CN111118060B

CN111118060B - BnALS1 mutant gene based on gene editing, protein and application thereof

Info

Publication number: CN111118060B
Application number: CN202010051263.0A
Authority: CN
Inventors: 吴健; 霍诗诗; 王幼平; 张辉; 倪萍
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-01-16
Filing date: 2020-01-16
Publication date: 2021-04-27
Anticipated expiration: 2040-01-16
Also published as: CN111118060A

Abstract

The invention discloses a BnALS1 mutant gene based on gene editing, a protein and application thereof. The invention also discloses a cytosine base editing vector, and the invention reports that single base editing is realized in rape for the first time. The base editing method provided by the invention can directionally improve the rape characters, is quicker, more efficient and more accurate compared with chemical mutagenesis, and enriches the germplasm resources of herbicide resistance of rape. The mutant materials R10 and R144 obtained by the invention have stronger resistance to tribenuron-methyl herbicide. After 20 days of spraying 15mg ai/L tribenuron-methyl 20ml (approximately equal to 5 times of the recommended amount of broadleaf weed control) on each plant in the 5-6 leaf stage, the R10 and R144 materials are not affected at all, and the transgenic receptor materials are seriously phytotoxicity (new leaves wither, old leaves stop growing and become purple).

Description

BnALS1 mutant gene based on gene editing, protein and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a brassica napus BnALS1 mutant gene based on gene editing, protein and application thereof.

Background

The field weeds are used as one of biotic stresses, compete with crops for nutrients and growth space, and seriously affect the growth and yield of the crops; on the other hand, artificial weeding and mechanical weeding increase the production cost of crops. The herbicide spraying is the most effective means for preventing and controlling weeds, and can promote the development of crop production towards high efficiency, low cost and light simplification.

The weeds in the rape fields in China are various, large in quantity and serious in harm, the yield of rapeseeds can be reduced by 15.8% according to statistics, the yield reduction range of serious fields can reach more than 50%, and the weed harm areas of winter rape areas and north spring rape areas in Yangtze river basin respectively reach 46.9% and 90%. The control of weeds in rape fields mainly depends on artificial weed removal and chemical herbicides. However, the artificial weeding increases the production cost, reduces the planting benefit and the enthusiasm of farmers, and simultaneously restricts the mechanized and large-scale planting of the rapes. Therefore, the cultivation of new herbicide-resistant rape varieties is an important measure for increasing the rape yield, improving the enthusiasm of farmers and realizing the large-scale rape planting.

Acetolactate synthase (ALS), or acetohydroxy acid synthase (AHAS), is a key enzyme in the biosynthesis of 3 branched-chain amino acids (valine, leucine, and isoleucine) in plants and microorganisms. ALS herbicides or ALS inhibitors are developed by taking ALS as a target, and spraying the ALS herbicides can reduce ALS activity in a plant body, so that synthesis of 3 branched chain amino acids is hindered, synthesis of protein is influenced, cell division is further inhibited, plant tissues are green and yellow, plant growth is inhibited, and finally the plant gradually dies. ALS herbicides have the advantages of strong selectivity, wide weed control spectrum, low dosage, low toxicity to mammals and the like, and are widely applied in production.

It is now known that alteration of certain amino acid residues of ALS can lead to a decrease in the sensitivity of the primer plant to ALS herbicides, thereby resulting in resistance to ALS herbicides. These include Ala122, Pro197, Ala205, Asp376, Arg377, Trp574, Ser653, Gly654 etc. (calculated for Arabidopsis ALS amino acid positions). Currently, herbicide-resistant materials are screened mainly by means of chemical mutagenesis, and herbicide-resistant mutants are screened from a large number of mutagenized offspring. Because of the randomness of the mutations generated by chemical mutagenesis, the screening effort is very large and blind.

The genome editing technology developed in recent years can carry out site-directed modification on biological DNA, and is widely applied to the genetic improvement of biomedicine and animals and plants. The CRISPR/Cas (Clustered regulated genomic repeats/CRISPR-associated) system is a novel genome site-directed editing technology developed in recent years, and the most mature type II CRISPR/Cas derived from Streptococcus pyogenes (Streptococcus pyogenes) in plants at present, namely the CRISPR/Cas9 system. CRISPR/Cas9 has been widely used for gene knockout and site-directed modification (including site-directed mutation and gene insertion). However, the efficiency of site-directed mutagenesis of genes based on homologous recombination is still low. Consequently, scientists have subsequently developed single base editing techniques that fuse cytosine deaminase or adenosine deaminase to nCas9(Cas9 nickase) or dCas9 (nuclear-dead Cas9) to achieve precise replacement of the single base C-T (G-a) or a-G (T-C) at a specific region of the target site that is complementarily paired to the sgRNA. Base editors based on CRISPR/Cas9 have been successfully used for a variety of plants including arabidopsis, rice, maize, wheat, and cotton, among others. However, no report that the base editing technology is applied to rape exists at present.

Disclosure of Invention

The purpose of the invention is as follows: the first technical problem to be solved by the invention is to provide a cytosine base editing vector.

The invention also aims to solve the technical problem of providing the BnALS1 mutant protein.

The invention also aims to solve the technical problem of providing a BnALS1 mutant gene or nucleic acid.

The technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector, a transgenic system or a cell.

The technical problem to be solved by the present invention is to provide a method for obtaining a plant having herbicide resistance based on gene editing technology.

The technical problem to be solved by the present invention is to provide a method for identifying plants obtained by said method based on gene editing technology.

It is therefore an object of the present invention to provide a vector and method useful for the single base editing of allotetraploid brassica napus. Pro197 of the cabbage type rape BnALS1 gene is mutated into Ser197 and Phe197 by the technology, so that the cabbage type rape material resisting sulfonylurea herbicide is obtained. The invention can provide a carrier and a method for rape base editing; provides a new resource for the breeding of the rape herbicide resistance.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a cytosine base editing vector is constructed based on a pCAMBIA1300 vector and comprises a hygromycin-resistant protein, a sgRNA expression cassette and an rAPOBEC1-nCas9-UGI fusion protein.

The sgRNA expression cassette is driven by an Arabidopsis U6 promoter, and the rAPOBEC1-nCas9-UGI fusion protein is driven by an Arabidopsis ubiqintin1 promoter.

The DNA sequence of the synthesized DNA containing the U6 promoter, the sgRNA expression cassette, the ubiqintin1 promoter, the NOs terminator and other elements is shown as SEQ ID NO:1 is shown.

The rAPOBEC1-nCas9-UGI fusion protein comprises D10ACas9 incision enzyme protein, rat cytosine deaminase and uracil DNA glucoamylase inhibitor.

Wherein, the nucleotide sequence of the cytosine base editing carrier is shown as SEQ ID NO:2, the cytosine base editing vector suitable for dicotyledonous plants such as rape is named as pCBE-Bn.

The invention also comprises a recombinant vector, wherein the recombinant vector is prepared by mixing the cytosine base editing vector with a target site sequence SEQ ID NO:3 are connected to obtain the compound.

The invention also discloses a BnALS1 mutant protein, wherein proline at position 182 of the amino acid sequence of the BnALS1 mutant protein is mutated into serine or phenylalanine, and the amino acid sequence is shown as SEQ ID NO:4 or SEQ ID NO:5, respectively.

