CN109652442A - Efficient CRISPR-CAS9 gene editing carrier and construction method in arabidopsis - Google Patents

Abstract

The present invention discloses in a kind of arabidopsis efficiently CRISPR-CAS9 gene editing carrier and construction method, and method is as follows, selects specific N₁₉NGG sequence, synthetic primer pair, using pCBC-DT1T2 sequence as template, PCR amplification, with digestion PCR product and pEntry-gRNA sequence, recycle target fragment, and connect recovery product, obtain pEntry-gRNA-CRIS- target gene plasmid, digestion target gene plasmid and pCambia-Cas9-amiR-RDR6 sequence, shown in SEQ ID NO.4, both connections recovery product, obtain ultimate sequence pCas9-amiR-R6-CRIS- target gene, the phenotype of similar rdr6 mutant can be presented containing the transgenic plant, the T2 without transgenosis plasmid can be filtered out for seed plant using this point, plant occur particular phenotype choose target gene edit and without turn The Mutants homozygous of gene plasmid.The present invention can significantly improve CRISPR-CAS9 editorial efficiency.

Description

Efficient CRISPR-CAS9 gene editing carrier and construction method in arabidopsis

Technical field

The present invention relates to CRISPR-CAS9 gene editing carrier efficient in a kind of arabidopsis and construction methods.

Background technique

CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated genne) it is that a kind of acquired immunity instructs Cas albumen to modify target gene by RNA Means, be widely present in the genome of bacterium and Archimycetes.CRISPR is with " repetitive sequence --- intervening sequences --- repeats sequence Form as column " is present in genome, and intervening sequences therein are not that prokaryotes itself are possessed, and are come from outer Source virus or plasmid, for identification foreign gene.And Cas is then a genoid relevant to CRISPR functional nucleotide sequence, is responsible for turning Record translates Cas albumen.CRISPR/Cas system is divided into the three classes of I, II, III according to the difference of Cas albumen, due to I class and III Class CRISPR/Cas system is excessively complicated, and II class CRISPR/Cas system is currently mainly used, and CRISPR/Cas9 belongs to II Class.CRISPR/Cas9 is used widely in worldwide as a kind of gene site-directed editing technique, is that molecule is educated The important means of the researchs such as kind, gene function.At present CRISPR-CAS9 human cell line and pig, monkey, drosophila, zebra fish, Accurate gene editing has been carried out in a variety of animals and plants model organisms such as nematode, arabidopsis.

CRISPR/Cas9 gene editing system has become the important means that mutant is obtained in animals and plants at present.Allusion quotation The CRISPR/Cas9 system of type by one section of artificial constructed particular sequence for identification sgRNA sequence (single-guide RNA) and an endonuclease Cas9 is formed.In plant, the higher strong promoter of expression efficiency such as Caulimovirus (CaMV) 35S promoter, ubiquitin (ubiqutin) promoter are all used to construct efficient CRISPR/Cas9 editing system.Separately Outside, also there is researcher and drive CRISPR/Cas9 system using the strong promoter specially expressed in blastaea, so as to improve Editorial efficiency of the CRISPR/Cas9 in biological cell mesoderm growing early stage.It is all at present to be with Agrobacterium even if there is these evolutionary approach The CRISPR/Cas9 transgenic method of medium also all suffers from the risk of transgene silencing.

Although also rarely having in the world at present there are many method of improvement CRISPR/Cas9 system through silencing after transcription The method that (Post-transcriptional gene silencing, PTGS) improves CRISPR/Cas9 system. PTGS phenomenon is most found early in the petunia of transgenosis (Petunia).Tiny RNA (the small generated from transgenic loci RNA, sRNA) it is the most important reason for causing transgene silencing phenomenon.Part mRNAs caused by foreign transgenes sequence can quilt The identification of RDR6 (RNA-dependent RNA polymerase 6) albumen, and RDR6 can be using the mRNA as one section of templated synthesis Double-stranded RNA (dsRNA).This section of dsRNA can be generated in turn by a series of DCL (Dicer-like) endonuclease restriction siRNAs.These last siRNA and AGO (ARGONAUTE) protein binding form silencing complex (RNA-induced Silencing complexes, RISCs) shearing is carried out to cause gene silencing to the mRNA containing exogenous plasmid sequence.

