CN110951742B - Method for realizing plant gene replacement without generating DNA double-strand break - Google Patents

Abstract

The invention provides a method for realizing gene replacement without generating DNA double-strand break. The method comprises the following steps: introducing the sgRNA, the Cas9 nickase and the donor DNA into a target plant; a sgRNA target DNA fragment A target sequence; the donor DNA sequentially comprises a DNA fragment A target sequence, a DNA fragment B and a DNA fragment A target sequence; the DNA fragment B is a DNA molecule obtained by mutating the DNA fragment A by one or more bases; under the guidance of sgRNA, the Cas9 nicking enzyme generates single-stranded DNA nicks at the target site sequence of the DNA fragment A in the target plant genome and the target site sequence of the DNA fragment A in the donor DNA, and replaces the DNA fragment A in the target plant genome with the DNA fragment B through a repair mechanism in a target plant body, so that the plant gene replacement is realized.

Description

Method for realizing plant gene replacement without generating DNA double-strand break

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for realizing plant gene replacement without generating DNA double-strand break.

Background

The probability of precise replacement of genes mediated by long-chain DNA templates in cells is very low, but the introduction of a DNA double strand break (DSDNA break, DSB) near the site to be replaced can significantly increase the probability of replacement. The CRISPR-Cas9 technology has become a powerful genome editing means and is widely applied to many tissues and cells. The CRISPR/Cas9 protein-RNA complex is positioned on a target point through a guide RNA (guide RNA), and the DNA generates DSB through cutting, so that the accurate replacement efficiency of the gene mediated by a long-chain DNA template is increased. After the DSB is produced, the organism will instigate DNA repair mechanisms. There are two general repair mechanisms, one is non-homologous end joining (NHEJ), the repair is mostly performed, and random indels (insertions or deletions) are generally generated after DNA repair. The other is homologous-directed repair (HDR), which uses sister chromatids or exogenous DNA donors (donors) as repair templates to achieve precise repair of genes. In animal cells, the specific principle of repair is: ctIP enzyme starts end-to-end cleavage at DSB, generating a protruding 3' -end single-stranded DNA (ssDNA) tail, the ssDNA is recognized by recombinase Rad51, combined into a complex, invaded into a donor DNA template, and annealed with homologous fragments thereof to synthesize a new DNA strand by using the donor DNA as the template, thereby completing repair. When the sequence between the homologous arms of the donor DNA carries an exogenous mutation, this mutation will be introduced into the DNA strand during repair, thus allowing a precise site-directed substitution. The HDR repair starts from the generation of DSB, and as the repair probability of NHEJ is far greater than that of HDR, in a sample subjected to precise replacement, more byproducts such as indels are introduced to cause large-fragment deletion, displacement and the like of DNA.

To increase the ratio of exact HDR to non-exact HDR in the product, one attempts to use an inactive mutant D10A of Cas9, resulting in a single-strand Nick (Nick) on the DNA. In animals, single-stranded nick-initiated HDR had fewer by-products than DSBs, but at the same time somewhat reduced the efficiency of HDR. In plants, DSB-induced HDR enabled precise replacement on different genes, but Cas 9D 10A-induced nicks enabled precise replacement of HDR, whether their efficiency was lower than DSB-induced HDR, and whether byproducts were reduced, were not reported.

Disclosure of Invention

The invention aims to provide a method for realizing plant gene replacement without generating DNA double-strand break.

The method for realizing plant gene replacement without generating DNA double-strand break provided by the invention comprises the following steps: introducing sgRNA, cas9 nickase (Cas 9 n) or a variant thereof, donor DNA into a plant of interest;

the sgRNA target DNA fragment A target sequence;

the donor DNA sequentially comprises the DNA fragment A target sequence, the DNA fragment B and the DNA fragment A target sequence;

the DNA fragment B is a DNA molecule obtained by mutating the DNA fragment A by one or more bases;

under the guidance of the sgRNA, the Cas9 nickase or the variant thereof generates single-stranded DNA nicks at a DNA fragment A target sequence in a target plant genome and a DNA fragment A target sequence in the donor DNA, and replaces the DNA fragment A in the target plant genome with the DNA fragment B through a repair mechanism in a target plant body, so that plant gene replacement is realized.

In the above method, the DNA fragment a may be any fragment on the genome of the target plant, and the DNA fragment b is obtained by mutating one or several bases of the DNA fragment a, and the DNA fragment b is a fragment on the donor DNA. The base mutation may be a base substitution and/or a base insertion and/or a base deletion.

In practical application, after a sgRNA/Cas9n system and a DNA fragment B with target point sequences corresponding to target points added at two ends, namely donor DNA, are introduced into a target plant, the DNA fragment A in a target plant genome can be replaced by the DNA fragment B, and further gene replacement is realized. The gene substitution can introduce the gene mutation site into the target plant genome, so as to realize the gene mutation (such as base substitution, base insertion or base deletion) on the target plant genome, thereby changing the amino acid functional site and/or the type and/or the activity and/or the content of the corresponding protein expressed in the target plant, and obtaining the plant mutant with a certain function or character. In a specific embodiment of the present invention, the base mutation may specifically be a base substitution.

Furthermore, the sizes of the DNA fragment A and the DNA fragment B can be 200-2000bp or 200-1500bp or 200-1000bp.

Furthermore, the sizes of the DNA fragment A and the DNA fragment B are 694bp.

The DNA fragment A is a DNA molecule shown in 1300 th-1993 th sites of a sequence 5.

The DNA fragment B is a DNA molecule shown in 10695-11388 th site of the sequence 1.

In a specific embodiment of the invention, the DNA fragment A consists of an ALS gene fragment with the size of 636bp and a fragment with the downstream size of 58bp in sequence. And the DNA fragment B is a DNA molecule obtained by mutating the 344 th site of the DNA fragment A from a base G to a base T, mutating the 581 st site from a base G to a base T, mutating the 336 nd site from a base G to a base C, mutating the 339 nd site from a base G to a base C, mutating the 342 nd site from a base A to a base G, and mutating the 396 th site from a base G to a base C. After the DNA fragment A in the rice genome is replaced by the DNA fragment B, the DNA fragment A in the rice genome is mutated from a base G to a base T at the 344 th position and from the base G to the base T at the 581 th position, the 548 th amino acid of an amino acid sequence of the ALS protein expressed in rice is mutated from tyrosine (Try) to leucine (Leu), and the 627 th amino acid is mutated from serine (Ser) to isoleucine (Ile), so that a precise editing plant with herbicide resistance is generated.

In the above method, the sgRNA structure is as follows: tRNA-RNA-sgRNA backbone transcribed from the DNA fragment A target sequence;

the tRNA is a 1) or a 2) or a 3):

a1 An RNA molecule obtained by replacing T at positions 474 to 550 of the sequence 1 with U;

a2 RNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides into the RNA molecules shown in a 1) and have the same functions;

a3 An RNA molecule with 75 percent or more than 75 percent of identity with the nucleotide sequence defined by a 1) or a 2) and with the same function;

the sgRNA backbone is b 1) or b 2) or b 3):

b1 RNA molecule obtained by replacing T in 571-646 th site of the sequence 1 with U;

b2 RNA molecules shown in b 1) are subjected to substitution and/or deletion and/or addition of one or more nucleotides and have the same functions;

b3 RNA molecules with 75% or more than 75% identity and the same function with the nucleotide sequences defined by b 1) or b 2).

In the method, the donor DNA sequentially consists of the DNA fragment A target sequence, the PAM sequence, the DNA fragment B, the DNA fragment A target sequence and the PAM sequence. The DNA fragment A target sequence is specifically an ST215 target sequence; the ST215 target sequence is 551-570 th site of the sequence 1. The donor DNA is particularly 10672-11411 th site of the sequence 1.

In the above method, the Cas9 nickase may be a Cas 9D 10A nickase or a Cas 9H 840A nickase;

the Cas9 nickase variants include Cas9 derived from bacteria (such as SaCas9, saCas9-KKH and the like), cas9 variants recognizing different PAMs (such as xCas9, cas9-NG, cas9-VQR, cas9-VRER and the like), cas9 high fidelity enzyme variants (such as HypaCas9, eSPcas9 (1.1), cas9-HF1 and the like) and the like.

