.1%. This shows that for NGC PAM targets, the SpRYn-ABE base editing system is better than the SpCas9n-NG-ABE base editing system and can achieve A·G base replacement in the rice genome.

Table 2. A·G base substitution efficiency

target name ABE system Total number of positive T0 seedlings Number of positive T0 seedlings with A·G base substitution A·G base substitution efficiency (%) NGC-A1 SpRYn-ABE 15 7 46.7 SpCas9n-NG-ABE twenty two 2 9.1 NGC-A2 SpRYn-ABE 18 2 11.1 SpCas9n-NG-ABE 30 0 0 NGC-A3 SpRYn-ABE twenty four 6 25 SpCas9n-NG-ABE 20 0 0

Example 2. The SpRYn-ABE base editing system can realize base editing of target sites in the rice genome whose PAM sequence is NGA or NGG.

1. Construction of recombinant expression vector

The following recombinant expression vectors are artificially synthesized: SpRYn-ABE-3 recombinant expression vector, SpRYn-ABE-4 recombinant expression vector, SpRYn-ABE-5 recombinant expression vector, SpRYn-ABE-6 recombinant expression vector and SpRYn-ABE-7 recombinant expression vector carrier. Each vector is a circular plasmid.

The sequence of the SpRYn-ABE-3 recombinant expression vector is to replace the NGC-A1 target sequence with the NGT-A3 target sequence and the NGC-A2 target sequence with the NGT-A4 target sequence in the SpRYn-ABE-1 recombinant expression vector sequence. sequence, and the sequence obtained by keeping other sequences unchanged. The NGT-A3 target sequence and NGT-A4 target sequence are shown in Table 3.

The sequence of the SpRYn-ABE-4 recombinant expression vector is the sequence obtained by replacing the NGC-A3 target sequence with the NGT-A2 target sequence in the SpRYn-ABE-2 recombinant expression vector sequence, while keeping other sequences unchanged. The NGT-A2 target sequence is shown in Table 3.

The sequence of the SpRYn-ABE-5 recombinant expression vector is to replace the NGC-A1 target sequence with the NGT-A1 target sequence and the NGC-A2 target sequence with the NGA-A1 target sequence in the SpRYn-ABE-1 recombinant expression vector sequence. sequence, and the sequence obtained by keeping other sequences unchanged. The NGT-A1 target sequence and NGA-A1 target sequence are shown in Table 3.

The sequence of the SpRYn-ABE-6 recombinant expression vector is to replace the NGC-A1 target sequence with the NGG-A1 target sequence and the NGC-A2 target sequence with the NGG-A2 target sequence in the SpRYn-ABE-1 recombinant expression vector sequence. sequence, and the sequence obtained by keeping other sequences unchanged. The NGG-A1 target sequence and NGG-A2 target sequence are shown in Table 3.

The sequence of the SpRYn-ABE-7 recombinant expression vector is to replace the NGC-A1 target sequence with the NGG-A3 target sequence and the NGC-A2 target sequence with the NGG-A4 target sequence in the SpRYn-ABE-1 recombinant expression vector sequence. sequence, and the sequence obtained by keeping other sequences unchanged. The NGG-A3 target sequence and NGG-A4 target sequence are shown in Table 3.

The target nucleotide sequence of esgRNA of each vector and the corresponding PAM sequence are shown in Table 3.

Table 3. Target nucleotide sequence of esgRNA of each vector and corresponding PAM sequence

2. Base editing of target sites in rice plants

1. Combine the SpRYn-ABE-3 recombinant expression vector, SpRYn-ABE-4 recombinant expression vector, SpRYn-ABE-5 recombinant expression vector, SpRYn-ABE-6 recombinant expression vector and SpRYn-ABE-7 recombinant expression vector constructed in step 1. Vector, operate according to steps 1-9 of step 2 of Example 1, respectively, to obtain rice positive TO seedlings.

2. Each vector takes the genomic DNA of the rice positive T0 seedlings obtained in step 1 as a template. For the NGT-A1 target, use primers to perform PCR amplification of NGT-A1 to obtain a PCR amplification product; for the NGT-A2 target For the NGT-A3 target point, use primers to perform PCR amplification of NGT-A2 to obtain a PCR amplification product; for the NGT-A3 target point, use primers to perform PCR amplification of NGT-A3 to obtain a PCR amplification product; for the NGT-A4 target point, Use primers to perform PCR amplification of NGT-A4 to obtain PCR amplification products; for the NGA-A1 target, use primers to perform PCR amplification of NGA-A1 to obtain PCR amplification products; for NGG-A1 target, use primers Perform PCR amplification of NGG-A1 to obtain a PCR amplification product; for the NGG-A2 target, use primers to PCR amplify NGG-A2 to obtain a PCR amplification product; for the NGG-A3 target, use primers to NGG -A3 is subjected to PCR amplification to obtain a PCR amplification product; for the NGG-A4 target, primers are used to perform PCR amplification of NGG-A4 to obtain a PCR amplification product.

