CN110964742A - Preparation method of herbicide-resistant rice - Google Patents

Abstract

The invention discloses a preparation method of herbicide-resistant rice. The method comprises the steps of expressing esgRNA, adenine deaminase, Cas9 nuclease and a nuclear localization signal bpNLS in receptor rice; under the guidance of the esgRNA, the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS can mutate the 7 th site of an OsACC1 gene target sequence in a receptor rice genome from A to G, so that the ACCase herbicide-inhibiting rice is obtained and has ACCase herbicide resistance.

Description

Preparation method of herbicide-resistant rice

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of herbicide-resistant rice.

Background

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 localized on the target by a guide RNA (guide RNA), cleaved to generate a DNA double strand break (dsDNA break, DSB), and the organism will then instinctively initiate a DNA repair mechanism to repair the DSB. Repair mechanisms are generally of two types, one being non-homologous end joining (NHEJ) and the other being homologous recombination (HDR). In general, NHEJ dominates, and repair produces random indels (insertions or deletions) much higher than precise repair. For base exact substitution, the application of using HDR to achieve base exact substitution is greatly limited because of the low efficiency of HDR and the need for a DNA template.

In 2017, a novel Adenine Base Editor (ABE) was reported by David Liu laboratories. Through seven rounds of evolution, researchers fuse tRNA adenine deaminase (tRNAadensonine deaminase, ecTadA) derived from Escherichia coli at the 5' end of Cas9 nickase (Cas9n), can directly realize the replacement of a single base A to G (Guanine, G) in cells, and greatly improve the base editing efficiency of A to G without generating DSB and starting HDR repair. The specific process is as follows: when sgRNA containing a genome targeting sequence binds to ecTadA & Cas9n, the complex is targeted to a target, ecTadA catalyzes adenine deamination of a on unpaired single stranded DNA to Inosine (Inosine, I), I is considered to be G during DNA repair, Cas9n introduces a cytosine C that pairs with I upon cleavage of the phosphodiester bond of the paired DNA strands. Finally, C-G pairing is generated in the following repair process, so that A-G conversion is realized.

The single-base editing system is widely applied to creation of crop single-base mutant materials. Acetyl-coenzyme a carboxylase (ACCase) is a key enzyme regulating fatty acid biosynthesis, and the ACCase inhibiting herbicides are increasing in variety since the 1975 development and are now important herbicides in the world. ACCase inhibiting herbicides include the earliest developed APP (aryloxyphenoxypropionic acid) and CHD (cyclohexenedione) and the later developed DEN (phenylpyrazoline), among three classes. With the use of ACCase inhibiting herbicides, resistance is developed in many weeds, and 8 amino acid substitutions within an ACCase amino acid control the resistance of weeds to different classes of ACCase inhibiting herbicides, glutamine 1756 → glutamic acid, isoleucine 1781 → leucine or valine, tryptophan 1999 → cysteine or serine, tryptophan 2027 → cysteine, isoleucine 2041 → asparagine or valine, asparagine 2078 → glycine, cysteine 2088 → arginine, glycine 2096 → alanine, respectively. Wherein the amino acid mutation at position 1781, 2078 or 2088 is resistant to all ACCase inhibiting herbicides. Cysteine (C) at position 2099 in the rice OsACC1 exactly corresponds to cysteine at position 2088 in the weeds, and is mutated into arginine (R), namely C2099R can enable the rice to generate ACCase herbicide resistance, and C2099R can be obtained by a single base editing technology, but most of base editing systems for generating ACCase herbicide resistance-inhibiting rice by mutation C209 2099R reported at present are low in editing efficiency, and the generated mutants not only contain mutation at position 2099 of OsACC1, but also contain mutation at other positions, so that the identification cost of mutant molecules is high.

Disclosure of Invention

The invention aims to provide a preparation method of ACCase herbicide-inhibiting resistant rice.

The preparation method of the ACCase inhibiting herbicide resistant rice provided by the invention comprises the steps of expressing esgRNA, adenine deaminase, Cas9 nuclease and a nuclear localization signal bpNLS in receptor rice;

the esgRNA targets an OsACC1 gene target sequence; the OsACC1 gene target sequence contains a target site;

the target site is a complementary base A of a base T shown in the 6295 th position of the sequence 14;

the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS can mutate the target site in the receptor rice genome from a base A to a base G under the guidance of the esgRNA, so that the ACCase herbicide-inhibiting resistant rice with the OsACC1 protein with the 2099 th position mutated from cysteine to arginine is obtained;

the amino acid sequence of the nuclear localization signal bpNLS is A1) or A2):

A1) the amino acid sequence is a protein shown in a sequence 7;

A2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 7 in the sequence table and has the same function.

In the above method, the number of the nuclear localization signals bpNLS may be 1 or 2 or more. In an embodiment of the present invention, the number of the nuclear localization signals bpNLS is 2.

The coding gene sequence of the nuclear localization signal bpNLS is a1) or a2) or a 3):

a1) a cDNA molecule or DNA molecule shown in 3796-3846 site of a sequence 1 in a sequence table;

a2) a cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined in a1) and encoding the nuclear localization signal bpNLS;

a3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in a1) or a2) under stringent conditions and codes for the nuclear localization signal bpNLS.

In the above method, the OsACC1 gene target site sequence is sequence 12, and the target site is base A shown in 7 th position of sequence 12.

In the above method, the esgRNA structure is as follows: an RNA-esgRNA backbone transcribed from the OsACC1 gene target sequence;

the esgRNA backbone is 1) or 2) or 3):

1) replacing T in the 617-702 th site of the sequence 1 with U to obtain an RNA molecule;

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

3) RNA molecule with 75% or more than 75% identity with the nucleotide sequence defined in 1) or 2) and with the same function.

In the above method, the Cas9 nuclease is Cas9n protein;

the Cas9n protein is C1) or C2):

C1) the amino acid sequence is a protein shown in a sequence 4;

C2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 4 in the sequence table and has the same function.

The adenine deaminase is an ecTadA protein and/or an ecTadA protein; in particular the ecTadA protein and the ecTadA protein;

the ecTadA protein is D1) or D2):

D1) the amino acid sequence is a protein shown in a sequence 2;

D2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

the ecTadA protein is E1) or E2):

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

E2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 in the sequence table and has the same function.

The protein of A2), C2), D2) or E2) is a protein having 75% or more or 75% or more identity to the amino acid sequence of the protein represented by SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 2 or SEQ ID NO. 3 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2), C2), D2) or E2) may be artificially synthesized, or may be obtained by synthesizing a gene encoding the protein and then performing biological expression.

The gene encoding the protein of A2), C2), D2) or E2) described above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in 3796-3846 (protein shown in coding sequence 7), 5035-9135 (protein shown in coding sequence 4), 3847-4344 (protein shown in coding sequence 2) or 4441-4938 (protein shown in coding sequence 3) of sequence 1, and/or by carrying out missense mutation of one or several base pairs and/or by linking a coding sequence of the tag shown in the above table to its 5 'end and/or 3' end.

Further, the coding gene of the Cas9n protein is c1) or c2) or c 3):

c1) a cDNA molecule or DNA molecule shown in position 5035-9135 of a sequence 1 in a sequence table;

c2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined by c1) and encoding the Cas9 n;

c3) a cDNA or DNA molecule hybridizing under stringent conditions with a nucleotide sequence defined in c1) or c2) and encoding the Cas9 n.

The coding gene of the ecTadA protein is d1) or d2) or d 3):

d1) a cDNA molecule or DNA molecule shown in 3847-4344 site of a sequence 1 in a sequence table;

d2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in d1) and encoding said ecTadA;

d3) a cDNA or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined by d1) or d2) and encodes said ecTadA;

the coding gene of the ecTadA protein is e1) or e2) or e 3):

e1) a cDNA molecule or DNA molecule shown in 4441-4938 site of a sequence 1 in a sequence table;

e2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in e1) and encoding said ecTadA;

e3) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in e1) or e2) and encodes said ecTadA.

The nucleotide sequence of the present invention encoding the nuclear localization signal bpNLS, the Cas9n, the ecTadA can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the nuclear localization signal bpNLS, the Cas9n, the ecTadA of the present invention are derived from and identical to the sequence of the present invention as long as they encode the nuclear localization signal bpNLS, the Cas9n, the ecTadA and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequences 7, 4, 2, 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

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

In the above method, the method for expressing esgRNA, adenine deaminase, Cas9 nuclease, and nuclear localization signal bpNLS in recipient rice is to introduce a DNA molecule for transcribing esgRNA, a gene encoding ecTadA protein, a gene encoding Cas9n protein, and a gene encoding nuclear localization signal bpNLS into recipient rice.

Furthermore, the DNA molecule for transcribing the esgRNA, the coding gene of the ecTadA protein, the coding gene of the Cas9n protein and the coding gene of the nuclear localization signal bpNLS are introduced into receptor rice through recombinant expression vectors.

Furthermore, the recombinant expression vector comprises an expression cassette which sequentially consists of a promoter, the coding gene of the nuclear localization signal bpNLS, the coding gene of the ecTadA protein, the coding gene of the Cas9n protein, the coding gene of the nuclear localization signal bpNLS and a terminator.

In a specific embodiment of the invention, the recombinant expression vector is specifically a sABE-1 recombinant expression vector.

The nucleotide sequence of the sABE-1 recombinant expression vector is sequence 1 in a sequence table.

In the above method, the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS are guided by the esgRNA to mutate the base a at position 690 (5 '-3' direction) on the non-coding strand (reverse complement of sequence 14) of the coding gene of the OsACC1 protein in the recipient rice genome to base G, further mutate the base T at position 6295 (5 '-3' direction) on the coding strand (sequence 14) of the coding gene of the OsACC1 protein to base C, cause the codon of cysteine (C) at position 2099 of the OsACC1 protein to be changed from "TGC" to "CGC", so that the position casc (C) at position 2099 of the OsACC1 protein is mutated from cysteine (C) to arginine (R), and the mutated rice mutant is the acacc herbicide-inhibiting rice of the present invention. The ACCase-inhibiting herbicide-resistant rice has ACCase-inhibiting herbicide resistance. The ACCase-inhibiting herbicide is specifically galapectium.

The ACCase inhibiting herbicide resistant rice is also the C2099R mutant or the A7> G7 mutant. The C2099R mutant is a rice mutant of OsACC1 protein (sequence 13) which is mutated into arginine (R) from cysteine (C) at position 2099. The A7> G7 mutant is a rice mutant in which the 7 th site of an OsACC1 gene target sequence (sequence 12) is mutated from a base A to a base G.

The application of the method in improving the A.G base substitution efficiency of the target site or preparing the ACCase inhibiting herbicide resistant rice with the A.G base substitution only at the target site also belongs to the protection scope of the invention; the target site is the 7 th site of the OsACC1 gene target sequence.

In the above application, the A.G base is replaced by a mutation from the base A to the base G.

In the method or the application, the amino acid sequence of the OsACC1 protein is shown as a sequence 13, and the gene sequence of the OsACC1 is shown as a sequence 14.