The invention also provides a brassica napus BnALS1 mutant protein. Sequence information of the wild-type BnALS1 gene was extracted from the reference genomic database (https:// www.genoscope.cns.fr/brassicana /) of winter rape variety Darmor-bzh. The target site is designed by using CRISPR-P2.0 software (https:// CRISPR. hzau. edu. cn/CRISPR2/), and the sequence of the target site is AGGTCCCTCGCCGGATGAT (SEQ ID NO: 3). The target site is connected to an sgRNA expression cassette, and an expression vector is constructed. Then carrying out genetic transformation on the rape to obtain transgenic plants, and finally screening to obtain mutant materials R10 and R144. The C of the R10 mutant material at nucleotide 543 and 544 is mutated to T, so that proline (Pro, CCT) at amino acid 182 (relative to position 197 of Arabidopsis) is mutated to serine (Ser, TCT). The C at the 543-545 position of the R144 mutant material nucleotide is mutated to T, so that the proline (Pro, CCT) at the 182 th position of the amino acid is mutated to phenylalanine (Phe, TTT).

The invention also comprises a BnALS1 mutant gene or nucleic acid, which encodes the mutant protein, and the nucleotide sequence of the mutant protein is shown as SEQ ID NO:6 or SEQ ID NO: shown at 7.

The present disclosure also includes expression cassettes, recombinant vectors, transgenic systems or cells containing the BnALS1 mutant gene or nucleic acid.

The invention also comprises the cytosine base editing vector, the recombinant vector, the Brassica napus BnALS1 mutant protein, the mutant gene or nucleic acid, and application of the expression cassette, the recombinant vector or the cell in herbicide resistance of plants.

The present disclosure also includes a method for obtaining a plant with herbicide resistance based on gene editing technology, comprising the steps of:

1) the cytosine base editing vector or the BnALS1 editing vector edits the BnALS1 gene, so that proline at the 197 th position of amino acid of the gene is mutated into serine and phenylalanine;

2) the cytosine base editing vector or the BnALS1 editing vector of claim 5 edits a BnALS1 gene so that the C at the 543 th site and the 544 th site of the nucleotide is mutated into T or the C at the 543 th site to the 545 th site of the nucleotide is mutated into T.

Specifically, the invention further discloses a method for editing the Brassica napus BnALS1 gene by using the cytosine base editing system, so that proline (Pro, CCT) at the 197 th site of the amino acid of the Brassica napus is mutated into serine (Ser, TCT) or phenylalanine (Phe, TTT), and the novel sulfonylurea herbicide-resistant Brassica napus materials R10 and R144 are obtained.

The invention also relates to a method for identifying plants obtained by said method, based on gene editing techniques, comprising the following steps:

1) determining whether said plant has said BnALS1 mutant gene or nucleic acid;

2) determining whether said plant has said BnALS1 mutant protein.

Has the advantages that: the invention reports that single base editing is realized in rape for the first time. The base editing method provided by the invention can directionally improve the rape characters, is quicker, more efficient and more accurate compared with chemical mutagenesis, and enriches the germplasm resources of herbicide resistance of rape. The mutant materials R10 and R144 obtained by the invention have stronger resistance to tribenuron-methyl herbicide. After 20 days of spraying 15mg ai/L tribenuron-methyl 20ml (approximately equal to 5 times of the recommended amount of broadleaf weed control) on each plant in the 5-6 leaf stage, the R10 and R144 materials are not affected at all, and the transgenic receptor materials are seriously phytotoxicity (new leaves wither, old leaves stop growing and become purple).

Drawings

FIG. 1 is a diagram of a base editing vector used in the present invention;

FIG. 2 shows a positive detection electrophoretogram constructed by the edited vector BnALS1, wherein M is 2000bpMarker, lanes 1-7 are 7 picked different colonies, and lane 8 is a negative control ddH₂O；

FIG. 3, Sanger sequencing peak chart, shows that 7T 0 transgenic plants have a mutation event at the target site;

FIG. 4, TA cloning to determine the specific mutation pattern of each mutant;

FIG. 5 shows the results of treatment of homozygous mutant material tribenuron-methyl.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 construction of cytosine base editing vector

The cytosine base editing vector pCBE-Bn suitable for dicotyledonous plants such as rape and the like provided by the invention is constructed based on a pCAMBIA1300 vector (figure 1). First, IDNA sequences (shown as SEQ ID NO: 1) comprising the U6 promoter, sgRNA expression cassette, ubiqintin1 promoter, NOs terminator and other elements were synthesized in Shanghai. The pCAMBIA1300 vector was digested with HindIII and Acc65I, and the digested vector was then ligated with the synthesized DNA sequence to obtain an intermediate vector. The rice nucleotide editing vector pH-nCas9-PBE (Addgene: #98164) is used as a template, and B-NcoI-F (GAAACACAAA)CCATGCCAAAGAAGAAGAGGAAGGTT) and BE-BamHI-R (CGATCAATCA)GGATCCCTACACCTTCCGCTTCTTCTTTG) is a primer for amplifying the rAPOBEC1-nCas9-UGI element sequence. The intermediate vector is cut by BamHI and NcoI and then is subjected to In-fusion with rAPOBEC1-nCas9-UGI amplified fragment

-Uni Seamless Cloning and analysis Kit) to construct a complete rape base editing vector, wherein the sequence of the vector is shown as SEQ ID NO:2, the vector diagram is shown in figure 1.

The modified editing vector contains hygromycin-resistant protein, sgRNA expression cassette, D10A Cas9 nickase protein (nCas9), rat cytosine deaminase (rAPOBEC1) and uracil DNA glucoamylase inhibitor (UGI). The sgRNA is driven by the Arabidopsis U6 promoter, and the rAPOBEC1-nCas9-UGI fusion protein is driven by the Arabidopsis ubiqintin1 promoter.

Example 2 construction and genetic transformation of BnALS1 editing vector

Sequence information for the wild-type BnALS1 gene was obtained from the reference genomic database (https:// www.genoscope.cns.fr/brassicana /) of winter rape variety Darmor-bzh. Combining known herbicide resistance site information with target site information designed by the CRISPR-P2.0 software (https:// CRISPR. hzau. edu. cn/CRISPR2/), it was found that there are 1 suitable target sites AGGTCCCTCGCCGGATGAT at the Pro197 site.

The rape base editing vector obtained in example 1 was digested with BsaI by synthesizing the following adapter-carrying primers sgRNA-F and sgRNA-R, annealing, and then the target site was ligated to the sgRNA expression cassette by the conventional fragment vector ligation method. The specific method comprises the following steps:

(1) the primer sequence is as follows:

sgRNA-F GATTGAGGTCCCTCGCCGGATGAT

sgRNA-R AAACATCATCCGGCGAGGGACCTC

(the plus base is a linker sequence)

(2) Annealing of the primer:

mu.l sgRNA-F (10. mu.M) + 1. mu.l sgRNA-R (10. mu.M) + 8. mu.M annex Buffer (TE +50mM NaCl) were mixed well, 10min at 95 ℃ and lowered to 20 ℃ at 0.1 ℃/s.

(3) And (3) carrying out enzyme digestion on the vector:

mu.l of the canola nucleotide editing vector plasmid obtained in example 1 (about 1. mu.g) + 10. mu.l of 10 XNEBuffer + 1. mu.l of Bsa I enzyme (NEB Co.) + 84. mu.l of water at 37 ℃ for 2-3h, and the vector was recovered.

(4) Connecting:

mu.l of the annealed product + 2. mu.l of the recovered digestion vector + 0.5. mu.l of 10 XT 4 buffer + 0.5. mu. l T4 ligase (200 units/. mu.l), 15min at room temperature.