3. Perform Sanger sequencing and analysis on the PCR amplification product obtained in step 2. Sequencing results are only analyzed for each target region. The number of positive T0 seedlings with A·G base substitution at each target site was counted respectively, and the A·G base substitution efficiency was calculated. The results are shown in Table 4.

The results show that the SpRYn-ABE base editing system cannot base edit target sequences with PAM sequences of NGT in the rice genome, but can effectively edit target sequences with PAM sequences of NGA and NGG, obtaining A·G bases. The base editing efficiency of the replaced T0 seedlings is 4.8%-19.1%. This shows that the SpRYn-ABE base editing system can base edit target sequences with PAM sequences of NGA and NGG in the rice genome to achieve A·G base replacement.

Table 4. Gene editing efficiency analysis results

target name Total number of positive T0 seedlings Number of positive T0 seedlings with A·G base substitution A·G base substitution efficiency (%) NGT-A1 6 0 0 NGT-A2 43 0 0 NGT-A3 twenty four 0 0 NGT-A4 twenty four 0 0 NGA-A1 6 1 16.7 NGG-A1 17 2 11.8 NGG-A2 twenty one 4 19.1 NGG-A3 18 2 11.1 NGG-A4 twenty one 1 4.8

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

sequence list

<110> Beijing Academy of Agriculture and Forestry Sciences

<120> A method of mutating A to G in target sequence of plant genome

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 16469

<212> DNA

<213> Artificial Sequence

<400> 1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt 180

ttaccatccg aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc 240

ccgtaaaaag cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca 300

ggctatcgag atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg 360

tcaggcgaaa tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag 420

ttggccggat aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag 480

cacttcgatt cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc 540

gcttagctag agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccggcac 600

cacggacatc tggagggttt cagagctatg ctggaaacag catagcaagt tgaaataagg 660

ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt gcaagaacga 720

actaagccgg acaaaaaaaa aaggagcaca tatacaaacc ggttttatattc atgaatggtc 780

acgatggatg atggggctca gacttgagct acgaggccgc aggcgagaga agcctagtgt 840

gctctctgct tgtttgggcc gtaacggagg atacggccga cgagcgtgta ctaccgcgcg 900

ggatgccgct gggcgctgcg ggggccgttg gatggggatc ggtgggtcgc gggagcgttg 960

aggggagaca ggtttagtac cacctcgcct accgaacaat gaagaaccca ccttataacc 1020

ccgcgcgctg ccgcttgtgt tgtggggatga aggttccatg ctgtttcaga gctatgctgg 1080

aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 1140

gtcggtgctttttttttttt aagcttacaa attcgggtca aggcggaagc cagcgcgcca 1200

ccccacgtca gcaaatacgg aggcgcgggg ttgacggcgt cacccggtcc taacggcgac 1260

caacaaacca gccagaagaa attacagtaa aaaaaaagta aattgcactt tgatccacct 1320

tttattacct aagtctcaat ttggatcacc cttaaaccta tcttttcaat ttgggccggg 1380

ttgtggtttg gactaccatg aacaactttt cgtcatgtct aacttccctt tcagcaaaca 1440

tatgaaccat atatagagga gatcggccgt atactagagc tgatgtgttt aaggtcgttg 1500

attgcacgag aaaaaaaaat ccaaatcgca acaatagcaa atttatctgg ttcaaagtga 1560

aaagatatgt ttaaaggtag tccaaagtaa aacttataga taataaaatg tggtccaaag 1620

cgtaattcac tcaaaaaaaa tcaacgagac gtgtaccaaa cggagacaaa cggcatcttc 1680

tcgaaatttc ccaaccgctc gctcgcccgc ctcgtcttcc cggaaaccgc ggtggtttca 1740

gcgtggcgga ttctccaagc agacggagac gtcacggcac gggactcctc ccaccaccca 1800

accgccataa ataccagccc cctcatctcc tctcctcgca tcagctccac ccccgaaaaa 1860

tttctcccca atctcgcgag gctctcgtcg tcgaatcgaa tcctctcgcg tcctcaaggt 1920

acgctgcttc tcctctcctc gcttcgtttc gattcgattt cggacgggtg aggttgtttt 1980

gttgctagat ccgattggtg gttagggttg tcgatgtgat tatcgtgaga tgtttagggg 2040

ttgtagatct gatggttgtg atttgggcac ggttggttcg ataggtggaa tcgtggttag 2100

gttttggggat tggatgttgg ttctgatgat tggggggaat ttttacggtt agatgaattg 2160