When knocking out specific gene acquisition plant Mutants homozygous using the CRISPR/Cas9 editing system of current mainstream, Need to carry out a large amount of DNA extraction and identification work, complex steps time-consuming is huge.Existing CRISPR-Cas9 plasmid is in place Expression can all be interfered by any PTGS mechanism in chief cell, lead to the reduction of CRISPR-Cas9 system editorial efficiency.As plant genetic Most important material --- the arabidopsis in research is learned, it is also deep during carrying out mutagenesis and obtaining mutant to be perplexed by it.

Summary of the invention

Based on above shortcoming, the present invention will provide in a kind of arabidopsis that efficiently CRISPR-CAS9 gene editing carries Body and construction method can effectively increase editor's intensity of CRISPR/Cas9 system in transgenic Arabidopsis plants body, improve and turn The intracorporal Cas9 expressing quantity of gene Arabidopsis plant, can improve CRISPR-CAS9 editorial efficiency significantly.

The technology used in the present invention is as follows: efficient CRISPR-CAS9 gene editing carrier in a kind of arabidopsis, including PCambia-Cas9-amiR-RDR6 sequence, as shown in sequence table SEQ ID NO.4.

The present invention provides the efficiently building of CRISPR-CAS9 gene editing carrier in a kind of arabidopsis there are also following purpose Method is subsidized as follows:

Step 1: the required gRNA of the target gene of selection CRISPR/Caa9 system editor selects specific N₁₉NGG sequence, Synthesize following templa-primer sequence group sequence:

CRIPSR-F:

GAATGGTCTCTATTG N₁₉NGG sequenceGTTTTAGAGCTAGAAATAGCAAGTT；

CRIPSR-R:

ATTATTGGTCTCTAAAC N₁₉NGG sequenceCAATCTCTTAGTCGACTCTACCA；

Step 2: using pCBC-DT1T2 sequence as template, sequence is drawn as shown in sequence table SEQ ID NO.1 with template Object group CRISPR-F/R carries out PCR amplification, purification and recovery PCR product；

Step 3: BsaI enzyme digestion PCR product and pEntry-gRNA sequence, sequence table SEQ ID NO.2 institute are used Show, recycle target fragment, with the recovery product of both T4DNA ligase connections, obtains pEntry-gRNA-CRIS- target gene matter Grain；

Step 4: two enzyme digestion pEntry-gRNA-CRIS- target gene plasmids of KpnI+EcoRI and pCambia- are used Cas9-amiR-RDR6 sequence, sequence recycle target fragment as shown in sequence table SEQ ID NO.4, are connected with T4DNA ligase The two recovery product is connect, ultimate sequence pCas9-amiR-R6-CRIS- target gene is obtained；

Step 5: utilizing Agrobacterium infestation method, pCas9-amiR-R6-CRIS- target gene plasmid be transferred in plant, to T1 is collected after plant is mature for seed, with the Screening of Media T1 containing hygromycin resistance for seed positive transgenic seedling, to T1 generation After seed positive transgenic plant seedling is mature, T2 is collected for seed；

Step 6: T2 can be presented particular vane phenotype, utilize this for the Arabidopsis plant containing transgenosis plasmid in seed The T2 without transgenosis plasmid can a little be filtered out for plant, plant occurs after editing target gene in conjunction with CRISPR/Cas9 Particular phenotype can choose target gene edited and be free of transgenosis plasmid Mutants homozygous.

The invention has the following advantages and beneficial effects: the present invention is able to suppress the work of any PTGS mechanism in Arabidopsis plant body With to improve the editorial efficiency of CRISPR-Cas9.CRISPR-Cas9 editing system after present invention improvement can be reduced A large amount of identification work, greatlys improve conventional efficient.The present invention can effectively increase in transgenic Arabidopsis plants body Editor's intensity of CRISPR/Cas9 system improves the intracorporal Cas9 expressing quantity of transgenic Arabidopsis plants, can significantly Improve CRISPR-CAS9 editorial efficiency.The present invention can greatly reduce the identification work amount tested when screening Mutants homozygous. When carrying out gene editing using traditional CRISPR-CAS9 technology, the Mutants homozygous for being free of CRISPR-CAS9 is if desired obtained, Experimenter is then needed to carry out a large amount of DNA identification work for plant to T2.Contain the present invention (CRISPR-CAS9-amiR-RDR6 Plasmid) transgenic Arabidopsis plants the phenotype of similar rdr6 mutant can be presented, rdr6 mutant blade crimps downwards, passes through The phenotype can determine whether T2 contains transgenosis plasmid in plant, in conjunction with the distinctive phenotype of purpose Mutants homozygous Judge whether plant is the Mutants homozygous without CRISPR-CAS9.Here by taking gl1 mutant as an example, as shown in figure 4, working as me When wanting from T2 for the Mutants homozygous without CRISPR-CAS9 plasmid is obtained in plant, we first with blade phenotype The plant without CRISPR-CAS9 plasmid is found, does not then have the characteristics of epidermal hair in conjunction with gl1 mutant, from being free of The plant of not epidermal hair is found in the plant of CRISPR-CAS9 plasmid, which is exactly the Mutants homozygous needed for us.