(5) And (3) transformation:

mu.l of the ligation product was added to E.coli competence for 30min on ice, heat shock at 42 ℃ for 45s, 2min on ice, 400. mu.l of nonreactive LB was added, 1h at 37 ℃, centrifugation at 5000rpm for 1min, most of the supernatant was aspirated off, 100. mu.l of liquid was left to aspirate and spread on LB + Kan plates.

(6) Colony PCR identification:

with PCR system, PCR mix 10. mu.l + M13F (19. mu.M, universal primer) 0.4. mu.l + sgRNA-R2 (19. mu.M) 0.4. mu.l + H₂O9.2. mu.l + A few bacteria were picked.

(7) And (3) PCR reaction:

7min at 95 ℃, 25s at 56 ℃, 25s at 72 ℃, 34 cycles, 5min at 72 ℃.

(8) Agarose electrophoresis detection, the electrophoretogram is shown in FIG. 2.

(9) Selecting positive bacterial colony, propagating, extracting plasmid by Beijing Quanjin biology corporation

The Plasmid MiniPrep Kit refers to the specification of the specific method, and the correct Cas9/sgRNA base editing vector is obtained through verification.

The successfully constructed Cas9/sgRNA base editing vector is transformed into the Agrobacterium GV3101 strain by a freeze-thaw method. Followed by genetic transformation of brassica napus. The specific method comprises the following steps:

(1) and (3) sterilization:

soaking Brassica napus receptor material J9712 (laboratory preservation strain) seeds with 75% alcohol for 1 min; then sterilizing with 2% sodium hypochlorite for 10-15min (time is determined according to seed contamination); finally, using sterile ddH₂And washing the seeds until the seeds have no smell of NaClO.

(2) Sowing:

1) the sterilized seeds are sown to M by tweezers burned by alcohol lamp₀20-30 granules per jar on the culture medium.

2) Placing the inoculated tissue culture tank in a tissue culture box, and culturing for 6-7d at 24 ℃ in dark light.

(3) Shaking the bacteria:

after 5-6 days of sowing, the strain of the transformation vector is activated by shaking, cultured for 14-24h at 28 ℃ and 220rpm until the OD of the bacterial liquid₆₀₀The value was 0.6.

(4) Explant preparation and infection:

1) cutting the seedling with sterile forceps and knife burned with alcohol, wherein the length of hypocotyl of each section is 0.8-1.0 cm.

2) Inoculum solution OD₆₀₀Adjusting the rotation speed of a centrifuge to 6000rpm, centrifuging for 10min, removing supernatant, resuspending the precipitate with DM solution with the same volume as the bacterial solution, and repeatedly washing the precipitate once. Then, an invasion solution is prepared according to the dilution ratio of 2mL of bacterial liquid and 20mL of LDM liquid.

3) The cut explants are placed into the prepared staining solution with the concentration, and the staining is carried out for 10min, and the following steps are taken: the infection time is not too long, otherwise the explant is dead. 20mL of bacterial liquid and preferably 150-200 explants.

(5) Placing the infected explants on filter paper for drying, and then transferring the explants to M₁In the culture medium, 20-25 explants per dish are generally suitable. Culturing at 24 ℃ in dark light.

(6) Transferring the explant to M after 36-48 h₂The culture medium was cultured in a light incubator (24 ℃ C. for 16h day/8 h night).

(7) After 20d from M₂Transfer of Medium to M₃In the medium, subculture was performed every 20d until green shoots appeared.

(8) Transfer of Green shoots into M₄In rooting culture medium. The rooting time is 2-4 weeks. After healthy and complete seedlings are grown, the seedlings can be transferred to a field or a greenhouse for normal growth and fructification.

The formula of the culture medium is as follows:

note: except that M₀The pH should be adjusted to 5.8-6.0, and the rest should be adjusted to 5.8.

Example 3 Positive identification and editing detection of tissue culture seedlings

After the regenerated seedlings of example 2 were transplanted into soil for normal production, young leaves were taken and genomic DNA was extracted using 2% CTAB method. PCR positive detection was then performed using vector specific primer M13F (GGTAACGCCAGGGTTTTCC) and sgRNA-R2 (GCCATTTGTCTGCAGAATTG). The detection shows that 217 positive plants in 230 tissue culture seedlings have a positive rate of 94.3%. The rape transgenic system used by the invention is more perfect.

We then performed editorial tests on the positive plants obtained. PCR amplification was performed using BnALS1 specific primers WJP198F (TCGACAAGAACAAGACTTTCG) and WJP198R (CACGGTCATCAAACCTAACAC), followed by direct Sanger sequencing of the amplified products. Based on the sequencing peak patterns, we found that 7 total T0 generation plants were mutated at the target site. Wherein base substitution events were present at #10, #127, #130, #144, and consecutive double peaks were present at #7, #8, and #34 from the target site, indicating the possible presence of insertion/deletion mutations (FIG. 3). We subsequently further determined the type of mutation editing the individual using TA clonal sequencing. Wherein #10, #127 and #130 are all C mutations at

positions

5 and 6 of the target site to T. The 5 th base mutation is in the third position of the codon and does not cause amino acid change; the mutation at position 6 mutated Pro to Ser (FIG. 4). #144 is a target site in which all of the C's at positions 5-7 were mutated to T, and Pro was mutated to Phe. Therefore we obtained mutants of both forms P197S and P197F.

Example 4 identification of resistance of mutants to tribenuron-methyl

The mutant strains obtained in example 3 are subjected to bagging selfing, leaf DNA of T1 plants is subjected to PCR amplification and Sanger sequencing by using BnALS1 specific primers, and finally homozygous mutant strains are separated. 10 plants of the homozygous mutant and WT are respectively selected for the treatment of tribenuron-methyl spraying. 15mg ai/L tribenuron-methyl (10% wettable powder, Henan Laienbane) is sprayed at the 5-6 leaf stage respectively, and 20ml of the wettable powder is sprayed on each plant. The using amount is approximately 3 times of the recommended concentration for controlling the broadleaf weeds in the wheat field. After 20 days, neither the P197S nor the P197F homozygous mutant material was affected at all, while its transgenic recipient material WT was severely phytologically damaged, as evidenced by new leaf withering, old leaf stopping growth and purple development (FIG. 5). The results show that the obtained homozygous mutant has stronger resistance to tribenuron-methyl herbicide. We named homozygous mutant progeny of individuals #10 and #144 as R10 and R144.

Sequence listing

<110> Yangzhou university

<120> gene editing-based BnALS1 mutant gene, protein and application thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1456

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aagcttcatt cggagttttt gtatcttgtt tcatagtttg tcccaggatt agaatgatta 60