In the method or the application, the amino acid sequence of the OsACC1 protein is shown as a sequence 13, and the coding gene sequence of the OsACC1 protein is shown as a sequence 14 (LOC 4338322).

In practical application, the OsACC1 gene in different rice varieties may contain different alleles, and particularly, the amino acid sequence of the OsACC1 protein may have 1 or 2 or more amino acid substitutions and/or deletions and/or additions with the amino acid sequence of the OsACC1 protein; in addition, there are differences in the annotation results of exon sequences of the OsACC1 gene by those skilled in the art, and it is specifically shown that the length of the amino acid sequence of the OsACC1 protein may be different from that of the OsACC1 protein of the present invention (e.g., position 2099 of the OsACC1 protein of the present invention, that is, position 2186 of the OsACC1 protein encoded by the OsACC1 encoding gene sequence of LOC _ Os05g22940, which are identical but different in amino acid sequence length). Therefore, the target site has the possibility of changing the nucleotide position of the OsACC1 gene corresponding to different rice varieties and the mutation position of the OsACC1 protein amino acid sequence caused by the mutation of the target site, namely, the No. 6295 position of the sequence 14 or the No. 2099 position of the sequence 13. However, any alteration of the target site and/or alteration of the amino acid mutation site due to the above-mentioned circumstances is within the scope of the present invention as long as the ACCase-inhibiting herbicide-resistant rice obtained by the method of the present invention is used.

In the method or the application, the rice variety can be Nipponbare.

The invention has the beneficial effects that: the method can improve the A.G base substitution efficiency of the target site (7 th site of the sequence 12), and the generated mutant has mutation of only one site of the target site, thereby greatly improving the creation efficiency of the C2099R mutant.

The invention provides a preparation method of ACCase inhibiting herbicide resistant rice, which comprises the steps of expressing esgRNA, adenine deaminase, Cas9 nuclease and a nuclear localization signal bpNLS in receptor rice; under the guidance of the esgRNA, the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS can mutate the 7 th site of an OsACC1 gene target sequence in a receptor rice genome from A to G, so that the ACCase herbicide-inhibiting rice is obtained and has ACCase herbicide resistance.

Drawings

FIG. 1 is a schematic diagram of a four-adenine base editor recombinant expression vector.

FIG. 2 shows the variation of the target site in the target sequence during the creation of the C2099R mutant.

FIG. 3 shows the sequencing result of the target sequence (peak diagram of the reverse complement sequence of the target sequence) in the rice positive T0 seedling with the mutation A7> G7.

FIG. 4 shows the effect of 1680ug/l Geranium on the growth of WT seedlings and rice positive T0 seedlings with the mutation A7> G7.

FIG. 5 shows the heading and seed setting of rice positive T0 seedlings with mutations A7> G7.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the following examples, unless otherwise specified, 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.

The primer pair T consists of a primer T-F: 5'-GCATTGCTGGACTTCAACC-3' and primer T-R: 5'-CAAACCGTATCGCAATCTGAG-3', and is used for amplifying target OsACC-T.

The efficiency of substitution of A.G bases was determined as the number of positive T0 seedlings in which substitution of A.G bases occurred/the number of total positive T0 seedlings analyzed × 100%.

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 Master Henan (Nature edition), 2017(2): 48-52.; the public is available from the agroforestry academy of sciences of Beijing.

Recovering the culture medium: n6 solid medium containing 200mg/L timentin.

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

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 medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.

The amino acid sequence of the OsACC1 protein in the following examples is shown as sequence 13, and the coding gene sequence of the OsACC1 protein is shown as sequence 14.

Example 1 preparation of ACCase inhibiting herbicide resistant Rice

Design and construction of recombinant expression vector

1. Design of recombinant expression vectors

The invention uses four types of adenine base editors to prepare ACCase inhibiting herbicide resistant rice, and the schematic structural diagram of the recombinant expression vector of the four types of adenine base editors is shown in figure 1. The method comprises the following specific steps:

the sABE system: the bpNLS nuclear localization signal was added before the ecTadA element in the ecTadA & Cas9n base editing system and after the Cas9n element. The amino acid sequence of the bpNLS nuclear localization signal is as follows: KRTADGSEFEPKKKRKV (SEQ ID NO: 7). This type of design is designated bpNLS-bpNLS.

FNLS-sABE system: in ecTadA&ecTadA*&Add 3 × Flag1 to the ecTadA element in Cas9n base editing System&1 × NLS1 nuclear localization signal, and 1 × NLS1 nuclear localization signal was added after the Cas9n element. 3 Flag1&1 the amino acid sequence of NLS1 is as follows:DYKDHDGDYKDHDIDYKDDDDKMAPKKKRKV(sequence 8), wherein the amino acid sequence of the 3 Flag1 tag protein is shown by underlining, and the amino acid sequence of the NLS1 protein is shown by wavy lines. The 1 × NLS1 nuclear localization signal includes 1 NLS1 protein, and the amino acid sequence of 1 × NLS1 is as follows: PKKKRKV (SEQ ID NO: 9). This design type is denoted as 3 Flag1&1*NLS1-1*NLS1(FNLS)。

F4NLS-sABE system: in ecTadA&ecTadA*&Add 3 × Flag2 to the ecTadA element in Cas9n base editing System&4N NLS2 nucleusLocalization signal, and 4 × NLS2 nuclear localization signal added after Cas9n element. 3 Flag2&The 4 × NLS2 nuclear localization signal sequentially comprises 13 × Flag2 tag protein and 4NLS 2 protein, 3 × Flag2&4NLS 2 the amino acid sequence of the nuclear localization signal is as follows:DYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKV(SEQ ID NO: 10); the amino acid sequence of the 3 Flag2 tag protein is shown by underlining, and the amino acid sequence of the NLS2 protein is shown by wavy lines. The 4 × NLS2 nuclear localization signal includes 4NLS 2 proteins, and the amino acid sequence of the 4 × NLS2 nuclear localization signal is as follows:PKKK RKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKV(SEQ ID NO: 11). This design type is denoted as 3 Flag2&4*NLS2-4*NLS2(F4NLS)。

4NLS-sABE system: the 4 × NLS2 nuclear localization signal was added after the Cas9n element in the ecTadA & Cas9n base editing system. This design type is denoted 4 × NLS2(4 NLS).

The recombinant expression vectors corresponding to the four adenine base editors all comprise esgRNA, the esgRNA targets the OsACC1 gene target sequence shown by the sequence 12, and the 7 th site of the target sequence is a target site. The four adenine base editors can mutate the 6295 th base A on the non-coding chain of the coding gene of the OsACC1 protein in the rice genome of the receptor into the G base, further mutate the 6295 th base T on the coding chain (SEQ ID NO: 14) of the coding gene of the OsACC1 protein into the C base, so that the codon of the 2099 th cysteine (C) of the OsACC1 protein is changed from 'TGC' to 'CGC' (FIG. 2), so that the 2099 th cysteine (C) of the OsACC1 protein is mutated into arginine (R), and the rice mutant with the mutation is the ACCase inhibitor herbicide-resistant rice of the invention, which is called as C2099R mutant, namely A7> G7 mutant. The C2099R mutant (A7> G7 mutant) can enable the rice to generate ACCase to inhibit herbicide resistance.

2. Construction of recombinant expression vectors

Artificially synthesizing recombinant expression vectors corresponding to four adenine base editors in the step 1, wherein each vector is a circular plasmid:

a recombinant expression vector comprising bpNLS-bpNLS: sABE-1;

a recombinant expression vector containing 3 Flag1&1 NLS1-1 NLS1 (FNLS): FNLS-sABE-1;

a recombinant expression vector containing 3 Flag2& 4NLS 2-4 NLS2(F4 NLS): f4 NLS-sABE-1;

a recombinant expression vector containing 4 × NLS2(4 NLS): 4 NLS-sABE-1.

The nucleotide sequence of the sABE-1 recombinant expression vector is sequence 1 in the sequence table. Wherein, the 131-596 site of the sequence 1 is a nucleotide sequence of OsU6a promoter, the 710-1090 site is a nucleotide sequence of OsU3 promoter, the 1204-1945 site is a nucleotide sequence of OsU6c promoter, the 597-702 site, the 1091-1196 site and the 1946-2051 site are espRNA nucleotide sequences, the 597-616 site, the 1091-1110 site and the 1946-1965 site are T1, T2 and OsACC-T target site sequences respectively, and the 617-702 site, the 1111-1196 site and the 1966-2051 site are espRNA skeleton nucleotide sequences; the nucleotide sequence of the OsUbq3 promoter at position 2070-3783 of the sequence 1, the bpNLS nucleotide sequence at position 3796-3846, the ecTadA protein coding sequence (without a stop codon) at position 3847-4344, and the ecTadA protein shown in the coding sequence 2; the sequence 1 has the coding sequence of the ecTadA protein (without a stop codon) at positions 4441-4938, and the ecTadA protein shown in the coding sequence 3; 5035-9135 of the sequence 1 is a coding sequence (without a stop codon) of the Cas9n protein, and the coding sequence 4 is a Cas9n protein; the 9136-9186 site of the sequence 1 is a bpNLS nucleotide sequence; no. 9529-9781 of the sequence 1 is a Nos terminator sequence; the nucleotide sequence of the ZmUbi1 promoter is positioned at the 9822-11814 site of the sequence 1, the coding sequence of hygromycin phosphotransferase is positioned at the 11821-12846 site, and the nucleotide sequence of CaMV35S polyA is positioned at the 12873-13088 site. Three targets in the sABE-1 recombinant expression vector are T1, T2 and OsACC-T respectively, and the sequences are shown in Table 1.

The FNLS-sABE-1 recombinant expression vector is a sequence obtained by replacing the bpNLS nucleotide sequence shown in 3796-3846 th site of the sequence 1 in the sequence of the sABE-1 recombinant expression vector with the sequence 5, replacing the bpNLS nucleotide sequence shown in 9186 rd site of the sequence 9136-9186 th site with the nucleotide sequence shown in 73-93 th site of the sequence 5, and keeping other sequences unchanged. Wherein, the 1 st to 66 th positions of the sequence 5 are 3 Flag1 nucleotide sequences, the 73 th to 93 th positions are NLS1 nucleotide sequences, and the sequence 5 totally contains 1 NLS1 nucleotide sequence.

The F4NLS-sABE-1 recombinant expression vector is a sequence obtained by replacing the bpNLS nucleotide sequence shown in 3796-3846 th site of the sequence 1 in the sequence of the sABE-1 recombinant expression vector with a sequence 6, replacing the bpNLS nucleotide sequence shown in 9136-9186 th site with a nucleotide sequence shown in 55-201 th site of the sequence 6, and keeping other sequences unchanged. Wherein, the 1 st to 66 th positions of the sequence 6 are 3 Flag2 nucleotide sequences, the 73 rd to 93 th positions, the 103 rd and 123 th positions, the 133 th and 153 th positions and the 163 th and 183 th positions are NLS2 nucleotide sequences, and the sequence 6 totally contains 4NLS 2 nucleotide sequences.