Further, the Cas 9D 10A nickase is SpCas9n protein;

the SpCas9n protein is C1) or C2):

c1 ) the amino acid sequence is the protein shown in the sequence 3;

c2 Protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table;

further, the encoding gene of the SpCas9n protein is c 1) or c 2) or c 3):

c1 A cDNA molecule or DNA molecule shown in 2877-6977 site of a sequence 1 in a sequence table;

c2 A cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in c 1) and encoding said SpCas9 n;

c3 A cDNA or DNA molecule hybridizing under stringent conditions with a nucleotide sequence defined in c 1) or c 2) and encoding said SpCas9 n.

In the above method, the Cas9 nickase or the variant thereof carries a nuclear localization signal. The nuclear localization signal may be BP NLS, virD2 NLS, or SV40 NLS. The number of nuclear localization signals may be 1 or 2 or more.

Further, the nuclear localization signal is SV40 NLS. The amino acid sequence of the SV40 NLS is a sequence 2. The number of the nuclear localization signals is 8.

Furthermore, the SV40 NLS has a coding sequence of 2742-2762 bits of the sequence 1. The Cas9 nickase or variant thereof carries 4 SV40 NLS at each end.

In the above method, the method for introducing the sgRNA, the Cas9 nickase or the variant thereof, and the donor DNA into the plant of interest comprises the steps of: introducing a DNA molecule that transcribes the sgRNA, a gene encoding the Cas9 nickase or a variant thereof, and a donor DNA into a plant of interest.

In the above method, the sgRNA is tRNA-sgRNA, and the tRNA-sgRNA obtained by transcribing the DNA molecule of the tRNA-sgRNA is an immature RNA precursor, and the tRNA in the RNA precursor is cleaved by two enzymes (RNase P and RNaseZ) to obtain mature RNA. And obtaining independent mature RNAs according to the number of targets in a recombinant expression vector, wherein each mature RNA consists of RNA transcribed by the target sequence and the sgRNA framework in sequence or consists of RNA transcribed by the target sequence, the sgRNA framework and residual individual bases of tRNA in sequence. In a specific embodiment of the invention, the recombinant expression vector contains a target.

Further, the sgRNA-transcribed DNA molecule, the gene encoding the Cas9 nickase or a variant thereof, and the donor DNA are introduced into the plant of interest via a recombinant expression vector. The sgRNA-transcribed DNA molecule, the gene encoding the Cas9 nickase or a variant thereof, and the donor DNA can be introduced into a target plant through the same recombinant expression vector, or can be introduced into a target plant through two or more recombinant expression vectors.

In a specific embodiment of the invention, the sgRNA-transcribed DNA molecule, the gene encoding the Cas9 nickase or a variant thereof and the donor DNA are introduced into the plant of interest via the same recombinant expression vector. The recombinant expression vector comprises an expression cassette consisting of a promoter, the DNA molecule for transcribing the sgRNA and a terminator in sequence, and an expression cassette consisting of a promoter, the coding gene of the Cas9 nickase or the variant thereof and a terminator in sequence.

Furthermore, the nucleotide sequence of the recombinant expression vector is shown as a sequence 1.

The application of the method in plant gene editing or plant mutant preparation or plant gene replacement efficiency improvement or by-product reduction in plant gene replacement also belongs to the protection scope of the invention.

The invention finally provides a method I or a method II or a method III or a method IV:

the first method is a method for editing plant genes; the method for plant gene editing comprises the following steps: and replacing the target gene segment in the plant genome according to the method so as to realize plant gene editing. The editing may specifically be a base substitution.

The second method is a method for preparing the plant mutant; the method for preparing the plant mutant comprises the following steps: and replacing the target gene segment in the plant genome according to the method to obtain the plant mutant. The plant mutant may specifically be a herbicide-resistant mutant.

The third method is a method for improving the gene replacement efficiency of the plant; the method for improving the gene replacement efficiency of the plant comprises the following steps: the target gene fragment in the plant genome is replaced according to the method. The replacement efficiency may specifically be an HDR replacement efficiency.

The fourth method is a method for reducing byproducts generated by plant gene replacement; the method for reducing the byproducts generated by the gene replacement of the plant comprises the following steps: the target gene fragment in the plant genome is replaced according to the method. The reduction of the by-products generated by the plant gene replacement is embodied in that no additional Indels are generated from the product obtained by replacing the target gene segment in the plant genome according to the method.

In the above method or use, the plant is any one of the following d 1) -d 3):

d1 A monocot or dicot;

d2 Gramineous plants;

d3 Rice (e.g., nipponbare).

The principle of the method for realizing gene replacement without generating DNA double-strand break provided by the invention is as follows: the Cas 9D 10A nickase initiates single-stranded DNA nicking at the genomic ALS target site under the guidance of the sgRNA; donor DNA on the vector contains a mutation site with herbicide resistance, and the 5 'end and the 3' end of the donor DNA respectively contain target sequences corresponding to 1 target point, and under the guidance of a Cas 9D 10A/sgRNA complex, a nicking site is respectively generated; precise replacement of donor DNA with genomic fragments is achieved under repair mechanisms in plants, resulting in precisely edited plants with herbicide resistance.

The invention has the following advantages:

1. the efficiency is high: the single strand Nick (Nick) induced HDR accurate replacement efficiency was 2-3 times higher than that induced by DSB.

2. And (3) few by-products: single-stranded Nick (Nick) -initiated HDR exact replacement plants do not contain random Indels, while DSB-initiated HDR exact replacement contains a large number of Indels.

3. The cost is low: the method for realizing gene replacement without generating DNA double-strand break only needs that the carrier contains Cas9n related elements and corresponding donor DNA, and can realize accurate replacement by an agrobacterium infection method.

The invention provides a method for realizing gene replacement without generating DNA double-strand break. Experiments prove that: the method for realizing gene replacement without generating DNA double-strand break realizes the accurate replacement of the endogenous acetolactate synthase ALS gene in rice, and obtains an accurate editing plant with herbicide resistance.

Drawings

FIG. 1 is a schematic diagram of the construction of an exact replacement vector.

FIG. 2 is a schematic diagram of the exact alternate working principle of Nick induced ALS.

FIG. 3 is a schematic diagram of the precise replacement of ALS by DSB.

FIG. 4 shows the result of amplification detection of specific primers of plants resulting from precise replacement of ALS by Nick-initiated ALS.

FIG. 5 shows DSB-initiated ALS precise replacement resulting in plants, specific primer amplification test results.

Fig. 6 shows an exact alternative embodiment of the Nick and DSB.

FIG. 7 is a schematic diagram of the precise replacement vector construction.

Fig. 8 shows sgRNA/Cas9n conventional vector transgenic plants, and specific primer amplification detection results.

FIG. 9 shows the results of amplification detection of specific primers of esgRNA/Cas9n-P2A-Hpt optimized vector transgenic plants.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples, the 1 st position of each nucleotide sequence in the sequence Listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA, unless otherwise specified.

The primer pair P1 consists of a primer HDR-F:5 'gcgccccgattctcttatgtc-3' and primer HDR-R:5 'acotatcctcacactggacg-3' for detecting whether the plant is accurately replaced.

Primer pair P2 consists of primer gALS-F:5 'atccccaggttacaaccacctg-3' and primer gALS-R:5 'and cacttaactcagagctattgcatag-3' for amplifying genome ALS sequence and sequencing.

HDR seedlings refer to T in obtention ₀ And in the seedling, plants containing the corresponding mutation sites brought by the donor are included.

Based on T ₀ HDR replacement efficiency of seedlings = T ₀ Number of seedlings in which precise replacement occurred/T obtained ₀ And (4) the total number of seedlings.

HDR replacement efficiency = T based on number of calli ₀ The number of shoots with precise placement/total number of calli from initial infestation occurred in the shoot.

Indels efficiency = exact in HDRAlternative T ₀ T with occurrence of indels in seedlings/precise replacement of seedlings ₀ And (4) the total number of seedlings.

Japanese fine rice: reference documents: the effects of sodium nitroprusside and its photolysis products on the growth of Nippon rice seedlings and the expression of 5 hormone marker genes [ J ]. Proceedings of university of south Henan (Nature edition), 2017 (2): 48-52.; the public is available from the agroforestry academy of sciences of Beijing.