ggcatcgaac cttcaagaat ttgattgaat aaaacatctt cattcttaag atatgaagat 120

aatcttcaaa aggcccctgg gaatctgaaa gaagagaagc aggcccattt atatgggaaa 180

gaacaatagt atttcttata taggcccatt taagttgaaa acaatcttca aaagtcccac 240

atcgcttaga taagaaaacg aagctgagtt tatatacagc tagagtcgaa gtagtgattg 300

ggagaccgag gtctcctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt 360

tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct agaaatagca 420

agttaaaata aggctagtcc gtagcgcgtg cgccaattct gcagacaaat cccgggatat 480

ttcacaaatt gaacatagac tacagaattt tagaaaacaa actttctctc tcttatctca 540

cctttatctt ttagagagaa aaagttcgat ttccggttga ccggaatgta tctttgtttt 600

ttttgttttg taacatattt cgttttccga tttagatcgg atctcctttt ccgttttgtc 660

ggaccttctt ccggtttatc cggatctaat aatatccatc ttagacttag ctaagtttgg 720

atctgttttt tggttagctc ttgtcaatcg cctcatcatc agcaagaagg tgaaattttt 780

gacaaataaa tcttagaatc atgtagtgtc tttggacctt gggaatgata gaaacgattt 840

gttatagcta ctctatgtat cagaccctga ccaagatcca acaatctcat aggttttgtg 900

catatgaaac cttcgactaa cgagaagtgg tcttttaatg agagagatat ctaaaatgtt 960

atcttaaaag cccactcaaa tctcaaggca taaggtagaa atgcaaattt ggaaagtggg 1020

ctgggccttt tgtggtaaag gcctgtaacc tagcccaata ttagcaaaac cctagacgcg 1080

tacattgaca tatataaacc cgcctcctcc ttgtttaggg tttctacgtg agagagacga 1140

aacacaaacc atggtctaga ggatcctgat tgatcgatag agctcgaatt tccccgatcg 1200

ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 1260

tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 1320

gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 1380

agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 1440

actagatcgg ggtacc 1456

<210> 2

<211> 15573

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 60

cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 120

ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 180

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg ctagagcagc 240

ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct 300

cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg 360

gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca 420

cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca 480

gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 540

ccacgtcttc aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct 600

actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 660

aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca 720

aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg 780

ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 840

gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 900

tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat 960

ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa 1020

tctatctctc tcgagctttc gcagatcccg gggggcaatg agatatgaaa aagcctgaac 1080

tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga 1140

tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat 1200

atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc 1260

actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggag tttagcgaga 1320

gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa 1380

ccgaactgcc cgctgttcta caaccggtcg cggaggctat ggatgcgatc gctgcggccg 1440

atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta 1500

catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga 1560

tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg 1620

aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga 1680

cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc 1740

aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga 1800

cgcgctactt cgagcggagg catccggagc ttgcaggatc gccacgactc cgggcgtata 1860

tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg 1920

cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc 1980

gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg 2040

ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaagaaatag agtagatgcc 2100

gaccggatct gtcgatcgac aagctcgagt ttctccataa taatgtgtga gtagttccca 2160

gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct 2220

tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca 2280

aaatccagta ctaaaatcca gatcccccga attaattcgg cgttaattca gtacattaaa 2340

aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc 2400

ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat 2460

acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact 2520

ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac 2580

ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg 2640

atcaccgcgg tttcaaaatc ggctccgtcg atactatgtt atacgccaac tttgaaaaca 2700

actttgaaaa agctgttttc tggtatttaa ggttttagaa tgcaaggaac agtgaattgg 2760

agttcgtctt gttataatta gcttcttggg gtatctttaa atactgtaga aaagaggaag 2820

gaaataataa atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata 2880

ccgctgcgta aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga 2940

aaatgaaaac ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt 3000

ggaacgggaa aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct 3060

gcactttgaa cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct 3120

ttgctcggaa gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga 3180

gtgcatcagg ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga 3240

cagccgctta gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga 3300

aaactgggaa gaagacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac 3360

ggaaaagccc gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt 3420

tgtgaaagat ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa 3480

gtggtatgac attgccttct gcgtccggtc gatcagggag gatatcgggg aagaacagta 3540

tgtcgagcta ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta 3600

tattttactg gatgaattgt tttagtacct agaatgcatg accaaaatcc cttaacgtga 3660

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 3720

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 3780

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 3840

gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 3900

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 3960

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 4020

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 4080

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 4140

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 4200

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 4260

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 4320

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 4380

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 4440

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt 4500

tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg 4560

ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg 4620

gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 4680

gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 4740

ccgtcatcac cgaaacgcgc gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg 4800

cgacggcgcg gcttgtccgc gccctggtag attgcctggc cgtaggccag ccatttttga 4860

gcggccagcg gccgcgatag gccgacgcga agcggcgggg cgtagggagc gcagcgaccg 4920

aagggtaggc gctttttgca gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc 4980

caggcgggtt ttaagagttt taataagttt taaagagttt taggcggaaa aatcgccttt 5040

tttctctttt atatcagtca cttacatgtg tgaccggttc ccaatgtacg gctttgggtt 5100

cccaatgtac gggttccggt tcccaatgta cggctttggg ttcccaatgt acgtgctatc 5160

cacaggaaag agaccttttc gacctttttc ccctgctagg gcaatttgcc ctagcatctg 5220

ctccgtacat taggaaccgg cggatgcttc gccctcgatc aggttgcggt agcgcatgac 5280

taggatcggg ccagcctgcc ccgcctcctc cttcaaatcg tactccggca ggtcatttga 5340

cccgatcagc ttgcgcacgg tgaaacagaa cttcttgaac tctccggcgc tgccactgcg 5400

ttcgtagatc gtcttgaaca accatctggc ttctgccttg cctgcggcgc ggcgtgccag 5460

gcggtagaga aaacggccga tgccgggatc gatcaaaaag taatcggggt gaaccgtcag 5520

cacgtccggg ttcttgcctt ctgtgatctc gcggtacatc caatcagcta gctcgatctc 5580

gatgtactcc ggccgcccgg tttcgctctt tacgatcttg tagcggctaa tcaaggcttc 5640

accctcggat accgtcacca ggcggccgtt cttggccttc ttcgtacgct gcatggcaac 5700

gtgcgtggtg tttaaccgaa tgcaggtttc taccaggtcg tctttctgct ttccgccatc 5760

ggctcgccgg cagaacttga gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt 5820

gtctcccttc ccttcccggt atcggttcat ggattcggtt agatgggaaa ccgccatcag 5880

taccaggtcg taatcccaca cactggccat gccggccggc cctgcggaaa cctctacgtg 5940

cccgtctgga agctcgtagc ggatcacctc gccagctcgt cggtcacgct tcgacagacg 6000

gaaaacggcc acgtccatga tgctgcgact atcgcgggtg cccacgtcat agagcatcgg 6060

aacgaaaaaa tctggttgct cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc 6120

tgccggcggt tgccgggatt ctttgcggat tcgatcagcg gccgcttgcc acgattcacc 6180

ggggcgtgct tctgcctcga tgcgttgccg ctgggcggcc tgcgcggcct tcaacttctc 6240

caccaggtca tcacccagcg ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgtc 6300

acgccgattc ctcgggcttg ggggttccag tgccattgca gggccggcag acaacccagc 6360

cgcttacgcc tggccaaccg cccgttcctc cacacatggg gcattccacg gcgtcggtgc 6420

ctggttgttc ttgattttcc atgccgcctc ctttagccgc taaaattcat ctactcattt 6480

attcatttgc tcatttactc tggtagctgc gcgatgtatt cagatagcag ctcggtaatg 6540

gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt gatcctccgc cggcaactga 6600

aagttgaccc gcttcatggc tggcgtgtct gccaggctgg ccaacgttgc agccttgctg 6660

ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc ttttgctcat tttctcttta 6720

cctcattaac tcaaatgagt tttgatttaa tttcagcggc cagcgcctgg acctcgcggg 6780

cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc ggcggcggca gtgcctgggt 6840

agctcacgcg ctgcgtgata cgggactcaa gaatgggcag ctcgtacccg gccagcgcct 6900

cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg cgacacgaca aaggccgctt 6960

gtagccttcc atccgtgacc tcaatgcgct gcttaaccag ctccaccagg tcggcggtgg 7020

cccatatgtc gtaagggctt ggctgcaccg gaatcagcac gaagtcggct gccttgatcg 7080

cggacacagc caagtccgcc gcctggggcg ctccgtcgat cactacgaag tcgcgccggc 7140

cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat gccgacaacg gttagcggtt 7200