The 4NLS-sABE-1 recombinant expression vector is a sequence obtained by deleting the bpNLS nucleotide sequence shown in 3796-3846 th site of the sequence 1 in the sequence of the sABE-1 recombinant expression vector, replacing the bpNLS nucleotide sequence shown in 9186 th site of the sequence 9136-9186 th site with the nucleotide sequence shown in 55-201 th site of the sequence 6 and keeping other sequences unchanged.

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

TABLE 1

Second, obtaining the Positive T0 Rice seedlings

Respectively operating the sABE-1 vector, the FNLS-sABE-1 vector, the F4NLS-sABE-1 vector and the 4NLS-sABE-1 vector obtained in the step one according to the following steps 1-9:

1. the vector was introduced into Agrobacterium EHA105 (product of Shanghai Diego Biotechnology Ltd., CAT #: AC1010) to obtain 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 OD₆₀₀At room temperature, centrifuging at 10000rpm for 1min, and treating with an infection solution (glucose and sucrose are substituted for N6 liquid culture medium), wherein the concentrations of glucose and sucrose in the infection solution are 10g/L and 10g/L respectively20g/L) resuspended cells and diluted to OD₆₀₀And the concentration is 0.2, and an agrobacterium tumefaciens 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 of the seeds facing downwards, and cultured in dark at 28 ℃ for 4-6 weeks to obtain the callus of the rice.

4. After the step 3 is completed, soaking the rice callus in an agrobacterium infection solution A (the agrobacterium infection solution A is a liquid obtained by adding acetosyringone into the agrobacterium infection solution, the addition amount of the acetosyringone meets the volume ratio of the acetosyringone to the agrobacterium infection solution of 25 mul: 50ml), soaking for 10min, then placing the rice callus on a culture dish (containing about 200ml of the agrobacterium-free infection solution) paved with two layers of sterilization filter paper, and performing dark culture at 21 ℃ for 1 day.

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, and performing dark culture at 28 ℃ for 2 weeks.

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

8. And (3) putting the rice resistant callus obtained in the step (7) on a differentiation culture medium, performing illumination culture at 25 ℃ for about 1 month, transplanting the differentiated plantlets on a rooting culture medium, and performing illumination culture at 25 ℃ for 2 weeks to obtain rice T0 seedlings.

9. Extracting genome DNA of rice T0 seedling, using the genome DNA as a template, and performing PCR amplification by using a primer pair consisting of a primer F (5'-CCGAGGAGACTATCACCCCT-3') and a primer R (5'-CGACCCATAACCTTGACAAGC-3') 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 a DNA fragment of about 853bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain the DNA fragment of about 853bp, the corresponding rice T0 seedling is not the rice positive T0 seedling.

Third, result analysis

1. And (3) taking the genomic DNA of the rice positive T0 seedling obtained in the second step as a template for each vector, and carrying out PCR amplification on the OsACC-T target by adopting a primer to obtain a PCR amplification product.

2. And (3) carrying out Sanger sequencing and analysis on the PCR amplification product obtained in the step (1). The sequencing results were analyzed only for the target region. Counting the number of positive T0 seedlings with A.G base substitution on the OsACC-T target spot, and calculating the A.G base substitution efficiency, wherein the result is shown in Table 2.

The nuclear localization signal used by the sABE system is bpNLS-bpNLS; the nuclear localization signal used by the FNLS-sccbe system was 3 Flag1&1 NLS1-1 NLS 1; the nuclear localization signal used by the F4 NLS-sccbe system was 3 Flag2& 4NLS 2-4 NLS 2; the nuclear localization signal used by the 4NLS-sABE system is 4 × NLS 2.

The results show that the four systems (the sABE system, the FNLS-sABE system, the F4NLS-sABE system and the 4NLS-sABE system) only generate a single A7> G7 mutant (figure 3), and the A.G base substitution efficiency is higher than 50 percent. Wherein, the A.G base substitution efficiency of the FNLS-sABE system is the highest and is as high as 78.3 percent, the A.G base substitution efficiency of the sABE system and the F4NLS-sABE system is the second highest and is respectively 66.7 percent and 61.9 percent, and the A.G base substitution efficiency of the F4NLS-sABE system is the lowest and is 52.2 percent. Therefore, the three systems of sABE, FNLS-sABE and F4NLS-sABE only generate a single A7> G7 mutant (only a target site is mutated, and the rest sites are not mutated), the A.G base substitution efficiency is high, and the creation efficiency of the A7> G7 mutant is greatly improved.

TABLE 2

Example 2, A7> ACCase inhibition of herbicide resistance assay of G7 mutant

Experiment of spraying herbicide

Randomly selecting 15 rice positive T0 seedlings with A7> G7 mutation and 15 rice positive T0 seedlings without A7> G7 mutation (marked as WT seedlings) obtained in the third step of example 1, and respectively carrying out the following steps 1-4:

1. when the positive T0 rice seedlings grow to about 15-20 cm in the rooting culture medium, the cover of the culture dish is opened, clear water is added for hardening seedlings, and the seedlings are cultured for 5 days at 25 ℃ by illumination.

2. And after hardening, taking out the T0 seedling from the culture medium, washing the residual culture medium in the root with clear water, transplanting the seedling into a small pot in a greenhouse, and transferring the seedling to a large pot for culture after 15 days.

3. At the 6-10 leaf stage, three rice positive T0 seedlings with mutation of A7> G7 and three rice positive T0 seedlings without mutation of A7> G7 are randomly selected as one group, and the total is five groups. Each group was sprayed with 1L of Geranium strictipes (69806-34-4, Yinong Dow, active ingredient haloxyfop-p-methyl 108 g/L) with concentrations of 0ug/L, 42ug/L, 84ug/L, 168ug/L, 1680ug/L, respectively, with a sprayer every two weeks for 3 times.

4. When the growth of the rice positive T0 seedlings which do not generate the A7 and G7 mutations stops or even withers, and the normal heading knots of the rice positive T0 seedlings which generate the A7 and G7 mutations are photographed in real time.

Second, result analysis

Gephyrocarbone belongs to a species of the APP class of ACCase inhibiting herbicides. For five concentrations of 0ug/l, 42ug/l, 84ug/l, 168ug/l and 1680ug/l, none of the first four concentrations had significant inhibition effect on the rice positive T0 seedlings without mutation of A7> G7, i.e., WT seedlings all grew normally, only 1680ug/l Geranium showed significant inhibition effect on WT seedlings, while the rice positive T0 seedlings with mutation of A7> G7 grew normally (FIG. 4), and the rice positive T0 seedlings with mutation of A7> G7 also showed normal spiked fruit (FIG. 5). Thus, it can be seen that the rice positive T0 seedling with the mutation A7> G7 has ACCase inhibiting herbicide resistance.

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> preparation method of herbicide-resistant rice