ttggatgatt cgattgggga aatcggtgta gatctgttgg ggaattgtgg aactagtcat 2220

gcctgagtga ttggtgcgat ttgtagcgtg ttccatcttg taggccttgt tgcgagcatg 2280

ttcagatcta ctgttccgct cttgattgag ttattggtgc catgggttgg tgcaaacaca 2340

ggctttaata tgttatatct gttttgtgtt tgatgtagat ctgtagggta gttcttctta 2400

gacatggttc aattatgtag cttgtgcgtt tcgatttgat ttcatatgtt cacagattag 2460

ataatgatga actcttttaa ttaattgtca atggtaaata ggaagtcttg tcgctatatc 2520

tgtcataatg atctcatgtt actatctgcc agtaatttat gctaagaact atattagaat 2580

atcatgttac aatctgtagt aatatcatgt tacaatctgt agttcatcta tataatctat 2640

tgtggtaatt tctttttat atctgtgtga agattattgc cactagttca ttctacttat 2700

ttctgaagtt caggatacgt gtgctgttac tacctatctg aatacatgtg tgatgtgcct 2760

gttactatct ttttgaatac atgtatgttc tgttggaata tgtttgctgt ttgatccgtt 2820

gttgtgtcct taatcttgtg ctagttctta ccctatctgt ttggtgatta tttcttgcag 2880

tacgtaagca tgaagaggac cgccgacggc agcgagttcg agccgaagaa gaagaggaag 2940

gtgtccgagg tggagttctc ccacgagtac tggatgaggc acgcactcac cctcgcaaag 3000

agggcatggg acgagaggga ggtgcctgtg ggagcagtgc tcgtgcacaa caacagggtg 3060

atcggagagg gatggaacag gcctatcgga aggcacgacc ctaccgcaca cgcagagatc 3120

atggcactca ggcagggagg cctcgtgatg cagaactaca ggctcatcga cgccaccctc 3180

tacgtgaccc tcgagccttg cgtgatgtgc gcaggagcca tgatccactc caggatcgga 3240

agggtggtgt tcggagcaag ggacgcaaag accggagcag ccggctccct catggacgtg 3300

ctccaccacc cgggcatgaa ccacagggtg gagatcaccg agggaatcct cgcagacgag 3360

tgcgcagccc tcctctccga cttcttcagg atgaggaggc aggagatcaa ggcccagaag 3420

aaggcccagt cctccaccga ctccggcggc tcatcaggcg gctcctccgg ctccgagaca 3480

ccgggcacct ccgagtccgc caccccggag tcctccggcg gctcctccgg cggctcctcc 3540

gaggtggagt tctcccacga gtactggatg aggcacgcac tcaccctcgc aaagagggca 3600

agggacgaga gggaggtgcc tgtgggagca gtgctcgtgc tcaacaacag ggtgatcgga 3660

gagggatgga acagggcaat cggcctccac gaccctaccg cacacgcaga gatcatggca 3720

ctcaggcagg gaggcctcgt gatgcagaac tacaggctca tcgacgccac cctctacgtg 3780

accttcgagc cttgcgtgat gtgcgcagga gccatgatcc actccaggat cggcagggtg 3840

gtgttcggcg tgaggaacgc aaagaccgga gcagcaggct ccctcatgga cgtgctccac 3900

tacccgggca tgaaccacag ggtggagatc accgagggaa tcctcgcaga cgagtgcgca 3960

gccctcctct gctacttctt caggatgccg aggcaggtgt tcaacgccca gaagaaggcc 4020

cagtcctcca ccgactccgg cggctcatca ggcggctcct ccggctccga gacaccgggc 4080

acctccgagt ccgccaccccc ggagtcctcc ggcggctcct ccggcggctc cgacaagaag 4140

tactccatcg gcctcgccat cggcaccaac agcgtcggct gggcggtgat caccgacgag 4200

tacaaggtcc cgtccaagaa gttcaaggtc ctgggcaaca ccgaccgcca ctccatcaag 4260

aagaacctca tcggcgcct cctcttcgac tccggcgaga cggcggagcg cacccgcctc 4320

aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctccaggag 4380

atcttctcca acgagatggc gaaggtcgac gactccttct tccaccgcct cgaggagtcc 4440

ttcctcgtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtcgac 4500

gaggtcgcct accacgagaa gtaccccact atctaccacc ttcgtaagaa gcttgttgac 4560

tctactgata aggctgatct tcgtctcatc taccttgctc tcgctcacat gatcaagttc 4620

cgtggtcact tccttatcga gggtgacctt aaccctgata actccgacgt ggacaagctc 4680

ttcatccagc tcgtccagac ctacaaccag ctcttcgagg agaaccctat caacgcttcc 4740

ggtgtcgacg ctaaggcgat cctttccgct aggctctcca agtccaggcg tctcgagaac 4800

ctcatcgccc agctccctgg tgagaagaag aacggtcttt tcggtaacct catcgctctc 4860

tccctcggtc tgacccctaa cttcaagtcc aacttcgacc tcgctgagga cgctaagctt 4920

cagctctcca aggataccta cgacgatgat ctcgacaacc tcctcgctca gattggagat 4980

cagtacgctg atctcttcct tgctgctaag aacctctccg atgctatcct cctttcggat 5040

atccttaggg ttaacactga gatcactaag gctcctcttt ctgcttccat gatcaagcgc 5100

tacgacgagc accaccagga cctcaccctc ctcaaggctc ttgttcgtca gcagctcccc 5160

gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ttacattgac 5220

ggtggagcta gccagggagga gttctacaag ttcatcaagc caatccttga gaagatggat 5280

ggtactgagg agcttctcgt taagcttaac cgtgaggacc tccttaggaa gcagaggact 5340

ttcgataacg gctctatccc tcaccagatc caccttggtg agcttcacgc catccttcgt 5400

aggcaggagg acttctaccc tttcctcaag gacaaccgtg agaagatcga gaagatcctt 5460