gatcttcccg cacggccgcc caatcgcggg cactgccctg gggatcggaa tcgactaaca 7260

gaacatcggc cccggcgagt tgcagggcgc gggctagatg ggttgcgatg gtcgtcttgc 7320

ctgacccgcc tttctggtta agtacagcga taaccttcat gcgttcccct tgcgtatttg 7380

tttatttact catcgcatca tatacgcagc gaccgcatga cgcaagctgt tttactcaaa 7440

tacacatcac ctttttagac ggcggcgctc ggtttcttca gcggccaagc tggccggcca 7500

ggccgccagc ttggcatcag acaaaccggc caggatttca tgcagccgca cggttgagac 7560

gtgcgcgggc ggctcgaaca cgtacccggc cgcgatcatc tccgcctcga tctcttcggt 7620

aatgaaaaac ggttcgtcct ggccgtcctg gtgcggtttc atgcttgttc ctcttggcgt 7680

tcattctcgg cggccgccag ggcgtcggcc tcggtcaatg cgtcctcacg gaaggcaccg 7740

cgccgcctgg cctcggtggg cgtcacttcc tcgctgcgct caagtgcgcg gtacagggtc 7800

gagcgatgca cgccaagcag tgcagccgcc tctttcacgg tgcggccttc ctggtcgatc 7860

agctcgcggg cgtgcgcgat ctgtgccggg gtgagggtag ggcgggggcc aaacttcacg 7920

cctcgggcct tggcggcctc gcgcccgctc cgggtgcggt cgatgattag ggaacgctcg 7980

aactcggcaa tgccggcgaa cacggtcaac accatgcggc cggccggcgt ggtggtgtcg 8040

gcccacggct ctgccaggct acgcaggccc gcgccggcct cctggatgcg ctcggcaatg 8100

tccagtaggt cgcgggtgct gcgggccagg cggtctagcc tggtcactgt cacaacgtcg 8160

ccagggcgta ggtggtcaag catcctggcc agctccgggc ggtcgcgcct ggtgccggtg 8220

atcttctcgg aaaacagctt ggtgcagccg gccgcgtgca gttcggcccg ttggttggtc 8280

aagtcctggt cgtcggtgct gacgcgggca tagcccagca ggccagcggc ggcgctcttg 8340

ttcatggcgt aatgtctccg gttctagtcg caagtattct actttatgcg actaaaacac 8400

gcgacaagaa aacgccagga aaagggcagg gcggcagcct gtcgcgtaac ttaggacttg 8460

tgcgacatgt cgttttcaga agacggctgc actgaacgtc agaagccgac tgcactatag 8520

cagcggaggg gttggatcaa agtactttga tcccgagggg aaccctgtgg ttggcatgca 8580

catacaaatg gacgaacgga taaacctttt cacgcccttt taaatatccg ttattctaat 8640

aaacgctctt ttctcttagg tttacccgcc aatatatcct gtcaaacact gatagtttaa 8700

actgaaggcg ggaaacgaca atctgatcca agctcaagct gctctagcat tcgccattca 8760

ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 8820

cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 8880

gacgttgtaa aacgacggcc agtgccaagc ttcattcgga gtttttgtat cttgtttcat 8940

agtttgtccc aggattagaa tgattaggca tcgaaccttc aagaatttga ttgaataaaa 9000

catcttcatt cttaagatat gaagataatc ttcaaaaggc ccctgggaat ctgaaagaag 9060

agaagcaggc ccatttatat gggaaagaac aatagtattt cttatatagg cccatttaag 9120

ttgaaaacaa tcttcaaaag tcccacatcg cttagataag aaaacgaagc tgagtttata 9180

tacagctaga gtcgaagtag tgattgggag accgaggtct cctgttttag agctagaaat 9240

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 9300

tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtag cgcgtgcgcc 9360

aattctgcag acaaatcccg ggatatttca caaattgaac atagactaca gaattttaga 9420

aaacaaactt tctctctctt atctcacctt tatcttttag agagaaaaag ttcgatttcc 9480

ggttgaccgg aatgtatctt tgtttttttt gttttgtaac atatttcgtt ttccgattta 9540

gatcggatct ccttttccgt tttgtcggac cttcttccgg tttatccgga tctaataata 9600

tccatcttag acttagctaa gtttggatct gttttttggt tagctcttgt caatcgcctc 9660

atcatcagca agaaggtgaa atttttgaca aataaatctt agaatcatgt agtgtctttg 9720

gaccttggga atgatagaaa cgatttgtta tagctactct atgtatcaga ccctgaccaa 9780

gatccaacaa tctcataggt tttgtgcata tgaaaccttc gactaacgag aagtggtctt 9840

ttaatgagag agatatctaa aatgttatct taaaagccca ctcaaatctc aaggcataag 9900

gtagaaatgc aaatttggaa agtgggctgg gccttttgtg gtaaaggcct gtaacctagc 9960

ccaatattag caaaacccta gacgcgtaca ttgacatata taaacccgcc tcctccttgt 10020

ttagggtttc tacgtgagag agacgaaaca caaaccatgc caaagaagaa gaggaaggtt 10080

tcatcggaga ccggccctgt tgctgttgac cccaccctgc ggcggagaat cgagccacac 10140

gagttcgagg tgttcttcga cccaagggag ctccgcaagg agacgtgcct cctgtacgag 10200

atcaactggg gcggcaggca ctccatctgg aggcacacca gccaaaacac caacaagcac 10260

gtggaggtca acttcatcga gaagttcacc accgagaggt acttctgccc aaacacccgc 10320

tgctccatca cctggttcct gtcctggagc ccatgcggcg agtgctccag ggccatcacc 10380

gagttcctca gccgctaccc acacgtcacc ctgttcatct acatcgccag gctctaccac 10440

cacgccgacc caaggaacag gcagggcctc cgcgacctga tctccagcgg cgtgaccatc 10500

caaatcatga ccgagcagga gtccggctac tgctggagga acttcgtcaa ctactcccca 10560

agcaacgagg cccactggcc aaggtaccca cacctctggg tgcgcctcta cgtgctcgag 10620

ctgtactgca tcatcctcgg cctgccacca tgcctcaaca tcctgaggcg caagcaacca 10680

cagctgacct tcttcaccat cgccctccaa agctgccact accagaggct cccaccacac 10740

atcctgtggg ctaccggcct caagtccggc agcgagacgc caggcacctc cgagagcgct 10800

acgcctgaac ttaaggacaa gaagtactcg atcggcctcg ccatcgggac gaactcagtt 10860

ggctgggccg tgatcaccga cgagtacaag gtgccctcta agaagttcaa ggtcctgggg 10920

aacaccgacc gccattccat caagaagaac ctcatcggcg ctctcctgtt cgacagcggg 10980

gagaccgctg aggctacgag gctcaagaga accgctaggc gccggtacac gagaaggaag 11040

aacaggatct gctacctcca agagattttc tccaacgaga tggccaaggt tgacgattca 11100

ttcttccacc gcctggagga gtctttcctc gtggaggagg ataagaagca cgagcggcat 11160

cccatcttcg gcaacatcgt ggacgaggtt gcctaccacg agaagtaccc tacgatctac 11220

catctgcgga agaagctcgt ggactccacc gataaggcgg acctcagact gatctacctc 11280

gctctggccc acatgatcaa gttccgcggc catttcctga tcgaggggga tctcaaccca 11340

gacaacagcg atgttgacaa gctgttcatc caactcgtgc agacctacaa ccaactcttc 11400

gaggagaacc cgatcaacgc ctctggcgtg gacgcgaagg ctatcctgtc cgcgaggctc 11460