Detailed description of the invention

Fig. 1 is rdr6 gene expression amount quantitative fluorescence analysis result figure；

Fig. 2 is containing transgenosis Plasmid samples group of the present invention and common CRISPR/Cas9 transgenosis Plasmid samples group Cas9 egg White expression quantity western blotting quantitatively schemes；

There is phenotype comparison diagram for transgenic plant for arabidopsis T1 in Fig. 3；

Fig. 4 is arabidopsis T2 for plant 4 kinds of phenotype comparison diagrams of appearance；

Specific embodiment

Below according to Figure of description citing, the present invention is described further:

Embodiment 1

Efficiently the construction method of CRISPR-CAS9 gene editing carrier, the plant of selection are brother's rival in a kind of arabidopsis Hypotype arabidopsis, target gene GL1, gene number are AT3G27920 (source TAIR databasehttps:// www.arabidopsis.org/),

Step 1: gRNA is carried out to GL1 gene using CRISPR-P (http://cbi.hzau.edu.cn/crispr/) Selection, selects N₁₉NGG sequence synthesizes following GL1 primer sets:

CRIS-GL1-F:

GAATGGTCTCTATTGTGGACTATGTTCTTAATCAGTTTTAGAGCTAGAAATAGCAAGTT；

CRIS-GL1-R:

ATTATTGGTCTCTAAACTTGTTCACATTAGGGCTCACAATCTCTTAGTCGACTCTACCA；

Step 2: using pCBC-DT1T2 sequence as template, sequence uses primer as shown in sequence table SEQ ID NO.1 CRISPR-GL1-F/R carries out PCR amplification (target fragment about 750bp), is produced using gel DNA QIAquick Gel Extraction Kit purification and recovery PCR Object；

Step 3: BsaI enzyme digestion PCR product and pEntry-gRNA sequence are used, such as sequence table SEQ ID NO.2 institute Show, recycling target fragment (recycling clip size is respectively 650bp and 1.2Kbp or so).It is connecting the two with T4DNA ligase Recovery product reuses DH5 α type Escherichia coli and carries out conversion to connection product and expands numerous, and the LB of coated plate Yu Kana chloramphenicol resistance is flat Bacterium colony screening is carried out on plate, selects positive bacterium colony, is sequenced and is identified by company, obtains pEntry-gRNA-CRIS-GL1 plasmid, As shown in sequence table SEQ ID NO.3.

Numerous step is expanded in wherein Escherichia coli conversion:

It thaws on ice 1. numerous type Escherichia coli will be expanded and be placed in.

2. in the Escherichia coli thawed plus 6ul converts plasmid, mixes gently, put 30min on ice.

3. being placed in water-bath 90s in 42 DEG C of water-baths again.

4. in placing 5min on ice.

5. the LB liquid medium of 1ml non-resistant is added into the Escherichia coli bacteria liquid after above-mentioned process, place Expand numerous completion after cultivating 1 hour in 37 DEG C, the shaking table of 200rpm.

Step 4: KpnI+EcoRI two enzyme digestions pEntry-gRNA-CRIS-GL1 and pCambia-Cas9- are used AmiR-RDR6, as shown in sequence table SEQ ID NO.4, recycling target fragment (recycling clip size is respectively 1.5K and 15K). With T4DNA ligase connect both recovery product, using DH5 α type Escherichia coli to connection product carry out conversion expand it is numerous, coated plate in Card receive chloramphenicol resistance LB plate on carry out bacterium colony screening, select positive bacterium colony, by company sequencing identification obtain ultimate sequence PCas9-amiR-R6-CRIS-GL1, as shown in sequence table SEQ ID NO.5.