actttccgta ttccttacta cgttggtcct cttgctcgtg gtaactcccg tttcgcttgg 5520

atgactagga agtccgagga gactatcacc ccttggaact tcgaggaggt tgttgacaag 5580

ggtgcttccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa cctccccaac 5640

gagaaggtcc tccccaagca ctccctcctc tacgagtact tcacggtcta caacgagctc 5700

accaaggtca agtacgtcac cgagggtatg cgcaagcctg ccttcctctc cggcgagcag 5760

aagaaggcta tcgttgacct cctcttcaag accaaccgca aggtcaccgt caagcagctc 5820

aaggaggact acttcaagaa gatcgagtgc ttcgactccg tcgagatcag cggcgttgag 5880

gaccgtttca acgcttctct cggtacctac cacgatctcc tcaagatcat caaggacaag 5940

gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtcct cactcttact 6000

ctcttcgagg atagggagat gatcgaggag aggctcaaga cttacgctca tctcttcgat 6060

gacaaggtta tgaagcagct caagcgtcgc cgttacaccg gttggggtag gctctcccgc 6120

aagctcatca acggtatcag ggataagcag agcggcaaga ctatcctcga cttcctcaag 6180

tctgatggtt tcgctaacag gaacttcatg cagctcatcc acgatgactc tcttaccttc 6240

aaggaggata ttcagaaggc tcaggtgtcc ggtcagggcg actctctcca cgagcacatt 6300

gctaaccttg ctggttcccc tgctatcaag aagggcatcc ttcagactgt taaggttgtc 6360

gatgagcttg tcaaggttat gggtcgtcac aagcctgaga acatcgtcat cgagatggct 6420

cgtgagaacc agactaccca gaagggtcag aagaactcga gggagcgcat gaagaggatt 6480

gaggagggta tcaaggagct tggttctcag atccttaagg agcaccctgt cgagaacacc 6540

cagctccaga acgagaagct ctacctctac tacctccaga acggtaggga tatgtacgtt 6600

gaccaggagc tcgacatcaa caggctttct gactacgacg tcgaccacat tgttcctcag 6660

tctttcctta aggatgactc catcgacaac aaggtcctca cgaggtccga caagaacagg 6720

ggtaagtcgg acaacgtccc ttccgaggag gttgtcaaga agatgaagaa ctactggagg 6780

cagcttctca acgctaagct cattacccag aggaagttcg acaacctcac gaaggctgag 6840

aggggtggcc tttccgagct tgacaaggct ggtttcatca agaggcagct tgttgagacg 6900

aggcagatta ccaagcacgt tgctcagatc ctcgattcta ggatgaacac caagtacgac 6960

gagaacgaca agctcatccg cgaggtcaag gtgatcaccc tcaagtccaa gctcgtctcc 7020

gacttccgca aggacttcca gttctacaag gtccgcgaga tcaacaacta ccaccacgct 7080

cacgatgctt accttaacgc tgtcgttggt accgctctta tcaagaagta ccctaagctt 7140

gagtccgagt tcgtctacgg tgactacaag gtctacgacg ttcgtaagat gatcgccaag 7200

tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa catcatgaac 7260

ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc tctttatcgag 7320

acgaacggtg agactggtga gatcgtttgg gacaagggtc gcgacttcgc tactgttcgc 7380

aaggtccttt ctatgcctca ggttaacatc gtcaagaaga ccgaggtcca gaccggtggc 7440

ttctccaagg agtctatccg cccaaagaga aactcggaca agctcatcgc taggaagaag 7500

gattgggacc ctaagaagta cggtggtttc ctgtggccta ctgtcgccta ctccgtcctc 7560

gtggtcgcca aggtggagaa gggtaagtcg aagaagctca agtccgtcaa ggagctcctc 7620

ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt cctcgaggcc 7680

aagggctaca aggaggtcaa gaaggacctc atcatcaagc tccccaagta ctctcttttc 7740

gagctcgaga acggtcgtaa gaggatgctg gcttccgcta agcagctcca gaagggtaac 7800

gagcttgctc ttccttccaa gtacgtgaac ttcctctacc tcgcctccca ctacgagaag 7860

ctcaagggtt cccctgagga taacgagcag aagcagctct tcgtggagca gcacaagcac 7920

tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtcat cctcgctgac 7980

gctaacctcg acaaggtcct ctccgcctac aacaagcacc gcgacaagcc catccgcgag 8040

caggccgaga acatcatcca cctcttcacg ctcacgcgcc tcggcgcccc tcgcgctttc 8100

aagtacttcg acaccaccat cgaccccaag cagtaccgct ccaccaagga ggttctcgac 8160

gctactctca tccaccagtc catcaccggt ctttacgaga ctcgtatcga cctttcccag 8220

cttggtggtg atgacgatga caaaatggca ccgaagaaaa aaaggaaggt cggcggctcc 8280

ccgaagaaaa aaaggaaggt cggcggctcc ccgaagaaaa aaaggaaggt cggcggctcc 8340

ccgaagaaaa aaaggaaggt cggaatccat ggcgttccag gatcaggagc caccaacttc 8400

tccctcctca agcaggccgg cgacgtggag gagaacccgg gcccaatgaa aaagcctgaa 8460

ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg 8520

atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga 8580

tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg 8640

cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga gtttagcgag 8700

agcctgacct attgcatctc ccgccgttca cagggtgtca cgttgcaaga cctgcctgaa 8760