tcgaagtcca ggaggctgga gaacctgatc gctcagctcc caggcgagaa gaagaacggc 11520

ctgttcggga acctcatcgc tctcagcctg gggctcaccc cgaacttcaa gtcgaacttc 11580

gatctcgctg aggacgccaa gctgcaactc tccaaggaca cctacgacga tgacctcgat 11640

aacctcctgg cccagatcgg cgatcaatac gcggacctgt tcctcgctgc caagaacctg 11700

tcggacgcca tcctcctgtc agatatcctc cgcgtgaaca ccgagatcac gaaggctcca 11760

ctctctgcct ccatgatcaa gcgctacgac gagcaccatc aggatctgac cctcctgaag 11820

gcgctggtcc gccaacagct cccggagaag tacaaggaga ttttcttcga tcagtcgaag 11880

aacggctacg ctgggtacat cgacggcggg gcctcacaag aggagttcta caagttcatc 11940

aagccaatcc tggagaagat ggacggcacg gaggagctcc tggtgaagct caacagggag 12000

gacctcctgc ggaagcagag aaccttcgat aacggcagca tcccccacca aatccatctc 12060

ggggagctgc acgccatcct gagaaggcaa gaggacttct accctttcct caaggataac 12120

cgggagaaga tcgagaagat cctgaccttc agaatcccat actacgtcgg ccctctcgcg 12180

cgggggaact caagattcgc ttggatgacc cgcaagtctg aggagaccat cacgccgtgg 12240

aacttcgagg aggtggtgga caagggcgct agcgctcagt cgttcatcga gaggatgacc 12300

aacttcgaca agaacctgcc caacgagaag gtgctcccta agcactcgct cctgtacgag 12360

tacttcaccg tctacaacga gctcacgaag gtgaagtacg tcaccgaggg catgcgcaag 12420

ccagcgttcc tgtccgggga gcagaagaag gctatcgtgg acctcctgtt caagaccaac 12480

cggaaggtca cggttaagca actcaaggag gactacttca agaagatcga gtgcttcgat 12540

tcggtcgaga tcagcggcgt tgaggaccgc ttcaacgcca gcctcgggac ctaccacgat 12600

ctcctgaaga tcatcaagga taaggacttc ctggacaacg aggagaacga ggatatcctg 12660

gaggacatcg tgctgaccct cacgctgttc gaggacaggg agatgatcga ggagcgcctg 12720

aagacgtacg cccatctctt cgatgacaag gtcatgaagc aactcaagcg ccggagatac 12780

accggctggg ggaggctgtc ccgcaagctc atcaacggca tccgggacaa gcagtccggg 12840

aagaccatcc tcgacttcct caagagcgat ggcttcgcca acaggaactt catgcaactg 12900

atccacgatg acagcctcac cttcaaggag gatatccaaa aggctcaagt gagcggccag 12960

ggggactcgc tgcacgagca tatcgcgaac ctcgctggct cccccgcgat caagaagggc 13020

atcctccaga ccgtgaaggt tgtggacgag ctcgtgaagg tcatgggccg gcacaagcct 13080

gagaacatcg tcatcgagat ggccagagag aaccaaacca cgcagaaggg gcaaaagaac 13140

tctagggagc gcatgaagcg catcgaggag ggcatcaagg agctggggtc ccaaatcctc 13200

aaggagcacc cagtggagaa cacccaactg cagaacgaga agctctacct gtactacctc 13260

cagaacggca gggatatgta cgtggaccaa gagctggata tcaaccgcct cagcgattac 13320

gacgtcgatc atatcgttcc ccagtctttc ctgaaggatg actccatcga caacaaggtc 13380

ctcaccaggt cggacaagaa ccgcggcaag tcagataacg ttccatctga ggaggtcgtt 13440

aagaagatga agaactactg gaggcagctc ctgaacgcca agctgatcac gcaaaggaag 13500

ttcgacaacc tcaccaaggc tgagagaggc gggctctcag agctggacaa ggccggcttc 13560

atcaagcggc agctggtcga gaccagacaa atcacgaagc acgttgcgca aatcctcgac 13620

tctcggatga acacgaagta cgatgagaac gacaagctga tcagggaggt taaggtgatc 13680

accctgaagt ctaagctcgt ctccgacttc aggaaggatt tccagttcta caaggttcgc 13740

gagatcaaca actaccacca tgcccatgac gcttacctca acgctgtggt cggcaccgct 13800

ctgatcaaga agtacccaaa gctggagtcc gagttcgtgt acggggacta caaggtttac 13860

gatgtgcgca agatgatcgc caagtcggag caagagatcg gcaaggctac cgccaagtac 13920

ttcttctact caaacatcat gaacttcttc aagaccgaga tcacgctggc caacggcgag 13980

atccggaaga gaccgctcat cgagaccaac ggcgagacgg gggagatcgt gtgggacaag 14040

ggcagggatt tcgcgaccgt ccgcaaggtt ctctccatgc cccaggtgaa catcgtcaag 14100

aagaccgagg tccaaacggg cgggttctca aaggagtcta tcctgcctaa gcggaacagc 14160

gacaagctca tcgccagaaa gaaggactgg gacccaaaga agtacggcgg gttcgacagc 14220

cctaccgtgg cctactcggt cctggttgtg gcgaaggttg agaagggcaa gtccaagaag 14280

ctcaagagcg tgaaggagct cctggggatc accatcatgg agaggtccag cttcgagaag 14340

aacccaatcg acttcctgga ggccaagggc tacaaggagg tgaagaagga cctgatcatc 14400

aagctcccga agtactctct cttcgagctg gagaacggca ggaagagaat gctggcttcc 14460

gctggcgagc tccagaaggg gaacgagctc gcgctgccaa gcaagtacgt gaacttcctc 14520

tacctggctt cccactacga gaagctcaag ggcagcccgg aggacaacga gcaaaagcag 14580

ctgttcgtcg agcagcacaa gcattacctc gacgagatca tcgagcaaat ctccgagttc 14640

agcaagcgcg tgatcctcgc cgacgcgaac ctggataagg tcctctccgc ctacaacaag 14700

caccgggaca agcccatcag agagcaagcg gagaacatca tccatctctt caccctgacg 14760

aacctcggcg ctcctgctgc tttcaagtac ttcgacacca cgatcgatcg gaagagatac 14820

acctccacga aggaggtcct ggacgcgacc ctcatccacc agtcgatcac cggcctgtac 14880

gagacgagga tcgacctctc acaactcggc ggggataaga gacccgcagc aaccaagaag 14940

gcagggcaag caaagaagaa gaagacgcgt gactccggcg gcagcaccaa cctgtccgac 15000

atcatcgaga aggagacggg caagcaactc gtgatccagg agagcatcct catgctgcca 15060

gaggaggtgg aggaggtcat cggcaacaag ccagagtccg acatcctggt gcacaccgcc 15120

tacgacgagt ccaccgacga gaacgtcatg ctcctgacca gcgacgcccc agagtacaag 15180

ccatgggccc tcgtcatcca ggacagcaac ggggagaaca agatcaagat gctgtcgggg 15240

gggagcccaa agaagaagcg gaaggtgtag ggatcctgat tgatcgatag agctcgaatt 15300

tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 15360

ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 15420

aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 15480

aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 15540

catctatgtt actagatcgg ggtaccgagc tcg 15573

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aggtccctcg ccggatgat 19

<210> 4

<211> 570

<212> PRT

<213> BnALS1 mutant protein (BnALS1)