Step 5: ultimate sequence pCas9-amiR-R6-CRIS-GL1 is transformed into Agrobacterium:

A, the final vector plasmid of 5-10ul is added in 100ul Agrobacterium competent cell.

B, competent cell is put on ice for 5 minutes.

C, competent cell is placed in liquid nitrogen 3 minutes.

D, competent cell is placed on 37 degree of water-baths 5 minutes.

E, competent cell is put on ice for 5 minutes.

F, after having carried out above step, 1ml LB liquid medium is added into competent cell, is placed in 28-30 ° 2h is cultivated in environment, then therefrom draws 100ul mixing liquid, and coated plate is in receiving the three anti-of mycin containing rifampin, gentamicin, card In LB culture medium.

G, plate is placed in 28 ° of incubators 36-40 hours, to obtain the Agrobacterium containing final carrier.

Step 6: arabidopsis is infected by the Agrobacterium containing final plasmid:

A, by the LB liquid medium of the Agrobacterium inoculation Yu Sankang containing final carrier, 28 °, 200rpm culture 10 it is small When.

B, the culture medium of previous step is collected into precipitating thallus with the pelleted by centrifugation 15min of 5000g.

C, thallus is resuspended with containing the solution of 0.03%Silwet and 5% sucrose.

D, arabidopsis bud 20-30s is infected with re-suspension liquid, when then the arabidopsis infected being placed in 24 in dark, Arabidopsis material will be infected from taking out normal growth under placement and light in dark.

E, after infecting, sowing is waited, then filters out T1 for transgenic plant with the MS culture plate containing hygromycin.

Step 7: T1 is for plant rdr6 gene expression quantitative fluorescence analysis

T1 is extracted for transgenic plant RNA, fluorescent quantitation qPCR analysis is carried out to the expression quantity of rdr6 gene in plant body.

Specific RNA extraction method is as follows:

A, the plant tissue for taking 100mg fresh is in clean 1.5ml centrifuge tube.

B, plant tissue is quick-frozen in liquid nitrogen, it is smashed to pieces with pipette tips.

C, 1ml TRIZOL is added to turn upside down mixing, stands 5min at room temperature.

D, 200ul chloroform is added, is mixed by inversion, is stored at room temperature 15min.

E, 12000X g is centrifuged 15min, draws supernatant 400ul.

F, 400ul isopropanol is added, is mixed by inversion, -20 DEG C of standing 30min shallow lake RNA.

G, 4 DEG C, 12000X g is centrifuged 15min, abandons supernatant.

H, the ethanol washing RNA of 1ml75% is added, 4 DEG C, 12000X g is centrifuged 5min.

I, drying at room temperature RNA is added 30ul ddH2O and dissolves RNA.

J, the RNA of extraction is deposited in into -80 DEG C of preservations.

Rdr6 gene expression amount quantitative fluorescence analysis result such as Fig. 1: the sample containing transgenosis plasmid of the present invention, rdr6 base Because expression quantity is far below wild type control group, illustrate that transgenosis plasmid of the present invention is capable of the table of strong inhibition plant rdr6 gene It reaches.

Step 8: Cas9 expressing quantity detection

The expression quantity of Cas9 albumen is the important indicator for measuring CRISPR/Cas9 system editor's intensity, usual Cas9 table amount Higher, CRISPR-Cas9 system editor's intensity is higher.Using western blot technology to the version CRISPR/Cas9 matter containing improvement The T1 of grain is detected for Cas9 expressing quantity in transgenic plant body.

Concrete operations mode is as follows:

A, SDS-PAGE is carried out according to method described in " molecular cloning ".With the protein molecular weight Marker of pre-dyed.

After electrophoresis, gel is put into electricity and turns to carry out transferring film after balancing 2min in buffer.

B, six 3mm thickness filter paper are sheared according to the size of gel, be soaked in transfering buffering liquid.Cut one it is more slightly larger than filter paper Pvdf membrane, methanol impregnate 5min or more, electricity turn liquid balance 10min after use.3 thickness are sequentially placed in half-dried transferring film instrument Filter paper, transfer film, gel, 3 layers of filter paper pay attention to driving each interlayer bubble away.15V electricity turns 20min.