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>19494

<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 ctgccgagtg 600

cacggtgtcc gtggccgttt cagagctatg ctggaaacag catagcaagt tgaaataagg 660

ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta ggaatcttta 720

aacatacgaa cagatcactt aaagttcttc tgaagcaact taaagttatc aggcatgcat 780

ggatcttgga ggaatcagat gtgcagtcag ggaccatagc acaagacagg cgtcttctac 840

tggtgctacc agcaaatgct ggaagccggg aacactgggt acgttggaaa ccacgtgtga 900

tgtgaaggag taagataaac tgtaggagaa aagcatttcg tagtgggcca tgaagccttt 960

caggacatgt attgcagtat gggccggccc attacgcaat tggacgacaa caaagactag 1020

tattagtacc acctcggcta tccacataga tcaaagctgg tttaaaagag ttgtgcagat 1080

gatccgtggc gttgatagca agataaaccc gtttcagagc tatgctggaa acagcatagc 1140

aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 1200

tttctcatta gcggtatgca tgttggtaga agtcggagat gtaaataatt ttcattatat 1260

aaaaaaggta cttcgagaaa aataaatgca tacgaattaa ttctttttat gttttttaaa 1320

ccaagtatat agaatttatt gatggttaaa atttcaaaaa tatgacgaga gaaaggttaa 1380

acgtacggca tatacttctg aacagagagg gaatatgggg tttttgttgc tcccaacaat 1440

tcttaagcac gtaaaggaaa aaagcacatt atccacattg tacttccaga gatatgtaca 1500

gcattacgta ggtacgtttt ctttttcttc ccggagagat gatacaataa tcatgtaaac 1560

ccagaattta aaaaatattc tttactataa aaattttaat tagggaacgt attatttttt 1620

acatgacacc ttttgagaaa gagggacttg taatatggga caaatgaaca atttctaaga 1680

aatgggcata tgactctcag tacaatggac caaattccct ccagtcggcc cagcaataca 1740

aagggaaaga aatgaggggg cccacaggcc acggcccact tttctccgtg gtggggagat 1800

ccagctagag gtccggccca caagtggccc ttgccccgtg ggacggtggg attgcagagc 1860

gcgtgggcgg aaacaacagt ttagtaccac ctcgctcacg caacgacgcg accacttgct 1920

tataagctgc tgcgctgagg ctcagcatag cactcaatgc ggtctgtttc agagctatgc 1980

tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac 2040

cgagtcggtg cttttttttt tttaagctta caaattcggg tcaaggcgga agccagcgcg 2100

ccaccccacg tcagcaaata cggaggcgcg gggttgacgg cgtcacccgg tcctaacggc 2160

gaccaacaaa ccagccagaa gaaattacag taaaaaaaaa gtaaattgca ctttgatcca 2220

ccttttatta cctaagtctc aatttggatc acccttaaac ctatcttttc aatttgggcc 2280

gggttgtggt ttggactacc atgaacaact tttcgtcatg tctaacttcc ctttcagcaa 2340

acatatgaac catatataga ggagatcggc cgtatactag agctgatgtg tttaaggtcg 2400

ttgattgcac gagaaaaaaa aatccaaatc gcaacaatag caaatttatc tggttcaaag 2460

tgaaaagata tgtttaaagg tagtccaaag taaaacttat agataataaa atgtggtcca 2520

aagcgtaatt cactcaaaaa aaatcaacga gacgtgtacc aaacggagac aaacggcatc 2580

ttctcgaaat ttcccaaccg ctcgctcgcc cgcctcgtct tcccggaaac cgcggtggtt 2640

tcagcgtggc ggattctcca agcagacgga gacgtcacgg cacgggactc ctcccaccac 2700

ccaaccgcca taaataccag ccccctcatc tcctctcctc gcatcagctc cacccccgaa 2760

aaatttctcc ccaatctcgc gaggctctcg tcgtcgaatc gaatcctctc gcgtcctcaa 2820

ggtacgctgc ttctcctctc ctcgcttcgt ttcgattcga tttcggacgg gtgaggttgt 2880

tttgttgcta gatccgattg gtggttaggg ttgtcgatgt gattatcgtg agatgtttag 2940

gggttgtaga tctgatggtt gtgatttggg cacggttggt tcgataggtg gaatcgtggt 3000

taggttttgg gattggatgt tggttctgat gattgggggg aatttttacg gttagatgaa 3060

ttgttggatg attcgattgg ggaaatcggt gtagatctgt tggggaattg tggaactagt 3120

catgcctgag tgattggtgc gatttgtagc gtgttccatc ttgtaggcct tgttgcgagc 3180

atgttcagat ctactgttcc gctcttgatt gagttattgg tgccatgggt tggtgcaaac 3240

acaggcttta atatgttata tctgttttgt gtttgatgta gatctgtagg gtagttcttc 3300

ttagacatgg ttcaattatg tagcttgtgc gtttcgattt gatttcatat gttcacagat 3360

tagataatga tgaactcttt taattaattg tcaatggtaa ataggaagtc ttgtcgctat 3420

atctgtcata atgatctcat gttactatct gccagtaatt tatgctaaga actatattag 3480

aatatcatgt tacaatctgt agtaatatca tgttacaatc tgtagttcat ctatataatc 3540

tattgtggta atttcttttt actatctgtg tgaagattat tgccactagt tcattctact 3600

tatttctgaa gttcaggata cgtgtgctgt tactacctat ctgaatacat gtgtgatgtg 3660

cctgttacta tctttttgaa tacatgtatg ttctgttgga atatgtttgc tgtttgatcc 3720

gttgttgtgt ccttaatctt gtgctagttc ttaccctatc tgtttggtga ttatttcttg 3780

cagtacgtaa gcatgaagag gaccgccgac ggcagcgagt tcgagccgaa gaagaagagg 3840

aaggtgtccg aggtggagtt ctcccacgag tactggatga ggcacgcact caccctcgca 3900

aagagggcat gggacgagag ggaggtgcct gtgggagcag tgctcgtgca caacaacagg 3960

gtgatcggag agggatggaa caggcctatc ggaaggcacg accctaccgc acacgcagag 4020

atcatggcac tcaggcaggg aggcctcgtg atgcagaact acaggctcat cgacgccacc 4080

ctctacgtga ccctcgagcc ttgcgtgatg tgcgcaggag ccatgatcca ctccaggatc 4140

ggaagggtgg tgttcggagc aagggacgca aagaccggag cagccggctc cctcatggac 4200

gtgctccacc acccgggcat gaaccacagg gtggagatca ccgagggaat cctcgcagac 4260

gagtgcgcag ccctcctctc cgacttcttc aggatgagga ggcaggagat caaggcccag 4320

aagaaggccc agtcctccac cgactccggc ggctcatcag gcggctcctc cggctccgag 4380

acaccgggca cctccgagtc cgccaccccg gagtcctccg gcggctcctc cggcggctcc 4440

tccgaggtgg agttctccca cgagtactgg atgaggcacg cactcaccct cgcaaagagg 4500

gcaagggacg agagggaggt gcctgtggga gcagtgctcg tgctcaacaa cagggtgatc 4560

ggagagggat ggaacagggc aatcggcctc cacgacccta ccgcacacgc agagatcatg 4620

gcactcaggc agggaggcct cgtgatgcag aactacaggc tcatcgacgc caccctctac 4680

gtgaccttcg agccttgcgt gatgtgcgca ggagccatga tccactccag gatcggcagg 4740

gtggtgttcg gcgtgaggaa cgcaaagacc ggagcagcag gctccctcat ggacgtgctc 4800

cactacccgg gcatgaacca cagggtggag atcaccgagg gaatcctcgc agacgagtgc 4860

gcagccctcc tctgctactt cttcaggatg ccgaggcagg tgttcaacgc ccagaagaag 4920

gcccagtcct ccaccgactc cggcggctca tcaggcggct cctccggctc cgagacaccg 4980

ggcacctccg agtccgccac cccggagtcc tccggcggct cctccggcgg ctccgacaag 5040

aagtactcca tcggcctcgc catcggcacc aacagcgtcg gctgggcggt gatcaccgac 5100

gagtacaagg tcccgtccaa gaagttcaag gtcctgggca acaccgaccg ccactccatc 5160

aagaagaacc tcatcggcgc cctcctcttc gactccggcg agacggcgga ggcgacccgc 5220

ctcaagcgca ccgcccgccg ccgctacacc cgccgcaaga accgcatctg ctacctccag 5280

gagatcttct ccaacgagat ggcgaaggtc gacgactcct tcttccaccg cctcgaggag 5340

tccttcctcg tggaggagga caagaagcac gagcgccacc ccatcttcgg caacatcgtc 5400

gacgaggtcg cctaccacga gaagtacccc actatctacc accttcgtaa gaagcttgtt 5460

gactctactg ataaggctga tcttcgtctc atctaccttg ctctcgctca catgatcaag 5520

ttccgtggtc acttccttat cgagggtgac cttaaccctg ataactccga cgtggacaag 5580

ctcttcatcc agctcgtcca gacctacaac cagctcttcg aggagaaccc tatcaacgct 5640

tccggtgtcg acgctaaggc gatcctttcc gctaggctct ccaagtccag gcgtctcgag 5700

aacctcatcg cccagctccc tggtgagaag aagaacggtc ttttcggtaa cctcatcgct 5760

ctctccctcg gtctgacccc taacttcaag tccaacttcg acctcgctga ggacgctaag 5820

cttcagctct ccaaggatac ctacgacgat gatctcgaca acctcctcgc tcagattgga 5880

gatcagtacg ctgatctctt ccttgctgct aagaacctct ccgatgctat cctcctttcg 5940

gatatcctta gggttaacac tgagatcact aaggctcctc tttctgcttc catgatcaag 6000

cgctacgacg agcaccacca ggacctcacc ctcctcaagg ctcttgttcg tcagcagctc 6060

cccgagaagt acaaggagat cttcttcgac cagtccaaga acggctacgc cggttacatt 6120

gacggtggag ctagccagga ggagttctac aagttcatca agccaatcct tgagaagatg 6180

gatggtactg aggagcttct cgttaagctt aaccgtgagg acctccttag gaagcagagg 6240

actttcgata acggctctat ccctcaccag atccaccttg gtgagcttca cgccatcctt 6300

cgtaggcagg aggacttcta ccctttcctc aaggacaacc gtgagaagat cgagaagatc 6360

cttactttcc gtattcctta ctacgttggt cctcttgctc gtggtaactc ccgtttcgct 6420

tggatgacta ggaagtccga ggagactatc accccttgga acttcgagga ggttgttgac 6480

aagggtgctt ccgcccagtc cttcatcgag cgcatgacca acttcgacaa gaacctcccc 6540

aacgagaagg tcctccccaa gcactccctc ctctacgagt acttcacggt ctacaacgag 6600

ctcaccaagg tcaagtacgt caccgagggt atgcgcaagc ctgccttcct ctccggcgag 6660

cagaagaagg ctatcgttga cctcctcttc aagaccaacc gcaaggtcac cgtcaagcag 6720

ctcaaggagg actacttcaa gaagatcgag tgcttcgact ccgtcgagat cagcggcgtt 6780

gaggaccgtt tcaacgcttc tctcggtacc taccacgatc tcctcaagat catcaaggac 6840

aaggacttcc tcgacaacga ggagaacgag gacatcctcg aggacatcgt cctcactctt 6900

actctcttcg aggataggga gatgatcgag gagaggctca agacttacgc tcatctcttc 6960

gatgacaagg ttatgaagca gctcaagcgt cgccgttaca ccggttgggg taggctctcc 7020

cgcaagctca tcaacggtat cagggataag cagagcggca agactatcct cgacttcctc 7080

aagtctgatg gtttcgctaa caggaacttc atgcagctca tccacgatga ctctcttacc 7140

ttcaaggagg atattcagaa ggctcaggtg tccggtcagg gcgactctct ccacgagcac 7200

attgctaacc ttgctggttc ccctgctatc aagaagggca tccttcagac tgttaaggtt 7260

gtcgatgagc ttgtcaaggt tatgggtcgt cacaagcctg agaacatcgt catcgagatg 7320

gctcgtgaga accagactac ccagaagggt cagaagaact cgagggagcg catgaagagg 7380

attgaggagg gtatcaagga gcttggttct cagatcctta aggagcaccc tgtcgagaac 7440

acccagctcc agaacgagaa gctctacctc tactacctcc agaacggtag ggatatgtac 7500

gttgaccagg agctcgacat caacaggctt tctgactacg acgtcgacca cattgttcct 7560

cagtctttcc ttaaggatga ctccatcgac aacaaggtcc tcacgaggtc cgacaagaac 7620

aggggtaagt cggacaacgt cccttccgag gaggttgtca agaagatgaa gaactactgg 7680

aggcagcttc tcaacgctaa gctcattacc cagaggaagt tcgacaacct cacgaaggct 7740

gagaggggtg gcctttccga gcttgacaag gctggtttca tcaagaggca gcttgttgag 7800

acgaggcaga ttaccaagca cgttgctcag atcctcgatt ctaggatgaa caccaagtac 7860

gacgagaacg acaagctcat ccgcgaggtc aaggtgatca ccctcaagtc caagctcgtc 7920

tccgacttcc gcaaggactt ccagttctac aaggtccgcg agatcaacaa ctaccaccac 7980

gctcacgatg cttaccttaa cgctgtcgtt ggtaccgctc ttatcaagaa gtaccctaag 8040

cttgagtccg agttcgtcta cggtgactac aaggtctacg acgttcgtaa gatgatcgcc 8100

aagtccgagc aggagatcgg caaggccacc gccaagtact tcttctactc caacatcatg 8160

aacttcttca agaccgagat caccctcgcc aacggcgaga tccgcaagcg ccctcttatc 8220

gagacgaacg gtgagactgg tgagatcgtt tgggacaagg gtcgcgactt cgctactgtt 8280

cgcaaggtcc tttctatgcc tcaggttaac atcgtcaaga agaccgaggt ccagaccggt 8340

ggcttctcca aggagtctat ccttccaaag agaaactcgg acaagctcat cgctaggaag 8400

aaggattggg accctaagaa gtacggtggt ttcgactccc ctactgtcgc ctactccgtc 8460

ctcgtggtcg ccaaggtgga gaagggtaag tcgaagaagc tcaagtccgt caaggagctc 8520

ctcggcatca ccatcatgga gcgctcctcc ttcgagaaga acccgatcga cttcctcgag 8580

gccaagggct acaaggaggt caagaaggac ctcatcatca agctccccaa gtactctctt 8640

ttcgagctcg agaacggtcg taagaggatg ctggcttccg ctggtgagct ccagaagggt 8700

aacgagcttg ctcttccttc caagtacgtg aacttcctct acctcgcctc ccactacgag8760

aagctcaagg gttcccctga ggataacgag cagaagcagc tcttcgtgga gcagcacaag 8820

cactacctcg acgagatcat cgagcagatc tccgagttct ccaagcgcgt catcctcgct 8880

gacgctaacc tcgacaaggt cctctccgcc tacaacaagc accgcgacaa gcccatccgc 8940

gagcaggccg agaacatcat ccacctcttc acgctcacga acctcggcgc ccctgctgct 9000

ttcaagtact tcgacaccac catcgacagg aagcgttaca cgtccaccaa ggaggttctc 9060

gacgctactc tcatccacca gtccatcacc ggtctttacg agactcgtat cgacctttcc 9120

cagcttggtg gtgataagag gaccgccgac ggcagcgagt tcgagccgaa gaagaagagg 9180

aaggtgtaga ctagttcagc cagtttggtg gagctgccga tgtgcctggt cgtcccgagc 9240

ctctgttcgt caagtatttg tggtgctgat gtctacttgt gtctggttta atggaccatc 9300

gagtccgtat gatatgttag ttttatgaaa cagtttcctg tgggacagca gtatgcttta 9360

tgaataagtt ggatttgaac ctaaatatgt gctcaatttg ctcatttgca tctcattcct 9420

gttgatgttt tatctgagtt gcaagtttga aaatgctgca tattcttatt aaatcgtcat 9480

ttacttttat cttaatgagc tttgcaatgg cctatgggat ataaaagaga tcgttcaaac 9540

atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 9600

taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 9660

atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 9720

aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 9780

cggcgcctgt ccgggcgcgc ctggtggatc gtccgcctag gctgcagtgc agcgtgaccc 9840

ggtcgtgccc ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac 9900

atattttttt tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa 9960

actttactct acgaataata taatctatag tactacaata atatcagtgt tttagagaat 10020

catataaatg aacagttaga catggtctaa aggacaattg agtattttga caacaggact 10080