And (3) recovering the culture medium: n6 solid medium containing 200mg/L timentin.

Screening medium 1: n6 solid medium containing 50mg/L hygromycin.

Screening medium 2: n6 solid medium containing 0.4uM/L bispyribac-sodium.

Differentiation medium: n6 solid culture medium containing 2mg/L KT, 0.2mg/L NAA, 0.5g/L glutamic acid and 0.5g/L proline.

Rooting culture medium: n6 solid culture medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline and 0.28uM/L bispyribac-sodium.

In the following embodiments, the amino acid sequence of ALS protein is shown as sequence 6 in the sequence table, and the coding gene sequence is shown as 1 st to 1935 th site of sequence 5 in the sequence table.

Example 1 construction of vector for realizing Gene replacement without generating DNA double-stranded breaks and application thereof in Rice Gene replacement

1. Description of the principles of construction and replacement of recombinant expression vectors

1. Construction of recombinant expression vectors

The following recombinant expression vectors were artificially synthesized, each of which was a circular plasmid: an sgRNA/Cas9 recombinant expression vector and an sgRNA/Cas9n recombinant expression vector. The sgRNA/Cas9 recombinant expression vector and the sgRNA/Cas9n recombinant expression vector are shown in a structural schematic diagram in FIG. 1. The specific structural descriptions are as follows:

the sequence of the sgRNA/Cas9n recombinant expression vector is sequence 1 in the sequence table. The 131 th to 467 th sites of the sequence 1 are OsU3 promoter sequences, the 474 th to 550 th sites are tRNA sequences, the 551 th to 570 th sites are ST215 target site sequences, the 571 th to 646 th sites are sgRNA framework sequences, and the 647 th to 937 th sites are OsU3 terminator sequences. The 944-2657 th site of the sequence 1 is an OsUbq3 promoter sequence, the 2742-2762 th site, the 2772-2792 th site, the 2802-2822 th site, the 2832-2852 th site, the 6996-7016 th site, the 7026-7046 th site, the 7056-7076 th site and the 7086-7106 th site are coding sequences of a nuclear localization signal SV40 (a nuclear localization signal SV40 shown in a coding sequence 2), and the 2877-6977 th site is a coding sequence of a SpCas9n protein (a SpCas9n protein shown in a coding sequence 3); the 7122-7376 bits of the sequence 1 are Nos terminator sequence; the 7405-9397 th site of the sequence 1 is ZmUbi1 promoter sequence, the 9404-10429 th site is hygromycin phosphotransferase coding sequence, and the 10456-10671 th site is CaMV35S terminator sequence. The 10672-10694 th site and the 11389-11411 th site are ST215 target site target sequences (consisting of an ST215 target site sequence and a PAM sequence); ALS donor DNA sequence is at positions 10695-11388.

The sequence of the sgRNA/Cas9 recombinant expression vector is obtained by replacing the 2877-6977 th site in the sequence 1 with the SpCas9 protein coding sequence shown in the sequence 4 and keeping other sequences unchanged.

2. Precise substitution principle of recombinant expression vector

1) Precise replacement principle of sgRNA/Cas9n recombinant expression vector

The sgRNA/Cas9n recombinant expression vector is based on a precise substitution by single-strand Nick (Nick) guide, and the schematic diagram is shown in fig. 2.

The sgRNA/Cas9n recombinant expression vector comprises the following elements: sgRNA, cas9n, donor DNA.

The sgRNA targets the ST215 target sequence.

Donor DNA (donor DNA): the donor DNA consists of, in order, an ST215 target sequence, an ALS donor DNA sequence, and an ST215 target sequence.

The ALS donor DNA sequence (10695-11388 th site of the sequence 1) is a DNA molecule obtained by mutating a DNA fragment A (the DNA fragment A is a 694bp fragment shown in the sequence 5, 1300-1993, and sequentially consists of a 636bp ALS gene fragment and a 58bp downstream fragment thereof, wherein the DNA fragment A is a fragment on a rice genome). Mutations include functional site mutations and synonymous site mutations.

Functional site mutation: the DNA fragment A was mutated from base G to base T at position 344 (where the base mutation resulted in the mutation of tyrosine (Try) to leucine (Leu) at position 548 in the amino acid sequence of ALS protein in rice) and from base G to base T at position 581 (where the base mutation resulted in the mutation of serine (Ser) to isoleucine (Ile) at position 627 in the amino acid sequence of ALS protein in rice). Tyrosine (Try) at 548 th position of an ALS protein amino acid sequence in rice is mutated into leucine (Leu), serine (Ser) at 627 th position is mutated into isoleucine (Ile) and then can resist double-herb ether herbicide, the 548 th position of the ALS protein amino acid sequence in rice is marked as a W548L functional mutation site, and the 627 th position of the ALS protein amino acid sequence in rice is marked as an S627I functional mutation site.

Mutation at the synonymous site: in order to facilitate later-stage design of specific detection primers to detect the accurate substitution mutant, the 336 th site of the DNA fragment A is mutated from a base G to a base C (the base mutation corresponds to the 545 th site of the ALS protein amino acid sequence of rice), the 339 th site is mutated from a base G to a base C (the base mutation corresponds to the 546 th site of the ALS protein amino acid sequence of rice), the 342 th site is mutated from a base A to a base G (the base mutation corresponds to the 547 th site of the ALS protein amino acid sequence of rice), and the 396 th site is mutated from a base G to a base C (the base mutation corresponds to the 565 th site of the ALS protein amino acid sequence of rice). The synonymous site mutation does not change the amino acid corresponding to the corresponding amino acid site on the rice ALS protein amino acid sequence, the 545 th site of the rice ALS protein amino acid sequence is marked as the 545 th synonymous mutation site, the 546 th site of the rice ALS protein amino acid sequence is marked as the 546 synonymous mutation site, the 547 th site of the rice ALS protein amino acid sequence is marked as the 547 synonymous mutation site, and the 565 th site of the rice ALS protein amino acid sequence is marked as the P565 synonymous mutation site.

Under the guidance of ST215 target, the Cas9n/sgRNA complex generates two single-chain Nick sites on a target sequence on a donor in a carrier, and simultaneously generates one single-chain Nick site on a target sequence on a rice genome DNA fragment A, and the scheme is recorded as triNicks (three nicks). Under the in vivo repair mechanism of rice, donor DNA is precisely replaced at a nicking site on a rice genome (a DNA fragment A on the rice genome is replaced by ALS donor DNA), so that a mutation site on the ALS donor DNA is introduced into the rice genome to obtain a plant after gene replacement, and the plant after gene replacement has herbicide resistance.

2) Precise replacement principle of sgRNA/Cas9 recombinant expression vector

The sgRNA/Cas9 recombinant expression vector is based on DSB-guided precise replacement, the schematic diagram is shown in fig. 3.

The sgRNA/Cas9 recombinant expression vector comprises the following elements: sgRNA, cas9, donor DNA.

sgRNA targets ST215 target sequences.

Donor DNA (donor DNA): the donor DNA consists of an ST215 target sequence, an ALS donor DNA sequence, and an ST215 target sequence, in that order.

The Cas9/sgRNA complex, guided by ST215 target, generates two DSB sites in the vector for the target sequence on the donor, and one DSB site in the rice genome, this protocol is denoted as triDSBs (three DSBs). Under the in vivo repair mechanism of rice, donor DNA is precisely replaced at a DSB site on a rice genome (a DNA fragment A on the rice genome is replaced by ALS donor DNA), so that a mutation site on the ALS donor DNA is introduced into the rice genome to obtain a plant after gene replacement, and the plant after gene replacement has herbicide resistance.

2. Obtaining of Rice Positive resistant callus

And (3) operating the sgRNA/Cas9n and sgRNA/Cas9 recombinant expression vectors obtained in the step one according to the following steps 1-7 respectively:

1. the vector was introduced into Agrobacterium EHA105 (product of Shanghai Diego Biotechnology Ltd., CAT #: AC 1010) to obtain a recombinant Agrobacterium.