accgaactgc ccgctgttct acaaccggtc gcggaggcta tggatgcgat cgctgcggcc 8820

gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact 8880

acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg 8940

atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc 9000

gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg 9060

acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc 9120

caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag 9180

acgcgctact tcgagcggag gcatccggag cttgcaggat cgccacgact ccgggcgtat 9240

atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat 9300

gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg 9360

cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc 9420

gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaagaaata gactagttca 9480

gccagtttgg tggagctgcc gatgtgcctg gtcgtcccga gcctctgttc gtcaagtatt 9540

tgtggtgctg atgtctactt gtgtctggtt taatggacca tcgagtccgt atgatatgtt 9600

agttttatga aacagtttcc tgtggggacag cagtatgctt tatgaataag ttggatttga 9660

acctaaatat gtgctcaatt tgctcatttg catctcattc ctgttgatgt tttatctgag 9720

ttgcaagttt gaaaatgctg catattctta ttaaatcgtc atttactttt atcttaatga 9780

gctttgcaat ggcctatggg atataaaaga gatcgttcaa acatttggca ataaagtttc 9840

ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac 9900

gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg 9960

attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac 10020

taggataaat tatcgcgcgc ggtgtcatct atgttactag atccctgcag gacgcgttta 10080

attaagtgca cgcggccgcc tacttagtca agagcctcgc acgcgactgt cacgcggcca 10140

ggatcgcctc gtgagcctcg caatctgtac ctagtgttta aactatcagt gtttgacagg 10200

atatattggc gggtaaacct aagagaaaag agcgtttatt agaataacgg atatttaaaa 10260

gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc atgccaacca cagggttccc 10320

ctcgggatca aagtactttg atccaacccc tccgctgcta tagtgcagtc ggcttctgac 10380

gttcagtgca gccgtcttct gaaaacgaca tgtcgcacaa gtcctaagtt acgcgacagg 10440

ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg ttttagtcgc ataaagtaga 10500

atacttgcga ctagaaccgg agacattacg ccatgaacaa gagcgccgcc gctggcctgc 10560

tgggctatgc ccgcgtcagc accgacgacc aggacttgac caaccaacgg gccgaactgc 10620

acgcggccgg ctgcaccaag ctgttttccg agaagatcac cggcaccagg cgcgaccgcc 10680

cggagctggc caggatgctt gaccacctac gccctggcga cgttgtgaca gtgaccaggc 10740

tagaccgcct ggcccgcagc acccgcgacc tactggacat tgccgagcgc atccaggagg 10800

ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga caccacccacg ccggccggcc 10860

gcatggtgtt gaccgtgttc gccggcattg ccgagttcga gcgttcccta atcatcgacc 10920

gcacccggag cgggcgcgag gccgccaagg cccgaggcgt gaagtttggc ccccgcccta 10980

ccctcacccc ggcacagatc gcgcacgccc gcgagctgat cgaccaggaa ggccgcaccg 11040

tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac cctgtaccgc gcacttgagc 11100

gcagcgagga agtgacgccc accgaggcca ggcggcgcgg tgccttccgt gaggacgcat 11160

tgaccgaggc cgacgccctg gcggccgccg agaatgaacg ccaagaggaa caagcatgaa 11220

accgcaccag gacggccagg acgaaccgtttttcattacc gaagagatcg aggcggagat 11280

gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc tcaaccgtgc ggctgcatga 11340

aatcctggcc ggtttgtctg atgccaagct ggcggcctgg ccggccagct tggccgctga 11400

agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt gagtaaaaca gcttgcgtca 11460

tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa atacgcaagg ggaacgcatg 11520

aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca agacgaccat cgcaacccat 11580

ctagcccgcg ccctgcaact cgccggggcc gatgttctgt tagtcgattc cgatccccag 11640

ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac cgctaaccgt tgtcggcatc 11700

gaccgcccga cgattgaccg cgacgtgaag gccatcggcc ggcgcgactt cgtagtgatc 11760

gacggagcgc cccaggcggc ggacttggct gtgtccgcga tcaaggcagc cgacttcgtg 11820

ctgattccgg tgcagccaag cccttacgac atatgggcca ccgccgacct ggtggagctg 11880

gttaagcagc gcattgaggt cacggatgga aggctacaag cggcctttgt cgtgtcgcgg 11940

gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg cgctggccgg gtacgagctg 12000