ctacagtttt atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc 10140

tatataatac ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt 10200

tatagactaa tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa 10260

ctaaaactct attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa 10320

gtgactaaaa attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc 10380

ttgtttcgag tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa 10440

ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg 10500

ctgcctctgg acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca 10560

tccagaaatt gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc 10620

ctctcacggc accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc 10680

ctcgcccgcc gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt 10740

tcggagcgca cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt 10800

caaggtacgc cgctcgtcct cccccccccc ccctctctac cttctctaga tcggcgttcc 10860

ggtccatggt tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt 10920

gtgttagatc cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc 10980

tgattgctaa cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg 11040

cagacgggat cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt 11100

tcctttattt caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt 11160

tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct 11220

gtttcaaact acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt 11280

catagttacg aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg 11340

atgcgggttt tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt 11400

ggtgtggttg ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac 11460

ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag 11520

tttaagatgg atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg 11580

atgcatatac atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat 11640

ctattataat aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg 11700

catatgcagc agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct 11760

tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcaggagctc 11820

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 11880

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 11940

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 12000

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 12060

ggggagttta gcgagagcct gacctattgc atctcccgcc gttcacaggg tgtcacgttg 12120

caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 12180

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 12240

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 12300

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 12360

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 12420

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 12480

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 12540

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 12600

cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 12660

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 12720

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 12780

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 12840

aaatagagta gatgccgacc gggatctgtc gatcgacaag ctcgagtttc tccataataa 12900

tgtgtgagta gttcccagat aagggaatta gggttcctat agggtttcgc tcatgtgttg 12960

agcatataag aaacccttagtatgtatttg tatttgtaaa atacttctat caataaaatt 13020

tctaattcct aaaaccaaaa tccagtacta aaatccagat cccccgaatt aattcggcgt 13080

taattcagcc tgcaggacgc gtttaattaa gtgcacgcgg ccgcctactt agtcaagagc 13140

ctcgcacgcg actgtcacgc ggccaggatc gcctcgtgag cctcgcaatc tgtacctagt 13200

gtttaaacta tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt 13260

ttattagaat aacggatatt taaaagggcg tgaaaaggtt tatccgttcg tccatttgta 13320

tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta ctttgatcca acccctccgc 13380

tgctatagtg cagtcggctt ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg 13440

cacaagtcct aagttacgcg acaggctgcc gccctgccct tttcctggcg ttttcttgtc 13500

gcgtgtttta gtcgcataaa gtagaatact tgcgactaga accggagaca ttacgccatg 13560

aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga cgaccaggac 13620

ttgaccaacc aacgggccga actgcacgcg gccggctgca ccaagctgtt ttccgagaag 13680

atcaccggca ccaggcgcga ccgcccggag ctggccagga tgcttgacca cctacgccct 13740

ggcgacgttg tgacagtgac caggctagac cgcctggccc gcagcacccg cgacctactg 13800

gacattgccg agcgcatcca ggaggccggc gcgggcctgc gtagcctggc agagccgtgg 13860

gccgacacca ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag 13920

ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga 13980

ggcgtgaagt ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag 14040

ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc 14100

tcgaccctgt accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg 14160

cgcggtgcct tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat 14220

gaacgccaag aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca 14280

ttaccgaaga gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc 14340

acgtctcaac cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg 14400

cctggccggc cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg 14460

tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata 14520

aacaaatacg caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc 14580

aggcaagacg accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt 14640

tctgttagtc gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga 14700

tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat 14760

cggccggcgc gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc 14820

cgcgatcaag gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg 14880

ggccaccgcc gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct 14940

acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc 15000

cgaggcgctg gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag 15060

ctacccaggc actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc 15120

tgcccgcgag gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga 15180

ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc 15240

agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac 15300

tttcagttgc cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag 15360

accattaccg agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga 15420

ataaatgagt agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc 15480

aggcaccgac gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag 15540

cggctgggtt gtctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc 15600

gtgacggtcg caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg 15660

tggagaagtt gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc 15720

ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg 15780

cagccggtgc gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg 15840

ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt 15900

tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg 15960

ggcacgtaga ggtttccgca gggccggccg gcatggccag tgtgtgggat tacgacctgg 16020

tactgatggc ggtttcccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag 16080

acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag 16140

ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc 16200

acgttgccat gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg 16260

gtgaagcctt gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca 16320

tcgagatcga gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg 16380

tgctgacggt tcaccccgat tactttttga tcgatcccgg catcggccgt tttctctacc 16440

gcctggcacg ccgcgccgca ggcaaggcag aagccagatg gttgttcaag acgatctacg 16500

aacgcagtgg cagcgccgga gagttcaaga agttctgttt caccgtgcgc aagctgatcg 16560

ggtcaaatga cctgccggag tacgatttga aggaggaggc ggggcaggct ggcccgatcc 16620

tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg 16680

agcagatgct agggcaaatt gccctagcag gggaaaaagg tcgaaaaggt ctctttcctg 16740

tggatagcac gtacattggg aacccaaagc cgtacattgg gaaccggaac ccgtacattg 16800

ggaacccaaa gccgtacatt gggaaccggt cacacatgta agtgactgat ataaaagaga 16860

aaaaaggcga tttttccgcc taaaactctt taaaacttat taaaactctt aaaacccgcc 16920

tggcctgtgc ataactgtct ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc 16980

ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc 17040

gctcaaaaat ggctggccta cggccaggca atctaccagg gcgcggacaa gccgcgccgt 17100

cgccactcga ccgccggcgc ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac 17160

ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat 17220

gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca 17280

gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag 17340

agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga 17400

gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 17460

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 17520

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 17580

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 17640

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 17700

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 17760

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 17820

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 17880

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 17940

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 18000

cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 18060

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 18120

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 18180

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 18240

actcacgtta agggattttg gtcatgcatt ctaggtacta aaacaattca tccagtaaaa 18300

tataatattt tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga 18360

catactgttc ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc 18420

acttgtccgc cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca 18480

caaagatgtt gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt 18540

ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa tggagtgtct tcttcccagt 18600

tttcgcaatc cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc 18660

tgtctaagct attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc 18720

actccgcata cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc 18780

aaaggacgcc atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt 18840

gcaggacctt tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt 18900

ccacatcata ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat 18960

tttctcccac cagcttatat accttagcag gagacattcc ttccgtatct tttacgcagc 19020

ggtatttttc gatcagtttt ttcaattccg gtgatattct cattttagcc atttattatt 19080

tccttcctct tttctacagt atttaaagat accccaagaa gctaattata acaagacgaa 19140

ctccaattca ctgttccttg cattctaaaa ccttaaatac cagaaaacag ctttttcaaa 19200

gttgttttca aagttggcgt ataacatagt atcgacggag ccgattttga aaccgcggtg 19260

atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc gcgagatcat 19320

ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg taacatgagc 19380

aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga tgggctgcct 19440

gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggct 19494

<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>1367

<212>PRT

<213>Artificial Sequence

<400>4

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 4045

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 Ser

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Gly Glu 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 Pro