2. Culturing the recombinant Agrobacterium with a medium (YEP medium containing 50. Mu.g/ml kanamycin and 25. Mu.g/ml rifampicin), shaking at 28 ℃ and 150rpm to OD ₆₀₀ At room temperature, centrifuging at 10000rpm for 1min, resuspending the thallus with infection solution (glucose and sucrose are substituted for N6 liquid culture medium, and the concentration of glucose and sucrose in the infection solution is 10g/L and 20g/L respectively) and diluting to OD ₆₀₀ And the concentration is 0.2, and an agrobacterium infection solution is obtained.

3. The mature seeds of the rice variety Nipponbare are shelled and threshed, placed in a 100mL triangular flask, added with 70% (v/v) ethanol water solution for soaking for 30sec, then placed in 25% (v/v) sodium hypochlorite water solution, sterilized by shaking at 120rpm for 30min, washed by sterile water for 3 times, sucked by filter paper to remove water, then placed on an N6 solid culture medium with the embryo downwards, and cultured in dark at 28 ℃ for 4-6 weeks to obtain the rice callus.

4. After the step 3 is completed, the rice callus is soaked in an agrobacterium infection solution A (the agrobacterium infection solution A is a liquid obtained by adding acetosyringone into an agrobacterium infection solution), the addition amount of the acetosyringone meets the volume ratio of the acetosyringone to the agrobacterium infection solution of 25 mul: 50 ml), and the rice callus is soaked for 10min, and then the rice callus is placed on a culture dish (containing about 200ml of the infection solution without agrobacterium) paved with two layers of sterilizing filter paper and is cultured in the dark for 1 day at the temperature of 21 ℃.

5. And (4) putting the rice callus obtained in the step (4) on a recovery culture medium, and performing dark culture at 25-28 ℃ for 3 days.

6. And (4) placing the rice callus obtained in the step (5) on a screening culture medium 1, and performing dark culture at 28 ℃ for 2 weeks.

7. And (4) transferring the rice callus obtained in the step (6) to a screening culture medium 2, and performing dark culture at 28 ℃ for 4 weeks to obtain the rice resistance callus.

3. Obtaining of T0 seedlings of rice

1. And (2) putting the rice resistant callus obtained in the step (1) on a differentiation culture medium, and culturing for about 1 month at 25 ℃ under illumination.

2. Transplanting the differentiated plantlets to a rooting culture medium, and performing illumination culture at 25 ℃ for 2 weeks to obtain herbicide-resistant rice T0 plantlets.

4. Accurate replacement plant identification

1. After screening by a rooting medium containing herbicide, respectively extracting genomic DNA of surviving rice T0 seedlings, taking the genomic DNA as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HDR-F (5 'gcgcgccgattctcttatgc-3') and a primer HDR-R (5 'acctatcctcacaggcaactgacg-3'), so as to obtain a PCR amplification product; the PCR amplification product was subjected to agarose gel electrophoresis, followed by judgment as follows: if the PCR amplification product contains DNA fragment of about 833bp, the corresponding rice T0 seedling is a positive T0 seedling which is accurately replaced; if the PCR amplification product does not contain the DNA fragment of about 833bp, the corresponding rice T0 seedling is the T0 seedling which is not accurately replaced.

2. PCR amplification is carried out on the genome ALS gene sequence of the positive T0 seedling accurately replaced and selected in the step 1 by using a primer pair consisting of a primer gALS-F (5-; and carrying out first-generation sequencing on the PCR amplification product, and analyzing whether the corresponding site is accurately replaced.

5. Analysis of results

1. Accurate replacement plant obtained by preliminary screening of primers in rice T0 seedling

32 transgenic positive seedlings (independent transformation events) are obtained by the triNicks scheme, and after the 32 seedlings are screened by a rooting medium containing herbicide, the 32 seedlings are all survived. After the screening of the HDR-F and HDR-R primers, 5 transgenic seedlings are accurate replacement plants, and the PCR detection result is shown in FIG. 4.

the triDSBs protocol yielded 23 transgenic positive shoots (independent transformation events) in total, and only 9 shoots survived after screening in rooting medium containing herbicide. After the screening of the HDR-F and HDR-R primers, 2 transgenic seedlings are accurate replacement plants, and the PCR detection result is shown in FIG. 5.

2. Accurate replacement plant confirmed by sequencing in rice T0 seedling and corresponding efficiency

The first generation sequencing results show: the plants with positive PCR detection result are accurately replaced on the corresponding sites. Thus, the probability of exact replacement in T0 shoots by the triNicks and the triDSBs regimens was 15.6% (5/32), 8.7% (2/23), respectively. From the point of view of overall transformation efficiency, the probability of precise replacement of triNicks and triDSBs protocols was 0.6% (5/840), 0.2% (2/840), respectively, calculated from the initial infected 840 pieces of resistant callus as the denominator.

Taken together, the precision replacement efficiency of the triNicks protocol in T0 shoots was 1.8 times that of the triDSBs, and 3 times that of the triNicks protocol if based on the initially infested calli. Demonstrating that the exact replacement efficiency of the Nick-induced triNicks protocol is significantly higher than the DSB-induced triDSBs protocol.

In addition, PCR amplification products of the samples with precise replacement are connected with pEASY-B (Beijing holotype gold organism), each sample is picked to select 4-6 positive clones for sequencing, the specific form of replacement is confirmed, and the specific sequencing result is shown in figure 6. the 5T 0 seedlings in the triNicks protocol were all heterozygous substitution mutants in the genome, causing no additional Indels production in the genome, while the 2T 0 seedlings in the triDSBs protocol all produced an insertion of one base G at the genomic ST215 target site. Indicating that the Nick-induced triNicks protocol produces fewer by-products on the genome than the DSB-induced triDSBs protocol.

Further analysis of the samples with precise substitutions in the triNicks protocol revealed that the substitution patterns at W548, P565 and S627 were not identical for the different samples, with substitutions occurring at three sites simultaneously (mutant pattern 1), at W548 and P565 (mutant pattern 2), at W548 and S627 (mutant pattern 3) and at W548 and S627 (mutant pattern 4). The efficiency for each mutant form is shown in table 1.

TABLE 1

Example 2 optimization of sgRNA/Cas9n recombinant expression vector and application thereof in rice gene replacement

1. Description of the principles of construction and replacement of recombinant expression vectors

1. Construction of recombinant expression vectors

The following recombinant expression vectors were artificially synthesized, each of which was a circular plasmid: sgRNA/Cas9n recombinant expression vector and esgRNA/Cas9n-P2A-Hpt recombinant expression vector. The sgRNA/Cas9n recombinant expression vector and the esgRNA/Cas9n-P2A-Hpt recombinant expression vector are shown in a structural schematic diagram in FIG. 7. The specific structural descriptions are as follows:

the sequence of the sgRNA/Cas9n recombinant expression vector is sequence 1 in the sequence table.

The sequence of the esgRNA/Cas9n-P2A-Hpt recombinant expression vector is obtained by replacing position 571-646 in the sequence 1 with an esgRNA framework sequence shown in a sequence 7, and replacing position 7107-10671 in the sequence 1 with a P2A-Hpt-tNos sequence shown in a sequence 8, and keeping other sequences unchanged. The 22 nd to 78 th positions of the sequence 8 are a coding sequence of a self-cutting oligopeptide P2A (the self-cutting oligopeptide P2A shown as a coding sequence 9), the 79 th to 1104 th positions of the sequence are coding sequences of hygromycin phosphotransferase (the hygromycin phosphotransferase shown as a coding sequence 10), and the 1111 nd to 1365 th positions of the sequence are Nos terminator sequences.

2. Explanation of the exact replacement principle for recombinant expression vectors

1) Precise replacement principle of sgRNA/Cas9n recombinant expression vector

The same precise replacement principle of sgRNA/Cas9n recombinant expression vector in example 1.

2) Precise replacement principle of esgRNA/Cas9n-P2A-Hpt recombinant expression vector

The precise replacement principle of the sgRNA/Cas9n-P2A-Hpt recombinant expression vector is the same as that of the sgRNA/Cas9n recombinant expression vector, only the sgRNA skeleton in the sgRNA/Cas9n recombinant expression vector is replaced by an sgRNA skeleton sequence, and an independent Cas9n coding gene expression cassette and an independent hygromycin phosphotransferase gene expression cassette in the sgRNA/Cas9n recombinant expression vector are replaced by an expression cassette co-expressed by Cas9n and hygromycin phosphotransferase connected by self-cleavage oligopeptide P2A.