cccattcttg agtcccgtat cacgcagcgc gtgagctacc caggcactgc cgccgccggc 12060

acaaccgttc ttgaatcaga acccgagggc gacgctgccc gcgaggtcca ggcgctggcc 12120

gctgaaatta aatcaaaact catttgagtt aatgaggtaa agagaaaatg agcaaaagca 12180

caaacacgct aagtgccggc cgtccgagcg cacgcagcag caaggctgca acgttggcca 12240

gcctggcaga cacgccagcc atgaagcggg tcaactttca gttgccggcg gaggatcaca 12300

ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat taccgagctg ctatctgaat 12360

acatcgcgca gctaccagag taaatgagca aatgaataaa tgagtagatg aattttagcg 12420

gctaaaggag gcggcatgga aaatcaagaa caaccaggca ccgacgccgt ggaatgcccc 12480

atgtgtggag gaacgggcgg ttggccaggc gtaagcggct gggttgtctg ccggccctgc 12540

aatggcactg gaacccccaa gcccgaggaa tcggcgtgac ggtcgcaaac catccggccc 12600

ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag aagttgaagg ccgcgcaggc 12660

cgccccagcgg caacgcatcg aggcagaagc acgccccggt gaatcgtggc aagcggccgc 12720

tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa 12780

gccgcccaag ggcgacgagc aaccagattt tttcgttccg atgctctatg acgtgggcac 12840

ccgcgatagt cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg 12900

agctggcgag gtgatccgct acgagcttcc agacgggcac gtagaggttt ccgcagggcc 12960

ggccggcatg gccagtgtgt gggattacga cctggtactg atggcggttt cccatctaac 13020

cgaatccatg aaccgatacc gggaagggaa gggagacaag cccggccgcg tgttccgtcc 13080

acacgttgcg gacgtactca agttctgccg gcgagccgat ggcggaaagc agaaagacga 13140

cctggtagaa acctgcattc ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa 13200

ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta gccgctacaa 13260

gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag ctgattggat 13320

gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc ccgattactt 13380

tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa 13440

ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg ccggagagtt 13500

caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc cggagtacga 13560

tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc gcaacctgat 13620

cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc aaattgccct 13680

agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca ttgggaaccc 13740

aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt acattgggaa 13800

ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa 13860

ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca 13920

gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc 13980

cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg gcctacggcc 14040

aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc ggcgcccaca 14100

tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 14160

tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 14220

gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata 14280

gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca 14340

tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 14400

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 14460

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 14520

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 14580

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 14640

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 14700

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 14760

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 14820

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 14880

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 14940

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 15000

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 15060

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 15120

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 15180

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 15240

gcattctagg tactaaaaca attcatccag taaaatataa tattttattt tctcccaatc 15300

aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc cgatatcctc 15360

cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc cgcttctccc 15420

aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt ctcccaggtc 15480

gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat catacagctc 15540

gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat cggccagatc 15600

gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg tatagggaca 15660

atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct cgataatctt 15720

ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg cctcactcat 15780

gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa caggcagctt 15840

tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg tccctttata 15900

ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct tatatacctt 15960

agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca gttttttcaa 16020

ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct acagtattta 16080

aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt ccttgcattc 16140

taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt ggcgtataac 16200

atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac gctctgtcat 16260

cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc ggcagcttag 16320

ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt acaacggctc 16380

tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat tttgtgccga 16440

gctgccggtc ggggagctgt tggctggct 16469

<210> 2

<211> 166

<212> PRT

<213> Artificial Sequence

<400> 2

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

165

<210> 3

<211> 166

<212> PRT

<213> Artificial Sequence

<400> 3

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 Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val

20 25 30

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

35 40 45

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

85 90 95

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

100 105 110

Ala Gly Ser Leu Met Asp Val Leu His Tyr 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

Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys

145 150 155 160

Ala Gln Ser Ser Thr Asp

165

<210> 4

<211> 1368

<212> PRT

<213> Artificial Sequence

<400> 4

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

1 5 10 15

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 Arg 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

210 215 220

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

245 250 255

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

1010 1015 1020

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

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

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

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala Pro Arg Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr Arg Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 5