C, it is sure not to make the transfer film of transfer to dry out, is quickly put in appropriate confining liquid and closes after transferring film.Room temperature jog 1h。

D, transfer film is put into hybridization bag, is separately added into according to 0.1ml/cm2 dosage with the suitably diluted mouse of confining liquid Anti- Flag primary antibody (Quan Shi King Company product, catalog number (Cat.No.): HT201-01,1:1000 dilution), internal reference albumen selects HSC70 (Enzo Life Sciences Products, catalog number (Cat.No.): ADI-SPA-818-F, 1:1000 dilution) bubble is eliminated, it seals.At room temperature It is placed in jog on decolorization swinging table and is incubated for 2h or so (or staying overnight in 4 DEG C of jogs), combine antibody sufficiently.It is put into after film is removed In TBST solution, film 3 times are washed, each 7min.

E, film is put into hybridization bag, addition prepares sheep anti-mouse igg secondary antibody with confining liquid.Bubble removing is caught up with to seal, room temperature vibration Swing 2h.

F, transfer film is taken out, is washed three times with TBST, each 10min.

G, luminescent solution (Tiangeng Products) A liquid, each 0.5ml of B liquid is taken to be uniformly mixed in equal volume.Transfer film is gently contacted Filter paper blots liquid, has the one side of protein to be placed on preservative film upward, and ECL mixed liquor is added drop-wise on transfer film, reaction 3min wraps up pvdf membrane with preservative film immediately, notices that surface wants smooth, be put into CLINX chemical gel imager and be imaged.

As a result such as Fig. 2: containing transgenosis Plasmid samples group of the present invention, compare common CRISPR/Cas9 non-transgenic control group Expression quantity, the sample sets Cas9 expression quantity containing transgenosis plasmid of the present invention are higher.

Step 9: T1 generation positive seedling phenotype statistics

It is as shown in Figure 3 that T1 for transgenic Arabidopsis plants phenotype occurs: when CRISPR system knocks out gl1 gene, plant Blade table fur can disappear, and different (blade epidermis approximate number amount number) according to editor's intensity, plant will appear following three kinds of tables Type, be divided into without, in, strong totally 3 kinds of phenotypes, wherein "None" epidermal hair is most, " in " take second place, " strong " type epidermal hair is minimum, so The intensity highest of CRISPR-Cas9 system editor's gene in the plant body of " strong " phenotype.In statistics T1 generation, contains common CRISPR/ respectively Cas9 transgenosis plasmid plants (CRISPR-GL1) and " strong " occurs with containing transgenosis plasmid plant (CRISPR-amiR-GL1) of the present invention Phenotype quantity, as a result such as table 1: there is the probability of " strong " phenotype much larger than common in the plant containing transgenosis plasmid of the present invention CRISPR-Cas9 transgenosis plasmid.

Table 1

	Strong phenotype	Positive seedling sum
			CRISPR-GL1	1 (5.8%)	17
CRISPR-amiR-GL1	5 (27%)	18

Step 10: Mutants homozygous plant is obtained

The T1 containing transgenosis plasmid of the present invention is collected for arabidopsis positive seedling plant seed, plantation obtains T2 for plant.It is quasi- Southern mustard genome is 2 times of bodies, and by the independent assortment of chromosome, T2 will appear 4 kinds of phenotypes as shown in Figure 4 for plant, wherein If containing transgenosis plasmid of the present invention, has the phenotype that blade crimps downwards and (phenotype as shown in a, b in Fig. 4) occur, if being free of Ellipticity is then presented in transgenosis plasmid of the present invention, blade (as shown in c, d in Fig. 4).Blade is ellipticity, and the plant without epidermal hair The Mutants homozygous plant of foreign transgenes is free from if strain (such as the d in Fig. 4).

The present invention be using the transgenosis of CRISPR/Cas9 in arabidopsis as object, by improvement CRISPR/Ca9 plasmid from And the expression of RDR6 albumen in organism is inhibited to weaken PTGS effect, to improve the editorial efficiency of CRISPR/Cas9 system.