2. Obtaining of Rice Positive resistant callus

And (3) respectively operating the sgRNA/Cas9n and the esgRNA/Cas9n-P2A-Hpt vectors obtained in the first step according to the method in the second step of the embodiment 1.

3. Obtaining of T0 seedling of rice

The same procedure as in step three of example 1.

4. Accurate replacement plant identification

The same procedure as in step four of example 1.

5. Analysis of results

1. Accurate replacement plant obtained by preliminary screening of primers in rice T0 seedling

32 transgenic positive seedlings (independent transformation events) are obtained by the sgRNA/Cas9n common vector, and the 32 seedlings survive after being screened by a rooting culture medium containing herbicide. After the screening of the HDR-F and HDR-R primers, 5 transgenic seedlings are accurate replacement plants, and the PCR detection result is shown in FIG. 8.

The esgRNA/Cas9n-P2A-Hpt optimized vector obtains 55 transgenic positive seedlings (independent transformation events) in total, and 24 seedlings survive after screening by a rooting culture medium containing a herbicide. After the screening of the HDR-F and HDR-R primers, 12 transgenic seedlings are accurate replacement plants, and the PCR detection result is shown in FIG. 9.

2. Accurate replacement plant confirmed by sequencing in rice T0 seedling and corresponding efficiency

The results of the first generation sequencing show: plants with positive PCR detection results are accurately replaced on corresponding sites, and no additional Indels bases are generated. Therefore, the probabilities of precise replacement of sgRNA/Cas9n ordinary vector and esgRNA/Cas9n-P2A-Hpt optimized vector in T0 vaccine are 15.6% (5/32) and 21.8% (12/55), respectively. From the point of view of overall transformation efficiency, the probabilities of precise replacement of sgRNA/Cas9n ordinary vector and esgRNA/Cas9n-P2A-Hpt optimized vector are 0.6% (5/840) and 1.4% (12/840), respectively, calculated by taking 840 resistant calli infected at the beginning as denominators.

In conclusion, the accurate replacement efficiency of the esgRNA/Cas9n-P2A-Hpt optimized vector in the T0 seedling is 1.4 times that of the sgRNA/Cas9n common vector, and if the initial infected callus is taken as the basis, the accurate replacement efficiency of the esgRNA/Cas9n-P2A-Hpt optimized vector is 2.3 times that of the sgRNA/Cas9n common vector. The accurate replacement efficiency of the sgRNA/Cas9n-P2A-Hpt optimized vector is higher than that of the sgRNA/Cas9n common vector.

TABLE 2

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

<120> a method for realizing gene replacement without causing DNA double strand break