<211> 584

<212> DNA

<213> Artificial Sequence

<400> 5

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaaga attgccacga 480

gaggaggttt cagagctatg ctggaaacag catagcaagt tgaaataagg ctagtccgtt 540

atcaacttga aaaagtggca ccgagtcggt gctttttttt tttt 584

<210> 6

<211> 4101

<212> DNA

<213> Artificial Sequence

<400> 6

gacaagaagt actccatcgg cctcgccatc ggcaccaaca gcgtcggctg ggcggtgatc 60

accgacgagt acaaggtccc gtccaagaag ttcaaggtcc tgggcaacac cgaccgccac 120

tccatcaaga agaacctcat cggcgccctc ctcttcgact ccggcgagac ggcggaggcg 180

acccgcctca agcgcaccgc ccgccgccgc tacacccgcc gcaagaaccg catctgctac 240

ctccaggaga tcttctccaa cgagatggcg aaggtcgacg actccttctt ccaccgcctc 300

gaggagtcct tcctcgtgga ggaggacaag aagcacgagc gccaccccat cttcggcaac 360

atcgtcgacg aggtcgccta ccacgagaag taccccacta tctaccacct tcgtaagaag 420

cttgttgact ctactgataa ggctgatctt cgtctcatct accttgctct cgctcacatg 480

atcaagttcc gtggtcactt ccttatcgag ggtgacctta accctgataa ctccgacgtg 540

gacaagctct tcatccagct cgtccagacc tacaaccagc tcttcgagga gaaccctatc 600

aacgcttccg gtgtcgacgc taaggcgatc ctttccgcta ggctctccaa gtccaggcgt 660

ctcgagaacc tcatcgccca gctccctggt gagaagaaga acggtctttt cggtaacctc 720

atcgctctct ccctcggtct gacccctaac ttcaagtcca acttcgacct cgctgaggac 780

gctaagcttc agctctccaa ggatacctac gacgatgatc tcgacaacct cctcgctcag 840

attggagatc agtacgctga tctcttcctt gctgctaaga acctctccga tgctatcctc 900

ctttcggata tccttagggt taacactgag atcactaagg ctcctctttc tgcttccatg 960

atcaagcgct acgacgagca ccaccaggac ctcaccctcc tcaaggctct tgttcgtcag 1020

cagctccccg agaagtacaa ggagatcttc ttcgaccagt ccaagaacgg ctacgccggt 1080

tacattgacg gtggagctag ccaggaggag ttctacaagt tcatcaagcc aatccttgag 1140

aagatggatg gtactgagga gcttctcgtt aagcttaacc gtgaggacct ccttaggaag 1200

cagaggactt tcgataacgg ctctatccct caccagatcc accttggtga gcttcacgcc 1260

atccttcgta ggcaggagga cttctaccct ttcctcaagg acaaccgtga gaagatcgag 1320

aagatcctta ctttccgtat tccttactac gttggtcctc ttgctcgtgg taactcccgt 1380

ttcgcttgga tgactaggaa gtccgaggag actatcaccc cttggaactt cgaggaggtt 1440

gttgacaagg gtgcttccgc ccagtccttc atcgagcgca tgaccaactt cgacaagaac 1500

ctccccaacg agaaggtcct ccccaagcac tccctcctct acgagtactt cacggtctac 1560

aacgagctca ccaaggtcaa gtacgtcacc gagggtatgc gcaagcctgc cttcctctcc 1620

ggcgagcaga agaaggctat cgttgacctc ctcttcaaga ccaaccgcaa ggtcaccgtc 1680

aagcagctca aggaggacta cttcaagaag atcgagtgct tcgactccgt cgagatcagc 1740

ggcgttgagg accgtttcaa cgcttctctc ggtacctacc acgatctcct caagatcatc 1800

aaggacaagg acttcctcga caacgaggag aacgaggaca tcctcgagga catcgtcctc 1860

actcttactc tcttcgagga tagggagatg atcgaggaga ggctcaagac ttacgctcat 1920

ctcttcgatg acaaggttat gaagcagctc aagcgtcgcc gttacaccgg ttggggtagg 1980

ctctcccgca agctcatcaa cggtatcagg gataagcaga gcggcaagac tatcctcgac 2040

ttcctcaagt ctgatggttt cgctaacagg aacttcatgc agctcatcca cgatgactct 2100

cttaccttca aggaggatat tcagaaggct caggtgtccg gtcagggcga ctctctccac 2160

gagcacattg ctaaccttgc tggttcccct gctatcaaga agggcatcct tcagactgtt 2220

aaggttgtcg atgagcttgt caaggttatg ggtcgtcaca agcctgagaa catcgtcatc 2280

gagatggctc gtgagaacca gactacccag aagggtcaga agaactcgag ggagcgcatg 2340

aagaggattg aggagggtat caaggagctt ggttctcaga tccttaagga gcaccctgtc 2400

gagaacaccc agctccagaa cgagaagctc tacctctact acctccagaa cggtagggat 2460

atgtacgttg accaggagct cgacatcaac aggctttctg actacgacgt cgaccacatt 2520

gttcctcagt ctttccttaa ggatgactcc atcgacaaca aggtcctcac gaggtccgac 2580

aagaacaggg gtaagtcgga caacgtccct tccgaggagg ttgtcaagaa gatgaagaac 2640

tactggaggc agcttctcaa cgctaagctc attacccaga ggaagttcga caacctcacg 2700

aaggctgaga ggggtggcctttccgagctt gacaaggctg gtttcatcaa gaggcagctt 2760

gttgagacga ggcagattac caagcacgtt gctcagatcc tcgattctag gatgaacacc 2820

aagtacgacg agaacgacaa gctcatccgc gaggtcaagg tgatcaccct caagtccaag 2880

ctcgtctccg acttccgcaa ggacttccag ttctacaagg tccgcgagat caacaactac 2940

caccacgctc acgatgctta ccttaacgct gtcgttggta ccgctcttat caagaagtac 3000

cctaagcttg agtccgagtt cgtctacggt gactacaagg tctacgacgt tcgtaagatg 3060