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Leu Ser Gln Leu Gly Gly Asp

1365

<210>5

<211>117

<212>DNA

<213>Artificial Sequence

<400>5

gactacaagg accacgacgg cgactacaag gatcatgaca tcgactacaa ggacgacgac 60

gacaagatgg ccccgaagaa gaagaggaaa gtgggcatcc acggcgtgcc ggccgcc 117

<210>6

<211>207

<212>DNA

<213>Artificial Sequence

<400>6

gactacaagg accacgacgg ggattacaaa gaccacgaca tagactacaa ggatgacgat 60

gacaaaatgg caccgaagaa aaaaaggaag gtcggcggct ccccgaagaa aaaaaggaag 120

gtcggcggct ccccgaagaa aaaaaggaag gtcggcggct ccccgaagaa aaaaaggaag 180

gtcggaatcc atggcgttcc agctgcc 207

<210>7

<211>17

<212>PRT

<213>Artificial Sequence

<400>7

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

1 5 10 15

Val

<210>8

<211>31

<212>PRT

<213>Artificial Sequence

<400>8

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val

20 25 30

<210>9

<211>7

<212>PRT

<213>Artificial Sequence

<400>9

Pro Lys Lys Lys Arg Lys Val

1 5

<210>10

<211>61

<212>PRT

<213>Artificial Sequence

<400>10

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val Gly

20 25 30

Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys

35 40 45

Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

50 55 60

<210>11

<211>37

<212>PRT

<213>Artificial Sequence

<400>11

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys

1 5 10 15

Val Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys

20 25 30

Lys Lys Arg Lys Val

35

<210>12

<211>20

<212>DNA

<213>Artificial Sequence

<400>12

catagcactc aatgcggtct 20

<210>13

<211>2327

<212>PRT

<213>Artificial Sequence

<400>13

Met Thr Ser Thr His Val Ala Thr Leu Gly Val Gly Ala Gln Ala Pro

1 5 10 15

Pro Arg His Gln Lys Lys Ser Ala Gly Thr Ala Phe Val Ser Ser Gly

20 25 30

Ser Ser Arg Pro Ser Tyr Arg Lys Asn Gly Gln Arg Thr Arg Ser Leu

35 40 45

Arg Glu Glu Ser Asn Gly Gly Val Ser Asp Ser Lys Lys Leu Asn His

50 55 60

Ser Ile Arg Gln Gly Leu Ala Gly Ile Ile Asp Leu Pro Asn Asp Ala

65 70 75 80

Ala Ser Glu Val Asp Ile Ser His Gly Ser Glu Asp Pro Arg Gly Pro

85 90 95

Thr Val Pro Gly Ser Tyr Gln Met Asn Gly Ile Ile Asn Glu Thr His

100 105 110

Asn Gly Arg His Ala Ser Val Ser Lys Val Val Glu Phe Cys Thr Ala

115 120 125

Leu Gly Gly Lys Thr Pro Ile His Ser Val Leu Val Ala Asn Asn Gly

130 135 140

Met Ala Ala Ala Lys Phe Met Arg Ser Val Arg Thr Trp Ala Asn Asp

145 150155 160

Thr Phe Gly Ser Glu Lys Ala Ile Gln Leu Ile Ala Met Ala Thr Pro

165 170 175

Glu Asp Leu Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe

180 185 190

Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln

195 200 205

Leu Ile Val Glu Ile Ala Glu Arg Thr Gly Val Ser Ala Val Trp Pro

210 215 220

Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Thr

225 230 235 240

Ala Lys Gly Ile Val Phe Leu Gly Pro Pro Ala Ser Ser Met His Ala

245 250 255

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

260 265 270

Pro Thr Leu Ala Trp Ser Gly Ser His Val Glu Val Pro Leu Glu Cys

275 280 285

Cys Leu Asp Ser Ile Pro Asp Glu Met Tyr Arg Lys Ala Cys Val Thr

290 295 300

Thr Thr Glu Glu Ala Val Ala Ser Cys Gln Val Val Gly Tyr Pro Ala

305 310315 320

Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Lys Val

325 330 335

His Asn Asp Asp Glu Val Arg Thr Leu Phe Lys Gln Val Gln Gly Glu

340 345 350

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

355 360 365

His Leu Glu Val Gln Leu Leu Cys Asp Gln Tyr Gly Asn Val Ala Ala

370 375 380

Leu His Ser Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile Ile

385 390 395 400

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

405 410 415

Glu Gln Ala Ala Arg Arg Leu Ala Lys Ala Val Gly Tyr Val Gly Ala

420 425 430

Ala Thr Val Glu Tyr Leu Tyr Ser Met Glu Thr Gly Glu Tyr Tyr Phe

435 440 445

Leu Glu Leu Asn Pro Arg Leu Gln Val Glu His Pro Val Thr Glu Trp

450 455 460

Ile Ala Glu Val Asn Leu Pro Ala Ala Gln Val Ala Val Gly Met Gly

465 470 475 480

Ile Pro Leu Trp Gln Ile Pro Glu Ile Arg Arg Phe Tyr Gly Met Asn

485 490 495

His Gly Gly Gly Tyr Asp Leu Trp Arg Lys Thr Ala Ala Leu Ala Thr

500 505 510

Pro Phe Asn Phe Asp Glu Val Asp Ser Lys Trp Pro Lys Gly His Cys

515 520 525

Val Ala Val Arg Ile Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro

530 535 540

Thr Gly Gly Lys Val Lys Glu Ile Ser Phe Lys Ser Lys Pro Asn Val

545 550 555 560

Trp Ala Tyr Phe Ser Val Lys Ser Gly Gly Gly Ile His Glu Phe Ala

565 570 575

Asp Ser Gln Phe Gly His Val Phe Ala Tyr Gly Thr Thr Arg Ser Ala

580 585 590

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

595 600 605

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

610 615 620

Asp Phe Arg Glu Asn Lys Ile His Thr Gly Trp Leu Asp Thr Arg Ile

625 630 635640

Ala Met Arg Val Gln Ala Glu Arg Pro Pro Trp Tyr Ile Ser Val Val

645 650 655

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

660 665 670

Asp Tyr Val Gly Tyr Leu Thr Lys Gly Gln Ile Pro Pro Lys His Ile

675 680 685

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

690 695 700

Thr Ile Asp Thr Val Arg Ser Gly His Gly Ser Tyr Arg Leu Arg Met

705 710 715 720

Asn Gly Ser Thr Val Asp Ala Asn Val Gln Ile Leu Cys Asp Gly Gly

725 730 735

Leu Leu Met Gln Leu Asp Gly Asn Ser His Val Ile Tyr Ala Glu Glu

740 745 750

Glu Ala Ser Gly Thr Arg Leu Leu Ile Asp Gly Lys Thr Cys Met Leu

755 760 765

Gln Asn Asp His Asp Pro Ser Lys Leu Leu Ala Glu Thr Pro Cys Lys

770 775 780

Leu Leu Arg Phe Leu Val Ala Asp Gly Ala His Val Asp Ala Asp Val

785 790 795800

Pro Tyr Ala Glu Val Glu Val Met Lys Met Cys Met Pro Leu Leu Ser

805 810 815

Pro Ala Ser Gly Val Ile His Val Val Met Ser Glu Gly Gln Ala Met

820 825 830

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

835 840 845

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

850 855 860

Pro Ile Ala Ala Ser Gly Gln Val His Lys Leu Cys Ala Ala Ser Leu

865 870 875 880

Asn Ala Cys Arg Met Ile Leu Ala Gly Tyr Glu His Asp Ile Asp Lys

885 890 895

Val Val Pro Glu Leu Val Tyr Cys Leu Asp Thr Pro Glu Leu Pro Phe

900 905 910

Leu Gln Trp Glu Glu Leu Met Ser Val Leu Ala Thr Arg Leu Pro Arg

915 920 925

Asn Leu Lys Ser Glu Leu Glu Gly Lys Tyr Glu Glu Tyr Lys Val Lys

930 935 940

Phe Asp Ser Gly Ile Ile Asn Asp Phe Pro Ala Asn Met Leu Arg Val

945 950 955 960

Ile Ile Glu Glu Asn Leu Ala Cys Gly Ser Glu Lys Glu Lys Ala Thr

965 970 975

Asn Glu Arg Leu Val Glu Pro Leu Met Ser Leu Leu Lys Ser Tyr Glu

980 985 990

Gly Gly Arg Glu Ser His Ala His Phe Val Val Lys Ser Leu Phe Glu

995 1000 1005

Glu Tyr Leu Tyr Val Glu Glu Leu Phe Ser Asp Gly Ile Gln Ser Asp

1010 1015 1020

Val Ile Glu Arg Leu Arg Leu Gln His Ser Lys Asp Leu Gln Lys Val

1025 1030 1035 1040

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

1045 1050 1055

Ile Leu Lys Leu Met Glu Ser Leu Val Tyr Pro Asn Pro Ala Ala Tyr

1060 1065 1070

Arg Asp Gln Leu Ile Arg Phe Ser Ser Leu Asn His Lys Ala Tyr Tyr

1075 1080 1085

Lys Leu Ala Leu Lys Ala Ser Glu Leu Leu Glu Gln Thr Lys Leu Ser

1090 1095 1100

Glu Leu Arg Ala Arg Ile Ala Arg Ser Leu Ser Glu Leu Glu Met Phe

1105 1110 1115 1120

Thr Glu Glu Ser Lys Gly Leu Ser Met His Lys Arg Glu Ile Ala Ile

1125 1130 1135

Lys Glu Ser Met Glu Asp Leu Val Thr Ala Pro Leu Pro Val Glu Asp

1140 1145 1150

Ala Leu Ile Ser Leu Phe Asp Cys Ser Asp Thr Thr Val Gln Gln Arg

1155 1160 1165

Val Ile Glu Thr Tyr Ile Ala Arg Leu Tyr Gln Pro His Leu Val Lys

1170 1175 1180

Asp Ser Ile Lys Met Lys Trp Ile Glu Ser Gly Val Ile Ala Leu Trp

1185 1190 1195 1200

Glu Phe Pro Glu Gly His Phe Asp Ala Arg Asn Gly Gly Ala Val Leu

1205 1210 1215

Gly Asp Lys Arg Trp Gly Ala Met Val Ile Val Lys Ser Leu Glu Ser

1220 1225 1230

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

1235 1240 1245

Ser Ser Glu Gly Asn Met Met His Ile Ala Leu Leu Gly Ala Asp Asn

1250 1255 1260

Lys Met His Ile Ile Gln Glu Ser Gly Asp Asp Ala Asp Arg Ile Ala

1265 1270 1275 1280

Lys Leu Pro Leu Ile Leu Lys Asp Asn Val Thr Asp Leu His Ala Ser

1285 1290 1295

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

1300 1305 1310

Thr Met Arg Arg Thr Phe Leu Trp Ser Asp Glu Lys Leu Ser Tyr Glu

1315 1320 1325

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

1330 1335 1340

Glu Leu Asp Lys Leu Lys Val Lys Gly Tyr Asn Glu Met Lys Tyr Thr

1345 1350 1355 1360

Pro Ser Arg Asp Arg Gln Trp His Ile Tyr Thr Leu Arg Asn Thr Glu

1365 1370 1375

Asn Pro Lys Met Leu His Arg Val Phe Phe Arg Thr Leu Val Arg Gln

1380 1385 1390

Pro Ser Val Ser Asn Lys Phe Ser Ser Gly Gln Ile Gly Asp Met Glu

1395 1400 1405

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

1410 1415 1420

Arg Ser Leu Met Thr Ala Ile Glu Glu Leu Glu Leu His Ala Ile Arg

1425 1430 1435 1440

Thr Gly His Ser His Met Tyr Leu His Val Leu Lys Glu Gln Lys Leu

1445 1450 1455

Leu Asp Leu Val Pro Val Ser Gly Asn Thr Val Leu Asp Val Gly Gln

1460 1465 1470

Asp Glu Ala Thr Ala Tyr Ser Leu Leu Lys Glu Met Ala Met Lys Ile

1475 1480 1485

His Glu Leu Val Gly Ala Arg Met His His Leu Ser Val Cys Gln Trp

1490 1495 1500

Glu Val Lys Leu Lys Leu Asp Cys Asp Gly Pro Ala Ser Gly Thr Trp

1505 1510 1515 1520

Arg Ile Val Thr Thr Asn Val Thr Ser His Thr Cys Thr Val Asp Ile

1525 1530 1535

Tyr Arg Glu Met Glu Asp Lys Glu Ser Arg Lys Leu Val Tyr His Pro