<160>10

<170>PatentIn version 3.5

<210>1

<211>11586

<212>DNA

<213>Artificial Sequence

<400>1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240

gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480

caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540

ggctggtgca atttgggtat ggtggtgcaa gttttagagc tagaaatagc aagttaaaat 600

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttttcgtt 660

ttgcattgag ttttctccgt cgcatgtttg cagttttatt ttccgttttg cattgaaatt 720

tctccgtctc atgtttgcag cgtgttcaaa aagtacgcag ctgtatttca cttatttacg 780

gcgccacatt ttcatgccgt ttgtgccaac tatcccgagc tagtgaatac agcttggctt 840

cacacaacac tggtgacccg ctgacctgct cgtacctcgt accgtcgtac ggcacagcat 900

ttggaattaa agggtgtgat cgatactgct tgctgctaag cttacaaatt cgggtcaagg 960

cggaagccag cgcgccaccc cacgtcagca aatacggagg cgcggggttg acggcgtcac 1020

ccggtcctaa cggcgaccaa caaaccagcc agaagaaatt acagtaaaaa aaaagtaaat 1080

tgcactttga tccacctttt attacctaag tctcaatttg gatcaccctt aaacctatct 1140

tttcaatttg ggccgggttg tggtttggac taccatgaac aacttttcgt catgtctaac 1200

ttccctttca gcaaacatat gaaccatata tagaggagat cggccgtata ctagagctga 1260

tgtgtttaag gtcgttgatt gcacgagaaa aaaaaatcca aatcgcaaca atagcaaatt 1320

tatctggttc aaagtgaaaa gatatgttta aaggtagtcc aaagtaaaac ttatagataa 1380

taaaatgtgg tccaaagcgt aattcactca aaaaaaatca acgagacgtg taccaaacgg 1440

agacaaacgg catcttctcg aaatttccca accgctcgct cgcccgcctc gtcttcccgg 1500

aaaccgcggt ggtttcagcg tggcggattc tccaagcaga cggagacgtc acggcacggg 1560

actcctccca ccacccaacc gccataaata ccagccccct catctcctct cctcgcatca 1620

gctccacccc cgaaaaattt ctccccaatc tcgcgaggct ctcgtcgtcg aatcgaatcc 1680

tctcgcgtcc tcaaggtacg ctgcttctcc tctcctcgct tcgtttcgat tcgatttcgg 1740

acgggtgagg ttgttttgtt gctagatccg attggtggtt agggttgtcg atgtgattat 1800

cgtgagatgt ttaggggttg tagatctgat ggttgtgatt tgggcacggt tggttcgata 1860

ggtggaatcg tggttaggtt ttgggattgg atgttggttc tgatgattgg ggggaatttt 1920

tacggttaga tgaattgttg gatgattcga ttggggaaat cggtgtagat ctgttgggga 1980

attgtggaac tagtcatgcc tgagtgattg gtgcgatttg tagcgtgttc catcttgtag 2040

gccttgttgc gagcatgttc agatctactg ttccgctctt gattgagtta ttggtgccat 2100

gggttggtgc aaacacaggc tttaatatgt tatatctgtt ttgtgtttga tgtagatctg 2160

tagggtagtt cttcttagac atggttcaat tatgtagctt gtgcgtttcg atttgatttc 2220

atatgttcac agattagata atgatgaact cttttaatta attgtcaatg gtaaatagga 2280

agtcttgtcg ctatatctgt cataatgatc tcatgttact atctgccagt aatttatgct 2340

aagaactata ttagaatatc atgttacaat ctgtagtaat atcatgttac aatctgtagt 2400

tcatctatat aatctattgt ggtaatttct ttttactatc tgtgtgaaga ttattgccac 2460

tagttcattc tacttatttc tgaagttcag gatacgtgtg ctgttactac ctatctgaat 2520

acatgtgtga tgtgcctgtt actatctttt tgaatacatg tatgttctgt tggaatatgt 2580

ttgctgtttg atccgttgtt gtgtccttaa tcttgtgcta gttcttaccc tatctgtttg 2640

gtgattattt cttgcagtac gtaagcatgg actacaagga ccacgacggg gattacaaag 2700

accacgacat agactacaag gatgacgatg acaaaatggc accgaagaaa aaaaggaagg 2760

tcggcggctc cccgaagaaa aaaaggaagg tcggcggctc cccgaagaaa aaaaggaagg 2820

tcggcggctc cccgaagaaa aaaaggaagg tcggaatcca tggcgttcca gctgccgaca 2880

agaagtactc catcggcctc gccatcggca ccaacagcgt cggctgggcg gtgatcaccg 2940

acgagtacaa ggtcccgtcc aagaagttca aggtcctggg caacaccgac cgccactcca 3000

tcaagaagaa cctcatcggc gccctcctct tcgactccgg cgagacggcg gaggcgaccc 3060

gcctcaagcg caccgcccgc cgccgctaca cccgccgcaa gaaccgcatc tgctacctcc 3120

aggagatctt ctccaacgag atggcgaagg tcgacgactc cttcttccac cgcctcgagg 3180

agtccttcct cgtggaggag gacaagaagc acgagcgcca ccccatcttc ggcaacatcg 3240

tcgacgaggt cgcctaccac gagaagtacc ccactatcta ccaccttcgt aagaagcttg 3300

ttgactctac tgataaggct gatcttcgtc tcatctacct tgctctcgct cacatgatca 3360

agttccgtgg tcacttcctt atcgagggtg accttaaccc tgataactcc gacgtggaca 3420

agctcttcat ccagctcgtc cagacctaca accagctctt cgaggagaac cctatcaacg 3480

cttccggtgt cgacgctaag gcgatccttt ccgctaggct ctccaagtcc aggcgtctcg 3540

agaacctcat cgcccagctc cctggtgaga agaagaacgg tcttttcggt aacctcatcg 3600

ctctctccct cggtctgacc cctaacttca agtccaactt cgacctcgct gaggacgcta 3660

agcttcagct ctccaaggat acctacgacg atgatctcga caacctcctc gctcagattg 3720

gagatcagta cgctgatctc ttccttgctg ctaagaacct ctccgatgct atcctccttt 3780

cggatatcct tagggttaac actgagatca ctaaggctcc tctttctgct tccatgatca 3840

agcgctacga cgagcaccac caggacctca ccctcctcaa ggctcttgtt cgtcagcagc 3900

tccccgagaa gtacaaggag atcttcttcg accagtccaa gaacggctac gccggttaca 3960

ttgacggtgg agctagccag gaggagttct acaagttcat caagccaatc cttgagaaga 4020

tggatggtac tgaggagctt ctcgttaagc ttaaccgtga ggacctcctt aggaagcaga 4080

ggactttcga taacggctct atccctcacc agatccacct tggtgagctt cacgccatcc 4140

ttcgtaggca ggaggacttc taccctttcc tcaaggacaa ccgtgagaag atcgagaaga 4200

tccttacttt ccgtattcct tactacgttg gtcctcttgc tcgtggtaac tcccgtttcg 4260

cttggatgac taggaagtcc gaggagacta tcaccccttg gaacttcgag gaggttgttg 4320

acaagggtgc ttccgcccag tccttcatcg agcgcatgac caacttcgac aagaacctcc 4380

ccaacgagaa ggtcctcccc aagcactccc tcctctacga gtacttcacg gtctacaacg 4440

agctcaccaa ggtcaagtac gtcaccgagg gtatgcgcaa gcctgccttc ctctccggcg 4500

agcagaagaa ggctatcgtt gacctcctct tcaagaccaa ccgcaaggtc accgtcaagc 4560

agctcaagga ggactacttc aagaagatcg agtgcttcga ctccgtcgag atcagcggcg 4620

ttgaggaccg tttcaacgct tctctcggta cctaccacga tctcctcaag atcatcaagg 4680

acaaggactt cctcgacaac gaggagaacg aggacatcct cgaggacatc gtcctcactc 4740

ttactctctt cgaggatagg gagatgatcg aggagaggct caagacttac gctcatctct 4800

tcgatgacaa ggttatgaag cagctcaagc gtcgccgtta caccggttgg ggtaggctct 4860

cccgcaagct catcaacggt atcagggata agcagagcgg caagactatc ctcgacttcc 4920

tcaagtctga tggtttcgct aacaggaact tcatgcagct catccacgat gactctctta 4980

ccttcaagga ggatattcag aaggctcagg tgtccggtca gggcgactct ctccacgagc 5040

acattgctaa ccttgctggt tcccctgcta tcaagaaggg catccttcag actgttaagg 5100

ttgtcgatga gcttgtcaag gttatgggtc gtcacaagcc tgagaacatc gtcatcgaga 5160

tggctcgtga gaaccagact acccagaagg gtcagaagaa ctcgagggag cgcatgaaga 5220

ggattgagga gggtatcaag gagcttggtt ctcagatcct taaggagcac cctgtcgaga 5280

acacccagct ccagaacgag aagctctacc tctactacct ccagaacggt agggatatgt 5340

acgttgacca ggagctcgac atcaacaggc tttctgacta cgacgtcgac cacattgttc 5400

ctcagtcttt ccttaaggat gactccatcg acaacaaggt cctcacgagg tccgacaaga 5460

acaggggtaa gtcggacaac gtcccttccg aggaggttgt caagaagatg aagaactact 5520

ggaggcagct tctcaacgct aagctcatta cccagaggaa gttcgacaac ctcacgaagg 5580

ctgagagggg tggcctttcc gagcttgaca aggctggttt catcaagagg cagcttgttg 5640

agacgaggca gattaccaag cacgttgctc agatcctcga ttctaggatg aacaccaagt 5700

acgacgagaa cgacaagctc atccgcgagg tcaaggtgat caccctcaag tccaagctcg 5760

tctccgactt ccgcaaggac ttccagttct acaaggtccg cgagatcaac aactaccacc 5820

acgctcacga tgcttacctt aacgctgtcg ttggtaccgc tcttatcaag aagtacccta 5880

agcttgagtc cgagttcgtc tacggtgact acaaggtcta cgacgttcgt aagatgatcg 5940

ccaagtccga gcaggagatc ggcaaggcca ccgccaagta cttcttctac tccaacatca 6000

tgaacttctt caagaccgag atcaccctcg ccaacggcga gatccgcaag cgccctctta 6060

tcgagacgaa cggtgagact ggtgagatcg tttgggacaa gggtcgcgac ttcgctactg 6120

ttcgcaaggt cctttctatg cctcaggtta acatcgtcaa gaagaccgag gtccagaccg 6180

gtggcttctc caaggagtct atccttccaa agagaaactc ggacaagctc atcgctagga 6240

agaaggattg ggaccctaag aagtacggtg gtttcgactc ccctactgtc gcctactccg 6300

tcctcgtggt cgccaaggtg gagaagggta agtcgaagaa gctcaagtcc gtcaaggagc 6360

tcctcggcat caccatcatg gagcgctcct ccttcgagaa gaacccgatc gacttcctcg 6420

aggccaaggg ctacaaggag gtcaagaagg acctcatcat caagctcccc aagtactctc 6480

ttttcgagct cgagaacggt cgtaagagga tgctggcttc cgctggtgag ctccagaagg 6540

gtaacgagct tgctcttcct tccaagtacg tgaacttcct ctacctcgcc tcccactacg 6600

agaagctcaa gggttcccct gaggataacg agcagaagca gctcttcgtg gagcagcaca 6660

agcactacct cgacgagatc atcgagcaga tctccgagtt ctccaagcgc gtcatcctcg 6720

ctgacgctaa cctcgacaag gtcctctccg cctacaacaa gcaccgcgac aagcccatcc 6780

gcgagcaggc cgagaacatc atccacctct tcacgctcac gaacctcggc gcccctgctg 6840

ctttcaagta cttcgacacc accatcgaca ggaagcgtta cacgtccacc aaggaggttc 6900

tcgacgctac tctcatccac cagtccatca ccggtcttta cgagactcgt atcgaccttt 6960

cccagcttgg tggtgatgac gatgacaaaa tggcaccgaa gaaaaaaagg aaggtcggcg 7020

gctccccgaa gaaaaaaagg aaggtcggcg gctccccgaa gaaaaaaagg aaggtcggcg 7080

gctccccgaa gaaaaaaagg aaggtcggaa tccatggcta gtcccgatcg ttcaaacatt 7140

tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 7200

tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 7260

aggtgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 7320

atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagaggcg 7380

cgcctggtgg atcgtccgcc taggctgcag tgcagcgtga cccggtcgtg cccctctcta 7440

gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt ttttgtcaca 7500

cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac tctacgaata 7560

atataatcta tagtactaca ataatatcag tgttttagag aatcatataa atgaacagtt 7620

agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt tttatctttt 7680

tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa tacttcatcc 7740

attttattag tacatccatt tagggtttag ggttaatggt ttttatagac taattttttt 7800

agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac tctattttag 7860

tttttttatt taataattta gatataaaat agaataaaat aaagtgacta aaaattaaac 7920

aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc gagtagataa 7980

tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga accagcagcg 8040

tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc tggacccctc 8100

tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa attgcgtggc 8160

ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac ggcaccggca 8220

gctacggggg attcctttcc caccgctcct tcgctttccc ttcctcgccc gccgtaataa 8280

atagacaccc cctccacacc ctctttcccc aacctcgtgt tgttcggagc gcacacacac 8340

acaaccagat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta cgccgctcgt 8400