<400> 4

Met Ala Ala Ala Thr Ser Ser Ser Pro Ile Ser Leu Thr Ala Lys Pro

1 5 10 15

Ser Ser Lys Ser Pro Leu Pro Ile Ser Arg Phe Ser Leu Pro Phe Ser

20 25 30

Leu Thr Pro Gln Lys Asp Ser Ser Arg Leu His Arg Pro Leu Ala Ile

35 40 45

Ser Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro Pro Ser Pro Glu

50 55 60

Lys Ile Asp Lys Asn Lys Thr Phe Val Ser Arg Tyr Ala Pro Asp Glu

65 70 75 80

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

85 90 95

Val Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His

100 105 110

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

115 120 125

Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly

130 135 140

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

145 150 155 160

Val Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Val Pro Leu Val Ala

165 170 175

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

180 185 190

Glu Thr Pro Ile Val Glu Val Thr Arg Pro Val Leu Tyr Val Gly Gly

195 200 205

Gly Ser Leu Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu Leu Thr

210 215 220

Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys

225 230 235 240

Asn Asp Glu Leu Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Thr Pro His Val Ser Val Cys Gly Asp Val Lys Leu Ala Leu Gln

305 310 315 320

Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys Leu Asp

325 330 335

Phe Gly Val Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys Phe Pro

340 345 350

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

355 360 365

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

370 375 380

Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr Arg Lys

385 390 395 400

Pro Arg Gln Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly

405 410 415

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

420 425 430

Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu

435 440 445

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

450 455 460

Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp Arg Phe Tyr Lys

465 470 475 480

Ala Asn Arg Ala His Thr Tyr Leu Gly Asp Pro Ala Arg Glu Asn Glu

485 490 495

Ile Phe Pro Asn Met Leu Gln Phe Ala Gly Ala Cys Gly Ile Pro Ala

500 505 510

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

515 520 525

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

530 535 540

Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Thr Phe Lys Asp Val

545 550 555 560

Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr

565 570

<210> 5

<211> 570

<212> PRT

<213> BnALS1 mutant protein (BnALS1)

<400> 5

Met Ala Ala Ala Thr Ser Ser Ser Pro Ile Ser Leu Thr Ala Lys Pro

1 5 10 15

Ser Ser Lys Ser Pro Leu Pro Ile Ser Arg Phe Ser Leu Pro Phe Ser

20 25 30

Leu Thr Pro Gln Lys Asp Ser Ser Arg Leu His Arg Pro Leu Ala Ile

35 40 45

Ser Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro Pro Ser Pro Glu

50 55 60

Lys Ile Asp Lys Asn Lys Thr Phe Val Ser Arg Tyr Ala Pro Asp Glu

65 70 75 80

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

85 90 95

Val Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His

100 105 110

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

115 120 125

Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly

130 135 140

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

145 150 155 160

Val Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Val Pro Leu Val Ala

165 170 175

Ile Thr Gly Gln Val Phe Arg Arg Met Ile Gly Thr Asp Ala Phe Gln

180 185 190

Glu Thr Pro Ile Val Glu Val Thr Arg Pro Val Leu Tyr Val Gly Gly

195 200 205

Gly Ser Leu Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu Leu Thr

210 215 220

Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys

225 230 235 240

Asn Asp Glu Leu Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Thr Pro His Val Ser Val Cys Gly Asp Val Lys Leu Ala Leu Gln

305 310 315 320

Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys Leu Asp

325 330 335

Phe Gly Val Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys Phe Pro

340 345 350

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

355 360 365

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

370 375 380

Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr Arg Lys

385 390 395 400

Pro Arg Gln Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly

405 410 415

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

420 425 430

Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu

435 440 445

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

450 455 460

Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp Arg Phe Tyr Lys

465 470 475 480

Ala Asn Arg Ala His Thr Tyr Leu Gly Asp Pro Ala Arg Glu Asn Glu

485 490 495

Ile Phe Pro Asn Met Leu Gln Phe Ala Gly Ala Cys Gly Ile Pro Ala

500 505 510

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

515 520 525

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

530 535 540

Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Thr Phe Lys Asp Val

545 550 555 560

Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr

565 570

<210> 6

<211> 1713

<212> DNA

<213> BnALS1 mutant protein (BnALS1)

<400> 6

atggcggcgg caacatcgtc ttctccgatc tccttaaccg ctaaaccttc ttccaaatcc 60

cctctaccca tttccagatt ctcccttccc ttctccttaa ccccacagaa agactcctcc 120

cgtctccacc gtcctctcgc catctccgcc gttctcaact cacccgtcaa tgtcgcacct 180

ccttcccctg aaaaaatcga caagaacaag actttcgtct cccgctacgc tcccgacgag 240

ccccgcaagg gtgctgatat cctcgtcgaa gccctcgagc gtcaaggcgt cgaaaccgtc 300

tttgcttatc ccggaggtgc ttccatggag atccaccaag ccttgactcg ctcctccacc 360

atccgtaacg tccttccccg tcacgaacaa ggaggagtct tcgccgccga gggttacgct 420

cgttcctccg gcaaaccggg aatctgcata gccacttcgg gtcccggagc taccaacctc 480

gtcagcgggt tagcagacgc gatgcttgac agtgttcctc ttgtcgccat tacaggacag 540

gtttctcgcc ggatgatcgg tactgacgcc ttccaagaga caccaatcgt tgaggtaacg 600

aggcctgttt tgtacgttgg tggtggaagc ttgaactcga gtgaagaact ggggagattt 660

gtcgagctta ctgggatccc cgttgcgagt actttgatgg ggcttggctc ttatccttgt 720

aacgatgagt tgtccctgca gatgcttggc atgcacggga ctgtgtatgc taactacgct 780

gtggagcata gtgatttgtt gctggcgttt ggtgttaggt ttgatgaccg tgtcacggga 840

aagctcgagg ctttcgctag cagggctaaa attgtgcaca tagacattga ttctgctgag 900

attgggaaga ataagacacc tcacgtgtct gtgtgtggtg atgtaaagct ggctttgcaa 960

gggatgaaca aggttcttga gaaccgagcg gaggagctca agcttgattt cggtgtttgg 1020

aggagtgagt tgagcgagca gaaacagaag ttccctttga gcttcaaaac gtttggagaa 1080

gccattcctc cgcagtacgc gattcagatc ctcgacgagc taaccgaagg gaaggcaatt 1140

atcagtactg gtgttggaca gcatcagatg tgggcggcgc agttttacaa gtacaggaag 1200

ccgagacagt ggctgtcgtc atcaggcctc ggagctatgg gttttggact tcctgctgcg 1260

attggagcgt ctgtggcgaa ccctgatgcg attgttgtgg atattgacgg tgatggaagc 1320

ttcataatga acgttcaaga gctggccaca atccgtgtag agaatcttcc tgtgaagata 1380

ctcttgttaa acaaccagca tcttgggatg gtcatgcaat gggaagatcg gttctacaaa 1440

gctaacagag ctcacactta tctcggggac ccggcaaggg agaacgagat cttccctaac 1500

atgctgcagt ttgcaggagc ttgcgggatt ccagctgcga gagtgacgaa gaaagaagaa 1560

ctccgagaag ctattcagac aatgctggat acaccaggac catacctgtt ggatgtgatc 1620

tgtccgcacc aagaacatgt gttaccgatg atcccaagtg gtggcacttt caaagatgta 1680

ataacagaag gggatggtcg cactaagtac tga 1713

<210> 7

<211> 1713

<212> DNA

<213> BnALS1 mutant protein (BnALS1)