1540 1545 1550

Ala Thr Pro Ala Ala Gly Pro Leu His Gly Val Ala Leu Asn Asn Pro

1555 1560 1565

Tyr Gln Pro Leu Ser Val Ile Asp Leu Lys Arg Cys Ser Ala Arg Asn

1570 1575 1580

Asn Arg Thr Thr Tyr Cys Tyr Asp Phe Pro Leu Ala Phe Glu Thr Ala

1585 1590 15951600

Val Arg Lys Ser Trp Ser Ser Ser Thr Ser Gly Ala Ser Lys Gly Val

1605 1610 1615

Glu Asn Ala Gln Cys Tyr Val Lys Ala Thr Glu Leu Val Phe Ala Asp

1620 1625 1630

Lys His Gly Ser Trp Gly Thr Pro Leu Val Gln Met Asp Arg Pro Ala

1635 1640 1645

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

1650 1655 1660

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

1665 1670 1675 1680

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

1685 1690 1695

Ala Val Thr Asn Leu Ala Cys Glu Lys Lys Leu Pro Leu Ile Tyr Leu

1700 1705 1710

Ala Ala Asn Ser Gly Ala Arg Ile Gly Ile Ala Asp Glu Val Lys Ser

1715 1720 1725

Cys Phe Arg Val Gly Trp Ser Asp Asp Gly Ser Pro Glu Arg Gly Phe

1730 1735 1740

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

1745 1750 17551760

Val Ile Ala His Lys Met Gln Leu Asp Ser Gly Glu Ile Arg Trp Val

1765 1770 1775

Ile Asp Ser Val Val Gly Lys Glu Asp Gly Leu Gly Val Glu Asn Ile

1780 1785 1790

His Gly Ser Ala Ala Ile Ala Ser Ala Tyr Ser Arg Ala Tyr Lys Glu

1795 1800 1805

Thr Phe Thr Leu Thr Phe Val Thr Gly Arg Thr Val Gly Ile Gly Ala

1810 1815 1820

Tyr Leu Ala Arg Leu Gly Ile Arg Cys Ile Gln Arg Leu Asp Gln Pro

1825 1830 1835 1840

Ile Ile Leu Thr Gly Tyr Ser Ala Leu Asn Lys Leu Leu Gly Arg Glu

1845 1850 1855

Val Tyr Ser Ser His Met Gln Leu Gly Gly Pro Lys Ile Met Ala Thr

1860 1865 1870

Asn Gly Val Val His Leu Thr Val Ser Asp Asp Leu Glu Gly Val Ser

1875 1880 1885

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

1890 1895 1900

Leu Pro Val Thr Thr Pro Leu Asp Pro Pro Asp Arg Pro Val Ala Tyr

1905 1910 19151920

Ile Pro Glu Asn Ser Cys Asp Pro Arg Ala Ala Ile Arg Gly Val Asp

1925 1930 1935

Asp Ser Gln Gly Lys Trp Leu Gly Gly Met Phe Asp Lys Asp Ser Phe

1940 1945 1950

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

1955 1960 1965

Lys Leu Gly Gly Ile Pro Val Gly Val Ile Ala Val Glu Thr Gln Thr

1970 1975 1980

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

1985 1990 1995 2000

Gln Ser Val Pro Arg Ala Gly Gln Val Trp Phe Pro Asp Ser Ala Thr

2005 2010 2015

Lys Thr Ala Gln Ala Leu Leu Asp Phe Asn Arg Glu Gly Leu Pro Leu

2020 2025 2030

Phe Ile Leu Ala Asn Trp Arg Gly Phe Ser Gly Gly Gln Arg Asp Leu

2035 2040 2045

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

2050 2055 2060

Thr Tyr Asn Gln Pro Ala Phe Val Tyr Ile Pro Met Ala Ala Glu Leu

2065 2070 20752080

Arg Gly Gly Ala Trp Val Val Val Asp Ser Lys Ile Asn Pro Asp Arg

2085 2090 2095

Ile Glu Cys Tyr Ala Glu Arg Thr Ala Lys Gly Asn Val Leu Glu Pro

2100 2105 2110

Gln Gly Leu Ile Glu Ile Lys Phe Arg Ser Glu Glu Leu Gln Asp Cys

2115 2120 2125

Met Ser Arg Leu Asp Pro Thr Leu Ile Asp Leu Lys Ala Lys Leu Glu

2130 2135 2140

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

2145 2150 2155 2160

Ile Glu Ala Arg Thr Lys Gln Leu Met Pro Leu Tyr Thr Gln Ile Ala

2165 2170 2175

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

2180 2185 2190

Gly Val Ile Lys Lys Val Val Asp Trp Glu Glu Ser Arg Ser Phe Phe

2195 2200 2205

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

2210 2215 2220

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

2225 2230 22352240

Leu Ile Lys Lys Trp Tyr Ser Ala Ser His Ala Ala Glu Trp Asp Asp

2245 2250 2255

Asp Asp Ala Phe Val Ala Trp Met Asp Asn Pro Glu Asn Tyr Lys Asp

2260 2265 2270

Tyr Ile Gln Tyr Leu Lys Ala Gln Arg Val Ser Gln Ser Leu Ser Ser

2275 2280 2285

Leu Ser Asp Ser Ser Ser Asp Leu Gln Ala Leu Pro Gln Gly Leu Ser

2290 2295 2300

Met Leu Leu Asp Lys Met Asp Pro Ser Arg Arg Ala Gln Leu Val Glu

2305 2310 2315 2320

Glu Ile Arg Lys Val Leu Gly

2325

<210>14

<211>6984

<212>DNA

<213>Artificial Sequence

<400>14

atgacatcca cacatgtggc gacattggga gttggtgccc aggcacctcc tcgtcaccag 60

aaaaagtcag ctggcactgc atttgtatca tctgggtcat caagaccctc ataccgaaag 120

aatggtcagc gtactcggtc acttagggaa gaaagcaatg gaggagtgtc tgattccaaa 180

aagcttaacc actctattcg ccaaggtctt gctggcatca ttgacctccc aaatgacgca 240

gcttcagaag ttgatatttc acatggttcc gaagatccca gggggcctac ggtcccaggt 300

tcctaccaaa tgaatgggat tatcaatgaa acacataatg ggaggcatgc ttcagtctcc 360

aaggttgttg agttttgtac ggcacttggt ggcaaaacac caattcacag tgtattagtg 420

gccaacaatg gaatggcagc agctaagttc atgcggagtg tccgaacatg ggctaatgat 480

acttttggat cagagaaggc aattcagctg atagctatgg caactccgga ggatctgagg 540

ataaatgcag agcacatcag aattgccgat caatttgtag aggtacctgg tggaacaaac 600

aacaacaact atgcaaatgt ccaactcata gtggagatag cagagagaac aggtgtttct 660

gctgtttggc ctggttgggg tcatgcatct gagaatcctg aacttccaga tgcgctgact 720

gcaaaaggaa ttgtttttct tgggccacca gcatcatcaa tgcatgcatt aggagacaag 780

gttggctcag ctctcattgc tcaagcagct ggagttccaa cacttgcttg gagtggatca 840

catgtggaag ttcctctgga gtgttgcttg gactcaatac ctgatgagat gtatagaaaa 900

gcttgtgtta ctaccacaga ggaagcagtt gcaagttgtc aggtggttgg ttatcctgcc 960

atgattaagg catcttgggg tggtggtggt aaaggaataa ggaaggttca taatgatgat 1020

gaggttagga cattatttaa gcaagttcaa ggcgaagtac ctggttcccc aatatttatc 1080

atgaggctag ctgctcagag tcgacatctt gaagttcagt tgctttgtga tcaatatggc 1140

aacgtagcag cacttcacag tcgagattgc agtgtacaac ggcgacacca aaagataatc 1200

gaggaaggac cagttactgt tgctcctcgt gagactgtga aagagcttga gcaggcagca 1260

cggaggcttg ctaaagctgt gggttatgtt ggtgctgcta ctgttgaata cctttacagc 1320

atggaaactg gtgaatatta ttttctggaa cttaatccac ggctacaggt tgagcatcct 1380

gtcactgagt ggatagctga agtaaatttg cctgcggctc aagttgctgt tggaatgggt 1440

ataccccttt ggcagattcc agagatcagg cgcttctacg gaatgaacca tggaggaggc 1500

tatgaccttt ggaggaaaac agcagctcta gcgactccat ttaactttga tgaagtagat 1560

tctaaatggc caaaaggcca ctgcgtagct gttagaataa ctagcgagga tccagatgat 1620

gggtttaagc ctactggtgg aaaagtaaag gagataagtt tcaagagtaa accaaatgtt 1680

tgggcctatt tctcagtaaa gtctggtgga ggcatccatg aattcgctga ttctcagttc 1740

ggacatgttt ttgcgtatgg aactactaga tcggcagcaa taactaccat ggctcttgca 1800

ctaaaagagg ttcaaattcg tggagaaatt cattcaaacg tagactacac agttgaccta 1860

ttaaatgcct cagattttag agaaaataag attcatactg gttggctgga taccaggata 1920

gccatgcgtg ttcaagctga gaggcctcca tggtatattt cagtcgttgg aggggcttta 1980

tataaaacag taactgccaa cacggccact gtttctgatt atgttggtta tcttaccaag 2040

ggccagattc caccaaagca tatatccctt gtctatacga ctgttgcttt gaatatagat 2100

gggaaaaaat atacaatcga tactgtgagg agtggacatg gtagctacag attgcgaatg 2160

aatggatcaa cggttgacgc aaatgtacaa atattatgtg atggtgggct tttaatgcag 2220

ctggatggaa acagccatgt aatttatgct gaagaagagg ccagtggtac acgacttctt 2280

attgatggaa agacatgcat gttacagaat gaccatgacc catcaaagtt attagctgag 2340

acaccatgca aacttcttcg tttcttggtt gctgatggtg ctcatgttga tgctgatgta 2400

ccatatgcgg aagttgaggt tatgaagatg tgcatgcccc tcttatcacc cgcttctggt 2460

gtcatacatg ttgtaatgtc tgagggccaa gcaatgcagg ctggtgatct tatagctagg 2520

ctggatcttg atgacccttc tgctgttaag agagctgagc cgttcgaaga tacttttcca 2580

caaatgggtc tccctattgc tgcttctggc caagttcaca aattatgtgc tgcaagtctg 2640

aatgcttgtc gaatgatcct tgcggggtat gagcatgata ttgacaaggt tgtgccagag 2700

ttggtatact gcctagacac tccggagctt cctttcctgc agtgggagga gcttatgtct 2760

gttttagcaa ctagacttcc aagaaatctt aaaagtgagt tggagggcaa atatgaggaa 2820

tacaaagtaa aatttgactc tgggataatc aatgatttcc ctgccaatat gctacgagtg 2880

ataattgagg aaaatcttgc atgtggttct gagaaggaga aggctacaaa tgagaggctt 2940

gttgagcctc ttatgagcct actgaagtca tatgagggtg ggagagaaag tcatgctcac 3000

tttgttgtca agtccctttt tgaggagtat ctctatgttg aagaattgtt cagtgatgga 3060

attcagtctg atgtgattga gcgtctgcgc cttcaacata gtaaagacct acagaaggtc 3120

gtagacattg tgttgtccca ccagagtgtt agaaataaaa ctaagctgat actaaaactc 3180

atggagagtc tggtctatcc aaatcctgct gcctacaggg atcaattgat tcgcttttct 3240

tcccttaatc acaaagcgta ttacaagttg gcacttaaag ctagtgaact tcttgaacaa 3300

acaaaactta gtgagctccg tgcaagaata gcaaggagcc tttcagagct ggagatgttt 3360

actgaggaaa gcaagggtct ctccatgcat aagcgagaaa ttgccattaa ggagagcatg 3420

gaagatttag tcactgctcc actgccagtt gaagatgcgc tcatttctttatttgattgt 3480

agtgatacaa ctgttcaaca gagagtgatt gagacttata tagctcgatt ataccagcct 3540

catcttgtaa aggacagtat caaaatgaaa tggatagaat cgggtgttat tgctttatgg 3600

gaatttcctg aagggcattt tgatgcaaga aatggaggag cggttcttgg tgacaaaaga 3660

tggggtgcca tggtcattgt caagtctctt gaatcacttt caatggccat tagatttgca 3720

ctaaaggaga catcacacta cactagctct gagggcaata tgatgcatat tgctttgttg 3780

ggtgctgata ataagatgca tataattcaa gaaagtggtg atgatgctga cagaatagcc 3840

aaacttccct tgatactaaa ggataatgta accgatctgc atgcctctgg tgtgaaaaca 3900

ataagtttca ttgttcaaag agatgaagca cggatgacaa tgcgtcgtac cttcctttgg 3960

tctgatgaaa agctttctta tgaggaagag ccaattctcc ggcatgtgga acctcctctt 4020

tctgcacttc ttgagttgga caagttgaaa gtgaaaggat acaatgaaat gaagtatacc 4080

ccatcacggg atcgtcaatg gcatatctac acacttagaa atactgaaaa ccccaaaatg 4140

ttgcaccggg tatttttccg aacccttgtc aggcaaccca gtgtatccaa caagttttct 4200

tcgggccaga ttggtgacat ggaagttggg agtgctgaag aacctctgtc atttacatca 4260

accagcatat taagatcttt gatgactgct atagaggaat tggagcttca cgcaattaga 4320

actggccatt cacacatgta tttgcatgta ttgaaagaac aaaagcttct tgatcttgtt 4380