cctccccccc cccccctctc taccttctct agatcggcgt tccggtccat ggttagggcc 8460

cggtagttct acttctgttc atgtttgtgt tagatccgtg tttgtgttag atccgtgctg 8520

ctagcgttcg tacacggatg cgacctgtac gtcagacacg ttctgattgc taacttgcca 8580

gtgtttctct ttggggaatc ctgggatggc tctagccgtt ccgcagacgg gatcgatttc 8640

atgatttttt ttgtttcgtt gcatagggtt tggtttgccc ttttccttta tttcaatata 8700

tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga 8760

tgtggtctgg ttgggcggtc gttctagatc ggagtagaat tctgtttcaa actacctggt 8820

ggatttatta attttggatc tgtatgtgtg tgccatacat attcatagtt acgaattgaa 8880

gatgatggat ggaaatatcg atctaggata ggtatacatg ttgatgcggg ttttactgat 8940

gcatatacag agatgctttt tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc 9000

gttcattcgt tctagatcgg agtagaatac tgtttcaaac tacctggtgt atttattaat 9060

tttggaactg tatgtgtgtg tcatacatct tcatagttac gagtttaaga tggatggaaa 9120

tatcgatcta ggataggtat acatgttgat gtgggtttta ctgatgcata tacatgatgg 9180

catatgcagc atctattcat atgctctaac cttgagtacc tatctattat aataaacaag 9240

tatgttttat aattattttg atcttgatat acttggatga tggcatatgc agcagctata 9300

tgtggatttt tttagccctg ccttcatacg ctatttattt gcttggtact gtttcttttg 9360

tcgatgctca ccctgttgtt tggtgttact tctgcaggag ctcatgaaaa agcctgaact 9420

caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat 9480

gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata 9540

tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca 9600

ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggagt ttagcgagag 9660

cctgacctat tgcatctccc gccgttcaca gggtgtcacg ttgcaagacc tgcctgaaac 9720

cgaactgccc gctgttctac aaccggtcgc ggaggctatg gatgcgatcg ctgcggccga 9780

tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac 9840

atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat 9900

ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga 9960

ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac 10020

ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca 10080

atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac 10140

gcgctacttc gagcggaggc atccggagct tgcaggatcg ccacgactcc gggcgtatat 10200

gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc 10260

agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg 10320

tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc 10380

cgatagtgga aaccgacgcc ccagcactcg tccgagggca aagaaataga gtagatgccg 10440

accgggatct gtcgatcgac aagctcgagt ttctccataa taatgtgtga gtagttccca 10500

gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct 10560

tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca 10620

aaatccagta ctaaaatcca gatcccccga attaattcgg cgttaattca gatttgggta 10680

tggtggtgca acgggaagag atcccaccgc aatatgccat tcaggtgctg gatgagctga 10740

cgaaaggtga ggcaatcatc gctactggtg ttgggcagca ccagatgtgg gcggcacaat 10800

attacaccta caagcggcca cggcagtggc tgtcttcggc tggtctgggc gcaatgggat 10860

ttgggctgcc tgctgcagct ggtgcttctg tggctaaccc aggtgtcaca gttgttgata 10920

ttgatgggga tggtagcttc ctcatgaaca ttcaggagct ggcattgatc cgcattgaga 10980

acctccctgt gaaggtgatg gtgttgaaca accaacattt gggtatggtc gtccagttgg 11040

aggataggtt ttacaaggcg aatagggcgc atacatactt gggcaacccc gaatgtgaga 11100

gcgagatata tccagatttt gtgactattg ctaaggggtt caatattcct gcagtccgtg 11160

taacaaagaa gagtgaagtc cgtgccgcca tcaagaagat gctcgagact ccagggccat 11220

acttgttgga tatcatcgtc ccgcaccagg agcatgtgct gcctatgatc ccaattgggg 11280

gcgcattcaa ggacatgatc ctggatggtg atggcaggac tgtgtattaa tctataatct 11340

gtatgttggc aaagcaccag cccggcctat gtttgacctg aatgacccat ttgggtatgg 11400

tggtgcaacg gcctgcagga cgcgtttaat taagtgcacg cggccgccta cttagtcaag 11460

agcctcgcac gcgactgtca cgcggccagg atcgcctcgt gagcctcgca atctgtacct 11520

agtgtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag 11580

cgttta 11586

<210>2

<211>7

<212>PRT

<213>Artificial Sequence

<400>2

Pro Lys Lys Lys Arg Lys Val

1 5

<210>3

<211>1367

<212>PRT

<213>Artificial Sequence

<400>3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

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

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

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

1355 1360 1365

<210>4

<211>4101

<212>DNA

<213>Artificial Sequence

<400>4

gacaagaagt actccatcgg cctcgacatc 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 ggggtggcct ttccgagctt 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 gtctatcctt ccaaagagaa actcggacaa gctcatcgct 3360

aggaagaagg attgggaccc taagaagtac ggtggtttcg actcccctac 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 cttccgctgg tgagctccag 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

gctgctttca agtacttcga caccaccatc gacaggaagc gttacacgtc caccaaggag 4020

gttctcgacg ctactctcat ccaccagtcc atcaccggtc tttacgagac tcgtatcgac 4080

ctttcccagc ttggtggtga t 4101

<210>5

<211>2936

<212>DNA

<213>Artificial Sequence

<400>5

atggctacga ccgccgcggc cgcggccgcc gccctgtccg ccgccgcgac ggccaagacc 60

ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120

gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180

ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240

ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300

caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360

gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420

acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480

gtcccgatgg tcgccatcac gggccaggtc ccccgccgca tgatcggcac cgacgccttc 540

caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600

gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660

cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc cgtgccggtc 720

tgggacacct cgatgaatct accagggtac atcgcacgcc tgcccaagcc acccgcgaca 780

gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840

ggtggtggct gctctgcatc tggtgacgaa ttgcgctggt ttgttgagct gactggtatc 900

ccagttacaa ccactctgat gggcctcggc aatttcccca gtgacgaccc gttgtccctg 960

cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg ccgtggataa ggctgacctg 1020