atcgccaagt ccgagcagga gatcggcaag gccaccgcca agtacttctt ctactccaac 3120

atcatgaact tcttcaagac cgagatcacc ctcgccaacg gcgagatccg caagcgccct 3180

cttatcgaga cgaacggtga gactggtgag atcgtttggg acaagggtcg cgacttcgct 3240

actgttcgca aggtcctttc tatgcctcag gttaacatcg tcaagaagac cgaggtccag 3300

accggtggct tctccaagga gtctatccgc ccaaagagaa actcggacaa gctcatcgct 3360

aggaagaagg attgggaccc taagaagtac ggtggtttcg tgtcccctac tgtcgcctac 3420

tccgtcctcg tggtcgccaa ggtggagaag ggtaagtcga agaagctcaa gtccgtcaag 3480

gagctcctcg gcatcaccat catggagcgc tcctccttcg agaagaaccc gatcgacttc 3540

ctcgaggcca agggctacaa ggaggtcaag aaggacctca tcatcaagct ccccaagtac 3600

tctcttttcg agctcgagaa cggtcgtaag aggatgctgg cttccgctcg cttcctccag 3660

aagggtaacg agcttgctct tccttccaag tacgtgaact tcctctacct cgcctcccac 3720

tacgagaagc tcaagggttc ccctgaggat aacgagcaga agcagctctt cgtggagcag 3780

cacaagcact acctcgacga gatcatcgag cagatctccg agttctccaa gcgcgtcatc 3840

ctcgctgacg ctaacctcga caaggtcctc tccgcctaca acaagcaccg cgacaagccc 3900

atccgcgagc aggccgagaa catcatccac ctcttcacgc tcacgaacct cggcgcccct 3960

cgcgctttca agtacttcga caccaccatc gacaggaagg tgtaccgctc caccaaggag 4020

gttctcgacg ctactctcat ccaccagtcc atcaccggtc tttacgagac tcgtatcgac 4080

ctttcccagc ttggtggtga t 4101

Claims (8)

1. A method of mutating A to G in a plant genome target sequence, as follows 1) or 2): Described 1) includes the following steps: introducing SpRYn, adenine deaminase and sgRNA into plants to mutate A to G in the plant genome target sequence; Described 2) includes the following steps: introducing the coding gene of SpRYn, the coding gene of adenine deaminase and the DNA molecule for transcribing sgRNA into the plant, so that the SpRYn, the adenine deaminase and the sgRNA are all obtained Expression to mutate A to G in the plant genome target sequence; The SpRYn is a protein whose amino acid sequence is shown in sequence 4; The adenine deaminase is ecTadA and ecTadA*; The ecTadA is a protein whose amino acid sequence is shown in sequence 2; The ecTadA* is a protein whose amino acid sequence is shown in sequence 3; The sgRNA is esgRNA; the esgRNA is as shown in Formula I: the RNA-esgRNA skeleton (Formula I) transcribed by the target sequence; the esgRNA skeleton is to replace T in positions 617-702 of Sequence 1 with U The resulting RNA molecule; The sgRNA targeting target sequence; The PAM sequence of the target sequence is NGC or NGA; N is A, T, C or G; The plant is rice. 2. The method according to claim 1, characterized in that: the coding gene of SpRYn is the DNA molecule shown at positions 4132-8232 of Sequence 1. 3. The method according to claim 1, characterized in that: the encoding gene of ecTadA is the DNA molecule shown at positions 2944-3441 of Sequence 1. 4. The method according to claim 1, characterized in that: the encoding gene of ecTadA* is the DNA molecule shown at positions 3538-4035 of Sequence 1. 5. Application of the method of any one of claims 1 to 4 in base editing of plant genomes; the plant is rice. 6. Application of the method of any one of claims 1 to 4 in improving the base editing efficiency of plant genomes; the plant is rice. 7. Application of the method of any one of claims 1 to 4 in preparing plant mutants; the plant is rice. 8. Application of complete set of reagents in any of the following T1)-T7): T1) Mute A to G in the plant genome target sequence; T2) Prepare products that mutate A to G in the plant genome target sequence; T3) Plant genome base editing; T4) Preparation of plant genome base editing products; T5) Improve plant genome base editing efficiency; T6) Prepare products that improve the efficiency of base editing in plant genomes; T7) Preparation of plant mutants; The set of reagents includes the SpRYn described in any one of claims 1-4, the adenine deaminase described in any one of claims 1-4, and the sgRNA described in any one of claims 1-4; The PAM sequence of the target sequence is NGC or NGA or NGG; N is A, T, C or G; The plant is rice.