ccagtttcag ggaatacagt tttggatgtt ggtcaagatg aagctactgc atattcactt 4440

ttaaaagaaa tggctatgaa gatacatgaa cttgttggtg caagaatgca ccatctttct 4500

gtatgccaat gggaagtgaa acttaagttg gactgcgatg gtcctgccag tggtacctgg 4560

aggattgtaa caaccaatgt tactagtcac acttgcactg tggatatcta ccgtgagatg 4620

gaagataaag aatcacggaa gttagtatac catcccgcca ctccggcggc tggtcctctg 4680

catggtgtgg cactgaataa tccatatcag cctttgagtg tcattgatct caaacgctgt 4740

tctgctagga ataatagaac tacatactgc tatgattttc cactggcatt tgaaactgca 4800

gtgaggaagt catggtcctc tagtacctct ggtgcttcta aaggtgttga aaatgcccaa 4860

tgttatgtta aagctacaga gttggtattt gcggacaaac atgggtcatg gggcactcct 4920

ttagttcaaa tggaccggcc tgctgggctc aatgacattg gtatggtagc ttggaccttg 4980

aagatgtcca ctcctgaatt tcctagtggt agggagatta ttgttgttgc aaatgatatt 5040

acgttcagag ctggatcatt tggcccaagg gaagatgcat tttttgaagc tgttaccaac 5100

ctagcctgtg agaagaaact tcctcttatt tatttggcag caaattctgg tgctcgaatt 5160

ggcatagcag atgaagtgaa atcttgcttc cgtgttgggt ggtctgatga tggcagccct 5220

gaacgtgggt ttcagtacat ttatctaagc gaagaagact atgctcgtat tggcacttct 5280

gtcatagcac ataagatgca gctagacagt ggtgaaatta ggtgggttat tgattctgtt 5340

gtgggcaagg aagatggact tggtgtggag aatatacatg gaagtgctgc tattgccagt 5400

gcttattcta gggcatataa ggagacattt acacttacat ttgtgactgg aagaactgtt 5460

ggaataggag cttatcttgc tcgacttggc atccggtgca tacagcgtct tgaccagcct 5520

attattctta caggctattc tgcactgaac aagcttcttg ggcgggaagt gtacagctcc 5580

cacatgcagt tgggtggtcc caaaatcatg gcaactaatg gtgttgtcca tcttactgtt 5640

tcagatgacc ttgaaggcgt ttctaatata ttgaggtggc tcagttatgt tcctgcctac 5700

attggtggac cacttccagt aacaacaccg ttggacccac cggacagacc tgttgcatac 5760

attcctgaga actcgtgtga tcctcgagcg gctatccgtg gtgttgatga cagccaaggg 5820

aaatggttag gtggtatgtt tgataaagac agctttgtgg aaacatttga aggttgggct 5880

aagacagtgg ttactggcag agcaaagctt ggtggaattc cagtgggtgt gatagctgtg 5940

gagactcaga ccatgatgca aactatccct gctgaccctg gtcagcttga ttcccgtgag 6000

caatctgttc ctcgtgctgg acaagtgtgg tttccagatt ctgcaaccaa gactgcgcag 6060

gcattgctgg acttcaaccg tgaaggatta cctctgttca tcctcgctaa ctggagaggc 6120

ttctctggtg gacaaagaga tctttttgaa ggaattcttc aggctggctc gactattgtt 6180

gagaacctta ggacatacaa tcagcctgcc tttgtctaca ttcccatggc tgcagagcta 6240

cgaggagggg cttgggttgt ggttgatagc aagataaacc cagaccgcat tgagtgctat 6300

gctgagagga ctgcaaaagg caatgttctg gaaccgcaag ggttaattga gatcaagttc 6360

aggtcagagg aactccagga ttgcatgagt cggcttgacc caacattaat tgatctgaaa 6420

gcaaaactcg aagtagcaaa taaaaatgga agtgctgaca caaaatcgct tcaagaaaat 6480

atagaagctc gaacaaaaca gttgatgcct ctatatactc agattgcgat acggtttgct 6540

gaattgcatg atacatccct cagaatggct gcgaaaggtg tgattaagaa agttgtggac 6600

tgggaagaat cacgatcttt cttctataag agattacgga ggaggatctc tgaggatgtt 6660

cttgcaaaag aaattagagc tgtagcaggt gagcagtttt cccaccaacc agcaatcgag 6720

ctgatcaaga aatggtattc agcttcacat gcagctgaat gggatgatga cgatgctttt 6780

gttgcttgga tggataaccc tgaaaactac aaggattata ttcaatatct taaggctcaa 6840

agagtatccc aatccctctc aagtctttca gattccagct cagatttgca agccctgcca 6900

cagggtcttt ccatgttact agataagatg gatccctcta gaagagctca acttgttgaa 6960

gaaatcagga aggtccttgg ttga 6984

Claims (10)

1. A method for preparing herbicide-resistant rice with ACCase inhibition comprises the steps of enabling a receptor rice to express esgRNA, adenine deaminase, Cas9 nuclease and a nuclear localization signal bpNLS;

the esgRNA targets an OsACC1 gene target sequence; the OsACC1 gene target sequence contains a target site;

the target site is a complementary base A of a base T shown in the 6295 th position of the sequence 14;

the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS can mutate the target site in the receptor rice genome from a base A to a base G under the guidance of the esgRNA, so that the ACCase herbicide-inhibiting resistant rice with the OsACC1 protein with the 2099 th position mutated from cysteine to arginine is obtained;

the amino acid sequence of the nuclear localization signal bpNLS is A1) or A2):

A1) the amino acid sequence is a protein shown in a sequence 7;

A2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 7 in the sequence table and has the same function.

2. The method of claim 1, wherein: the number of the nuclear localization signals bpNLS is 2;

and/or the coding gene sequence of the nuclear localization signal bpNLS is a1) or a2) or a 3):

a1) a cDNA molecule or DNA molecule shown in 3796-3846 site of a sequence 1 in a sequence table;

a2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in a1) and encoding the nuclear localization signal bpNLS as claimed in claim 1;

a3) a cDNA or DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under a1) or a2) and which codes for the nuclear localization signal bpNLS as claimed in claim 1;

and/or the OsACC1 gene target point sequence is sequence 12, and the target point is base A shown in 7 th position of sequence 12.

3. The method according to claim 1 or 2, characterized in that: the esgRNA structure is as follows: an RNA-esgRNA backbone transcribed from the OsACC1 gene target sequence;

the esgRNA backbone is 1) or 2) or 3):

1) replacing T in the 617-702 th site of the sequence 1 with U to obtain an RNA molecule;

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

3) RNA molecule with 75% or more than 75% identity with the nucleotide sequence defined in 1) or 2) and with the same function.

4. A method according to any one of claims 1 to 3, wherein: the Cas9 nuclease is a Cas9n protein;

the Cas9n protein is C1) or C2):

C1) the amino acid sequence is a protein shown in a sequence 4;

C2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 4 in the sequence table and has the same function.

5. The method according to any one of claims 1 to 4, wherein: the coding gene of the Cas9n protein is c1) or c2) or c 3):

c1) a cDNA molecule or DNA molecule shown in position 5035-9135 of a sequence 1 in a sequence table;

c2) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in c1) and encoding for Cas9n as claimed in claim 4;

c3) a cDNA molecule or a DNA molecule hybridizing under stringent conditions with a nucleotide sequence defined in c1) or c2) and encoding Cas9n as claimed in claim 4.

6. The method according to any one of claims 1 to 5, wherein: the adenine deaminase is an ecTadA protein and/or an ecTadA protein;

the ecTadA protein is D1) or D2):

D1) the amino acid sequence is a protein shown in a sequence 2;

D2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

the ecTadA protein is E1) or E2):

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

E2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 in the sequence table and has the same function.

7. The method according to any one of claims 1 to 6, wherein: the coding gene of the ecTadA protein is d1) or d2) or d 3):

d1) a cDNA molecule or DNA molecule shown in 3847-4344 site of a sequence 1 in a sequence table;

d2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in d1) and encoding said ecTadA according to claim 6;

d3) a cDNA molecule or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined by d1) or d2) and which encodes the ecTadA according to claim 6;

the coding gene of the ecTadA protein is e1) or e2) or e 3):

e1) a cDNA molecule or DNA molecule shown in 4441-4938 site of a sequence 1 in a sequence table;

e2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in e1) and encoding said ecTadA according to claim 6;

e3) hybridizes under stringent conditions with a nucleotide sequence defined in e1) or e2) and encodes a cDNA molecule or a DNA molecule of the ecTadA) according to claim 6.

8. The method according to any one of claims 1 to 7, wherein: the method for expressing the esgRNA, the adenine deaminase, the Cas9 nuclease and the nuclear localization signal bpNLS in the receptor rice is characterized in that a DNA molecule for transcribing the esgRNA, an encoding gene of the ecTadA protein, an encoding gene of the Cas9n protein and an encoding gene of the nuclear localization signal bpNLS are introduced into the receptor rice.

9. The method of claim 8, wherein: the DNA molecule for transcribing the esgRNA, the coding gene of the ecTadA protein, the coding gene of the Cas9n protein and the coding gene of the nuclear localization signal bpNLS are introduced into receptor rice through recombinant expression vectors;

the recombinant expression vector comprises an expression cassette which consists of a promoter, the coding gene of the nuclear localization signal bpNLS, the coding gene of the ecTadA protein, the coding gene of the Cas9n protein, the coding gene of the nuclear localization signal bpNLS and a terminator in sequence.

10. Use of the method of any one of claims 1 to 9 for increasing the efficiency of a.g base substitutions at a target site;

or, the use of the method according to any one of claims 1 to 9 for preparing ACCase inhibiting herbicide resistant rice in which only the target site is substituted with the a.g base;

the target site is a complementary base A of a base T shown in the 6295 th position of the sequence 14;

the A.G base is replaced by a base A which is mutated into a base G.

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