ttgcttgcgt ttggtgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080

agcagggcca agattgtgca cattgacatt gatccagcag agattggaaa gaacaagcaa 1140

ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200

caacagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260

cagaagaggg agtttcctct ggggtacaaa acttttggtg aagagatccc accgcaatat 1320

gccattcagg tgctggatga gctgacgaaa ggtgaggcaa tcatcgctac tggtgttggg 1380

cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440

tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500

aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560

gagctggcat tgatccgcat tgagaacctc cctgtgaagg tgatggtgtt gaacaaccaa 1620

catttgggta tggtggtgca atgggaggat aggttttaca aggcgaatag ggcgcataca 1680

tacttgggca acccggaatg tgagagcgag atatatccag attttgtgac tattgctaag 1740

gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800

aagatgctcg agactccagg gccatacttg ttggatatca tcgtcccgca ccaggagcat 1860

gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920

aggactgtgt attaatctat aatctgtatg ttggcaaagc accagcccgg cctatgtttg 1980

acctgaatga cccataaaga gtggtatgcc tatgatgttt gtatgtgctc tatcaataac 2040

taaggtgtca actatgaacc atatgctctt ctgttttact tgtttgatgt gcttggcatg 2100

gtaatcctaa ttagcttcct gctgtctagg tttgtagtgt gttgttttct gtaggcatat 2160

gcatcacaag atatcatgta agtttcttgt cctacatatc aataataaga gaataaagta 2220

cttctatgca atagctctga gttaagtgtt tcaacaattt ctgaacttct gaacttatgt 2280

ttgctcaact gtcatcacac gaagtactct ccttgtaact acattttccc caagacttta 2340

aatcccctca gttacagcaa aaaataaact ttgcatctac tgttttccct ctcttcggtc 2400

gatcttattg ggtactacta tagagagagg ctgcatgaag tatttccttt ttctgtttag 2460

ttatgccgtg taaattagca tccatgcaaa atagatgaaa aatcaagcta ttcctgactg 2520

ctaaggatta tttttggcat aatgtattct tatatactcc ctccgtccca tattataagg 2580

gattttgagt ttttgtttat actgtttgac cactcgtctt attcaaaaaa ttttagaatt 2640

attatttatt ttttttgtga cttactttat tatctaaagt actttaagca caattttcgt 2700

attttatatt tgcacaaatt ttttgaataa gacgaatggt caaacaatac aaataaaaat 2760

tcaaaatccc ttataatatg ggacggaggt atgatagttg gtgaactgct acgtattgcc 2820

atttgacatt ttttggatta tgcaattttg ctgtctatag tgctctaatc aattcgcaat 2880

cccgaccttg gagtattggt ctcatggaac ccctcatctg agtaatctcc atattt 2936

<210>6

<211>644

<212>PRT

<213>Artificial Sequence

<400>6

Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala

1 5 10 15

Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro

20 25 30

Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser

35 40 45

Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro

50 55 60

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

65 70 75 80

Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala

85 90 95

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

100 105 110

His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr

115 120 125

Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro

130 135 140

Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser

145 150 155 160

Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly

165 170 175

Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile

180 185 190

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

195 200 205

Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val

210 215 220

Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val

225 230 235 240

Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys

245 250 255

Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu

260 265 270

Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly

275 280 285

Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr

290 295 300

Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu

305 310 315 320

Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp

325 330 335

Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val

340 345 350

Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile

355 360 365

Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser

370 375 380

Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu

385 390 395 400

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

405 410 415

Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe

420 425 430

Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu

435 440 445

Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met

450 455 460

Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser

465 470 475 480

Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly

485 490 495

Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp

500 505 510

Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu

515 520 525

Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met

530 535 540

Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr

545 550 555 560

Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val

565 570 575

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

580 585 590

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

595 600 605

Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met

610 615 620

Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly

625 630 635 640

Arg Thr Val Tyr

<210>7

<211>86

<212>DNA

<213>Artificial Sequence

<400>7

gtttcagagc tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac 60

ttgaaaaagt ggcaccgagt cggtgc 86

<210>8

<211>1373

<212>DNA

<213>Artificial Sequence

<400>8

ggaatccatg gcggatcagg agccaccaac ttctccctcc tcaagcaggc cggcgacgtg 60

gaggagaacc cgggcccaat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt 120

ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct 180

cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc 240

gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt 300

ccggaagtgc ttgacattgg ggagtttagc gagagcctga cctattgcat ctcccgccgt 360

tcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctacaaccg 420

gtcgcggagg ctatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc 480

ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt 540

gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc 600

gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc 660

gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc 720

attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc 780

tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg 840

gagcttgcag gatcgccacg actccgggcg tatatgctcc gcattggtct tgaccaactc 900

tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac 960

gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg 1020

gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc 1080

actcgtccga gggcaaagaa atagactagt tcccgatcgt tcaaacattt ggcaataaag 1140

tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 1200

ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga ggtgggtttt 1260

tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 1320

aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagaggcgc gcc 1373

<210>9

<211>19

<212>PRT

<213>Artificial Sequence

<400>9

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210>10

<211>341

<212>PRT

<213>Artificial Sequence

<400>10

Met Lys Lys Pro Glu Leu Thr Ala Thr Ser Val Glu Lys Phe Leu Ile

1 5 10 15

Glu Lys Phe Asp Ser Val Ser Asp Leu Met Gln Leu Ser Glu Gly Glu

20 25 30

Glu Ser Arg Ala Phe Ser Phe Asp Val Gly Gly Arg Gly Tyr Val Leu

35 40 45

Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr

50 55 60

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

65 70 75 80

Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg Arg Ser Gln

85 90 95

Gly Val Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu Pro Ala Val Leu

100 105 110

Gln Pro Val Ala Glu Ala Met Asp Ala Ile Ala Ala Ala Asp Leu Ser

115 120 125

Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro Gln Gly Ile Gly Gln Tyr

130 135 140

Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile Ala Asp Pro His Val Tyr

145 150 155 160

His Trp Gln Thr Val Met Asp Asp Thr Val Ser Ala Ser Val Ala Gln

165 170 175

Ala Leu Asp Glu Leu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg

180 185 190

His Leu Val His Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn

195 200 205

Gly Arg Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp

210 215 220

Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu Ala

225 230 235 240

Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg His Pro Glu Leu

245 250 255

Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met Leu Arg Ile Gly Leu Asp

260 265 270

Gln Leu Tyr Gln Ser Leu Val Asp Gly Asn Phe Asp Asp Ala Ala Trp

275 280 285

Ala Gln Gly Arg Cys Asp Ala Ile Val Arg Ser Gly Ala Gly Thr Val

290 295 300

Gly Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly

305 310 315 320

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

325 330 335

Pro Arg Ala Lys Lys

340

Claims (15)

1. A method for realizing plant gene replacement without generating DNA double-strand break, comprising the following steps: introducing the sgRNA, the Cas9 nickase and the donor DNA into a target plant;

the sgRNA target DNA fragment A target sequence;

the donor DNA sequentially comprises the DNA fragment A target sequence, the DNA fragment B and the DNA fragment A target sequence;

the DNA fragment B is a DNA molecule obtained by mutating the DNA fragment A by one or more bases;

under the guidance of the sgRNA, the Cas9 nicking enzyme generates single-stranded DNA nicks at a DNA fragment A target sequence in a target plant genome and a DNA fragment A target sequence in the donor DNA, and replaces the DNA fragment A in the target plant genome with the DNA fragment B through a repair mechanism in a target plant body to realize plant gene replacement;

the DNA fragment A is a DNA molecule shown in 1300 th-1993 th sites of a sequence 5;

the DNA fragment B is a DNA molecule shown in 10695-11388 th site of the sequence 1;

the sgRNA structure is as follows: tRNA-the RNA-sgRNA backbone transcribed from the DNA fragment a target sequence;

the tRNA is an RNA molecule obtained by replacing T in 474 th-550 th positions of the sequence 1 with U;

the sgRNA framework is an RNA molecule obtained by replacing T in 571-646 of a sequence 1 with U;

the first target sequence of the DNA fragment is 551-570 th site of the sequence 1;

the sequence of the donor DNA is 10672-11411 th site of the sequence 1;

the Cas9 nickase is a Cas 9D 10A nickase;

the Cas 9D 10A nickase is a SpCas9n protein;

the amino acid sequence of the SpCas9n protein is shown as sequence 3.

2. The method of claim 1, wherein:

the encoding gene of the SpCas9n protein is a cDNA molecule or a DNA molecule shown in 2877-6977 site of a sequence 1 in a sequence table.

3. The method according to claim 1 or 2, characterized in that:

the Cas9 nickase carries a nuclear localization signal;

the nuclear localization signal is SV40 NLS;

the amino acid sequence of the SV40 NLS is a sequence 2.

4. The method according to claim 1 or 2, characterized in that: the method for introducing the sgRNA, the Cas9 nickase and the donor DNA into the target plant comprises the following steps:

the sgRNA-transcribed DNA molecule, the Cas9 nickase-encoding gene and the donor DNA are introduced into a target plant through a recombinant expression vector;

the recombinant expression vector comprises an expression cassette consisting of a promoter, the DNA molecule for transcribing the sgRNA and a terminator in sequence, and an expression cassette consisting of the promoter, the coding gene of the Cas9 nickase and the terminator in sequence;

the nucleotide sequence of the recombinant expression vector is shown as a sequence 1.

5. The method according to claim 1 or 2, characterized in that: the plant is rice.

6. Use of the method of any one of claims 1-4 for gene editing in plants.

7. Use of the method of any of claims 1-4 for the preparation of plant mutants.

8. Use of the method of any one of claims 1-4 for increasing the efficiency of gene replacement in plants.

9. Use of the method of any one of claims 1 to 4 for reducing the production of by-products of gene replacement in plants.

10. Use according to any one of claims 6 to 9, characterized in that: the plant is rice.

11. A method of plant gene editing comprising the steps of: a method according to any one of claims 1 to 4, wherein the plant gene is edited by replacing the gene segment of interest in the plant genome.

12. A method of making a plant mutant comprising the steps of: a plant mutant obtained by replacing a desired gene fragment in the genome of a plant according to the method of any one of claims 1 to 4.

13. A method for improving the gene replacement efficiency of a plant comprises the following steps: a method according to any one of claims 1 to 4 for replacing a gene segment of interest in the genome of a plant.

14. A method for reducing byproducts produced by gene replacement in plants comprising the steps of: a method according to any one of claims 1 to 4 for replacing a gene segment of interest in the genome of a plant.

15. The method according to any one of claims 11-14, wherein: the plant is rice.

Images