<400> 7

atggcggcgg caacatcgtc ttctccgatc tccttaaccg ctaaaccttc ttccaaatcc 60

cctctaccca tttccagatt ctcccttccc ttctccttaa ccccacagaa agactcctcc 120

cgtctccacc gtcctctcgc catctccgcc gttctcaact cacccgtcaa tgtcgcacct 180

ccttcccctg aaaaaatcga caagaacaag actttcgtct cccgctacgc tcccgacgag 240

ccccgcaagg gtgctgatat cctcgtcgaa gccctcgagc gtcaaggcgt cgaaaccgtc 300

tttgcttatc ccggaggtgc ttccatggag atccaccaag ccttgactcg ctcctccacc 360

atccgtaacg tccttccccg tcacgaacaa ggaggagtct tcgccgccga gggttacgct 420

cgttcctccg gcaaaccggg aatctgcata gccacttcgg gtcccggagc taccaacctc 480

gtcagcgggt tagcagacgc gatgcttgac agtgttcctc ttgtcgccat tacaggacag 540

gtttttcgcc ggatgatcgg tactgacgcc ttccaagaga caccaatcgt tgaggtaacg 600

aggcctgttt tgtacgttgg tggtggaagc ttgaactcga gtgaagaact ggggagattt 660

gtcgagctta ctgggatccc cgttgcgagt actttgatgg ggcttggctc ttatccttgt 720

aacgatgagt tgtccctgca gatgcttggc atgcacggga ctgtgtatgc taactacgct 780

gtggagcata gtgatttgtt gctggcgttt ggtgttaggt ttgatgaccg tgtcacggga 840

aagctcgagg ctttcgctag cagggctaaa attgtgcaca tagacattga ttctgctgag 900

attgggaaga ataagacacc tcacgtgtct gtgtgtggtg atgtaaagct ggctttgcaa 960

gggatgaaca aggttcttga gaaccgagcg gaggagctca agcttgattt cggtgtttgg 1020

aggagtgagt tgagcgagca gaaacagaag ttccctttga gcttcaaaac gtttggagaa 1080

gccattcctc cgcagtacgc gattcagatc ctcgacgagc taaccgaagg gaaggcaatt 1140

atcagtactg gtgttggaca gcatcagatg tgggcggcgc agttttacaa gtacaggaag 1200

ccgagacagt ggctgtcgtc atcaggcctc ggagctatgg gttttggact tcctgctgcg 1260

attggagcgt ctgtggcgaa ccctgatgcg attgttgtgg atattgacgg tgatggaagc 1320

ttcataatga acgttcaaga gctggccaca atccgtgtag agaatcttcc tgtgaagata 1380

ctcttgttaa acaaccagca tcttgggatg gtcatgcaat gggaagatcg gttctacaaa 1440

gctaacagag ctcacactta tctcggggac ccggcaaggg agaacgagat cttccctaac 1500

atgctgcagt ttgcaggagc ttgcgggatt ccagctgcga gagtgacgaa gaaagaagaa 1560

ctccgagaag ctattcagac aatgctggat acaccaggac catacctgtt ggatgtgatc 1620

tgtccgcacc aagaacatgt gttaccgatg atcccaagtg gtggcacttt caaagatgta 1680

ataacagaag gggatggtcg cactaagtac tga 1713

<210> 8

<211> 36

<212> DNA

<213> B-NcoI-F(Artificial Sequence)

<400> 8

gaaacacaaa ccatgccaaa gaagaagagg aaggtt 36

<210> 9

<211> 39

<212> DNA

<213> BE-BamHI-R(Artificial Sequence)

<400> 9

cgatcaatca ggatccctac accttccgct tcttctttg 39

<210> 10

<211> 24

<212> DNA

<213> sgRNA-F(Artificial Sequence)

<400> 10

gattgaggtc cctcgccgga tgat 24

<210> 11

<211> 24

<212> DNA

<213> sgRNA-R(Artificial Sequence)

<400> 11

aaacatcatc cggcgaggga cctc 24

<210> 12

<211> 19

<212> DNA

<213> M13F(Artificial Sequence)

<400> 12

ggtaacgcca gggttttcc 19

<210> 13

<211> 20

<212> DNA

<213> sgRNA-R2(Artificial Sequence)

<400> 13

gccatttgtc tgcagaattg 20

<210> 14

<211> 21

<212> DNA

<213> WJP198F(Artificial Sequence)

<400> 14

tcgacaagaa caagactttc g 21

<210> 15

<211> 21

<212> DNA

<213> WJP198R(Artificial Sequence)

<400> 15

cacggtcatc aaacctaaca c 21

Claims

1. A method for obtaining a plant having herbicide resistance based on a gene editing technique, comprising the steps of: editing the BnALS1 gene by using a BnALS1 editing vector to mutate proline at the 182 th site of the amino acid into serine or phenylalanine, wherein the amino acid sequence of the proline is shown as SEQ ID NO. 4 or SEQ ID NO. 5; editing a BnALS1 gene by using a BnALS1 editing vector, so that the 543 th and 544 th nucleotides of the gene are all mutated into T or the 543 th to 545 th nucleotides of the gene are all mutated into T, and the nucleotide sequence of the gene is shown as SEQ ID NO. 6 or SEQ ID NO. 7; the specific steps of the construction of the BnALS1 editing vector are as follows: synthesizing a DNA sequence shown as SEQ ID NO. 1, carrying out enzyme digestion on the pCAMBIA1300 vector by using Hind III and Acc65I to obtain an enzyme digestion vector, and then connecting the enzyme digestion vector with the synthesized DNA sequence to obtain an intermediate vector; taking a rice base editing vector pH-nCas9-PBE as a template and B-NcoI-F and BE-BamHI-R is a primer for amplifying an element sequence of rAPOBEC1-nCas9-UGI in the HI-R, and an intermediate carrier is usedBamHI andNcoi, after enzyme digestion, connecting the I with an element sequence amplification fragment In an In-fusion mode to construct and finish a rape base editing vector, wherein the sequence of the vector is shown as SEQ ID NO. 2; synthesizing primers sgRNA-F and sgRNA-R with joints, annealing, digesting a rape base editing vector by BsaI, and then connecting a target site sequence SEQ ID NO. 3 into a sgRNA expression box by a conventional fragment vector connection method to obtain a Cas9/sgRNA base editing vector, namely a BnALS1 editing vector; said B-NcoThe sequence of I-F is GAAACACAAACCATGCCAAAGAAGAAGAGGAAGGTT and BE-BamThe HI-R sequence is CGATCAATCAGGATCCCTACACCTTCCGCTTCTTCTTTG; the sequence of the sgRNA-F is as follows: GATTGAGGTCCCTCGCCGGATGAT, the sgRNA-R sequence is AAACATCATCCGGCGAGGGACCTC; the specific editing steps are as follows: transforming the successfully constructed Cas9/sgRNA base editing vector by a freeze-thaw methodEntering agrobacterium GV3101 strain, then carrying out the genetic transformation of cabbage type rape to obtain positive plant, and then carrying out the editing detection on the positive plant, wherein the editing detection comprises the following steps: use ofBnALS1Specific primer WJP 198F: TCGACAAGAACAAGACTTTCG and WJP 198R: CACGGTCATCAAACCTAACAC, carrying out PCR amplification, and then directly carrying out Sanger sequencing on the amplification product to finally obtain two forms of mutant plants; wherein the plant is Brassica napus winter rape variety Darmor-bzhThe herbicide is tribenuron-methyl.