Sequence table

<110>Shenzhen University

<120>efficient CRISPR-CAS9 gene editing carrier and construction method in arabidopsis

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 553

<212> DNA

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gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt 120

cttcggcgtt caatttctgg ggttttctct tcgttttctg taactgaaac ctaaaatttg 180

acctaaaaaa aatctcaaat aatatgattc agtggttttg tacttttcag ttagttgagt 240

tttgcagttc cgatgagata aaccaatatt aatccaaact actgcagcct gacagacaaa 300

tgaggatgca aacaatttta aagtttatct aacgctagct gttttgtttc ttctctctgg 360

tgcaccaacg acggcgtttt ctcaatcata aagaggcttg ttttacttaa ggccaataat 420

gttgatggat cgaaagaaga gggcttttaa taaacgagcc cgtttaagct gtaaacgatg 480

tcaaaaacat cccacatcgt tcagttgaaa atagaagctc tgtttatata ttggtagagt 540

cgactaagag att 553

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ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600

ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660

agcaggctcc gcggccgccc ccttcaccga attccgactt gccttccgca caatacatca 720

tttcttctta gctttttttc ttcttcttcg ttcatacagt ttttttttgt ttatcagctt 780

acattttctt gaaccgtagc tttcgttttc ttctttttaa ctttccattc ggagtttttg 840

tatcttgttt catagtttgt cccaggatta gaatgattag gcatcgaacc ttcaagaatt 900

tgattgaata aaacatcttc attcttaaga tatgaagata atcttcaaaa ggcccctggg 960

aatctgaaag aagagaagca ggcccattta tatgggaaag aacaatagta tttcttatat 1020

aggcccattt aagttgaaaa caatcttcaa aagtcccaca tcgcttagat aagaaaacga 1080

agctgagttt atatacagct agagtcgaag tagtgattgg gagaccaacc cagtggacat 1140

aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc aactggtcca 1200

gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg 1260

actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg cgttacgccg 1320

tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc agtcgcccta 1380

aaacaaagtt aaacatcatg ggggaagcgg tgatcgccga agtatcgact caactatcag 1440

aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg 1500

gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga 1560

ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg 1620

cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg 1680

acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca 1740

atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc 1800

tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg 1860

atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact 1920

cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt 1980

acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc 2040

gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag 2100

aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag 2160

gcgagatcac caaggtagtc ggcaaataat gtctagctag aaattcgttc aagccgacgc 2220

cgcttcgcgg cgcggcttaa ctcaagcgtt agatgcacta agcacataat tgctcacagc 2280

caaactatca ggtcaagtct gcttttatta tttttaagcg tgcataataa gccggtctcg 2340

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 2400

ggcaccgagt cggtgctttt ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt 2460

cttcggcgtt caatttctgg ggttttctct tcgttttctg taactgaaac ctaaaatttg 2520

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tttgcagttc cgatgagata aaccaatggt accaagggtg ggcgcgccga cccagctttc 2640

ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc 2700

actatcagtc aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg 2760

tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt 2820

tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac 2880

agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat 2940

tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca 3000

ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat 3060

ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg 3120

gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta 3180

ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca 3240

ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt 3300

tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg 3360

aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa 3420

caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat 3480

ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat 3540

gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc 3600

ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct 3660

gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt 3720

aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga 3780

ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag 3840

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 3900

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 3960

aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 4020

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 4080

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 4140

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 4200

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc 4260

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 4320

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 4380

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 4440

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 4500

atgtt 4505

<210> 3

<211> 3876

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600

ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660

agcaggctcc gcggccgccc ccttcaccga attccgactt gccttccgca caatacatca 720

tttcttctta gctttttttc ttcttcttcg ttcatacagt ttttttttgt ttatcagctt 780

acattttctt gaaccgtagc tttcgttttc ttctttttaa ctttccattc ggagtttttg 840

tatcttgttt catagtttgt cccaggatta gaatgattag gcatcgaacc ttcaagaatt 900

tgattgaata aaacatcttc attcttaaga tatgaagata atcttcaaaa ggcccctggg 960

aatctgaaag aagagaagca ggcccattta tatgggaaag aacaatagta tttcttatat 1020

aggcccattt aagttgaaaa caatcttcaa aagtcccaca tcgcttagat aagaaaacga 1080

agctgagttt atatacagct agagtcgaag tagtgattgt ggactatgtt cttaatcagt 1140

tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 1200

caccgagtcg gtgctttttt ttgcaaaatt ttccagatcg atttcttctt cctctgttct 1260

tcggcgttca atttctgggg ttttctcttc gttttctgta actgaaacct aaaatttgac 1320

ctaaaaaaaa tctcaaataa tatgattcag tggttttgta cttttcagtt agttgagttt 1380

tgcagttccg atgagataaa ccaatattaa tccaaactac tgcagcctga cagacaaatg 1440

aggatgcaaa caattttaaa gtttatctaa cgctagctgt tttgtttctt ctctctggtg 1500

caccaacgac ggcgttttct caatcataaa gaggcttgtt ttacttaagg ccaataatgt 1560

tgatggatcg aaagaagagg gcttttaata aacgagcccg tttaagctgt aaacgatgtc 1620

aaaaacatcc cacatcgttc agttgaaaat agaagctctg tttatatatt ggtagagtcg 1680

actaagagat tgtgagccct aatgtgaaca agttttagag ctagaaatag caagttaaaa 1740

taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttttgcaaa 1800

attttccaga tcgatttctt cttcctctgt tcttcggcgt tcaatttctg gggttttctc 1860

ttcgttttct gtaactgaaa cctaaaattt gacctaaaaa aaatctcaaa taatatgatt 1920

cagtggtttt gtacttttca gttagttgag ttttgcagtt ccgatgagat aaaccaatgg 1980

taccaagggt gggcgcgccg acccagcttt cttgtacaaa gttggcatta taagaaagca 2040

ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg 2100

ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca 2160

gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc 2220

atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat 2280

tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 2340

taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa 2400

gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 2460

agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca 2520

ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc 2580

attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 2640

gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 2700

atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 2760

tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt 2820

gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 2880

tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 2940

ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 3000

gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 3060

gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac 3120

gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc 3180

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 3240

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 3300

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 3360

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 3420

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 3480

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 3540

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 3600

atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3660

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3720

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3780

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3840

gttcctggcc ttttgctggc cttttgctca catgtt 3876

<210> 4

<211> 16238

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag 60

gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa 120

gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc 180

cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc 240

ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact 300

agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 360

gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct 420

gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca 480

ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg 540

cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc 600

tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga 660

ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg 720

ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg 780

cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg 840

ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag 900

tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg 960

acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 1020

cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg 1080

gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg 1140

tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 1200

ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 1260

gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 1320

gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 1380

ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 1440

gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 1500

attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 1560

caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 1620

cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 1680

attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 1740

acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 1800

tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 1860

gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 1920

tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa 1980

gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 2040

cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 2100

tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc 2160

taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc 2220

ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 2280

acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 2340

acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 2400

cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 2460

acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 2520

caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 2580

cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 2640

cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 2700

gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 2760

cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 2820

ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 2880

cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 2940

ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 3000

ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 3060

cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 3120

cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 3180

cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 3240

atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 3300

tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 3360

ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 3420

atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 3480

aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 3540

tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 3600

gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 3660

tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 3720

agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 3780

tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 3840

agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 3900

gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 3960

tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 4020

gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 4080

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 4140

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 4200

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4260

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4320

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4380

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4440

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4500

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4560

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4620

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 4680

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 4740

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 4800

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 4860

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 4920

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 4980

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta 5040

ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc 5100

cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg 5160

gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa 5220

gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa 5280

gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt 5340

aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa 5400

gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc 5460

gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg 5520

ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct 5580

ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca 5640

tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt 5700

catttttaaa tataggtttt cattttctcc caccagctta tataccttag caggagacat 5760

tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat 5820

tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa 5880

gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa 5940

taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg 6000

gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa 6060

catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc 6120

cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc 6180

cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg 6240

ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac 6300

aacttaataa cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg 6360

ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt 6420

attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc 6480

ctataggaac cctaattccc ttatctggga actactcaca cattattatg gagaaactcg 6540

agcttgtcga tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg 6600

ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc 6660

gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg 6720

tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag 6780

agttggtcaa gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc 6840

ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag 6900

aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg 6960

accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg 7020

aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg 7080

acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca 7140

atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg 7200

ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc 7260

ggttgtagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca 7320

cggcgggaga tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga 7380

atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg 7440

gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga 7500

gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga 7560

aacttctcga cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga 7620

tctgcgaaag ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct 7680

ctccaaatga aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt 7740

gcgtcatccc ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt 7800

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 7860

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 7920

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 7980

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 8040

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat 8100

caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg 8160

ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct 8220

ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag 8280

tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa 8340

gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga 8400

gtgtcgtgct ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg 8460

cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 8520

gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 8580

ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 8640

acagctatga catgattacg aattgagctt ggcactggcc gtcgttttac aacgtcgtga 8700

ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 8760

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 8820

tggcgaatgc tagagcagct tgagcttgga tcagattgtc gtttcccgcc ttcagtttag 8880

cttcatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 8940

acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 9000

ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 9060

ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 9120

agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 9180

tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 9240

tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 9300

gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 9360

ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 9420

tcttgaccat ggtagatctg actagtgatt acaaggacga tgatgacaag aaagactata 9480

aagatgacga tgataagcat atggacaaga agtacagcat cggcctggac atcggcacga 9540

actcggtggg ctgggcggtg atcacggacg agtacaaggt gccctccaag aagttcaagg 9600

tgctgggcaa caccgaccgc cactcgatca agaagaacct gatcggcgcc ctgctgttcg 9660

actccggcga gaccgccgag gcgacgcgcc tgaagcgcac cgcgcgtcgc cgctacacgc 9720

gtcgcaagaa ccgcatctgc tacctgcagg agatcttcag caacgagatg gccaaggtgg 9780

acgactcgtt cttccaccgc ctggaggagt ccttcctggt ggaggaagac aagaagcacg 9840

agcgccaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccga 9900

cgatctacca cctgcgcaag aagctggtgg acagcaccga caaggcggac ctgcgcctga 9960

tctacctggc cctggcgcac atgatcaagt tccgcggcca cttcctgatc gagggcgacc 10020

tgaaccccga caactcggac gtggacaagc tgttcatcca gctggtgcag acctacaacc 10080

agctgttcga ggagaacccg atcaacgcct ccggcgtgga cgccaaggcg atcctgagcg 10140

cgcgcctgtc caagagccgt cgcctggaga acctgatcgc ccagctgccc ggcgagaaga 10200

agaacggcct gttcggcaac ctgatcgcgc tgtcgctggg cctgacgccg aacttcaagt 10260

ccaacttcga cctggccgag gacgcgaagc tgcagctgag caaggacacc tacgacgacg 10320

acctggacaa cctgctggcc cagatcggcg accagtacgc ggacctgttc ctggccgcga 10380

agaacctgtc ggacgccatc ctgctgtccg acatcctgcg cgtgaacacc gagatcacga 10440

aggcccccct gtcggcgtcc atgatcaagc gctacgacga gcaccaccag gacctgaccc 10500

tgctgaaggc gctggtgcgc cagcagctgc cggagaagta caaggagatc ttcttcgacc 10560

agagcaagaa cggctacgcc ggctacatcg acggcggcgc gtcgcaagag gagttctaca 10620

agttcatcaa gcccatcctg gagaagatgg acggcacgga ggagctgctg gtgaagctga 10680

accgcgagga cctgctgcgc aagcagcgca ccttcgacaa cggcagcatc ccccaccaga 10740

tccacctggg cgagctgcac gccatcctgc gtcgccaaga ggacttctac ccgttcctga 10800

aggacaaccg cgagaagatc gagaagatcc tgacgttccg catcccctac tacgtgggcc 10860

cgctggcccg cggcaacagc cgcttcgcgt ggatgacccg caagtcggag gagaccatca 10920

cgccctggaa cttcgaggaa gtggtggaca agggcgccag cgcgcagtcg ttcatcgagc 10980

gcatgaccaa cttcgacaag aacctgccca acgagaaggt gctgccgaag cactccctgc 11040

tgtacgagta cttcaccgtg tacaacgagc tgacgaaggt gaagtacgtg accgagggca 11100

tgcgcaagcc cgccttcctg agcggcgagc agaagaaggc gatcgtggac ctgctgttca 11160

agaccaaccg caaggtgacg gtgaagcagc tgaaagagga ctacttcaag aagatcgagt 11220

gcttcgacag cgtggagatc tcgggcgtgg aggaccgctt caacgccagc ctgggcacct 11280

accacgacct gctgaagatc atcaaggaca aggacttcct ggacaacgag gagaacgagg 11340

acatcctgga ggacatcgtg ctgaccctga cgctgttcga ggaccgcgag atgatcgagg 11400

agcgcctgaa gacgtacgcc cacctgttcg acgacaaggt gatgaagcag ctgaagcgtc 11460

gccgctacac cggctggggc cgcctgagcc gcaagctgat caacggcatc cgcgacaagc 11520

agtccggcaa gaccatcctg gacttcctga agagcgacgg cttcgcgaac cgcaacttca 11580

tgcagctgat ccacgacgac tcgctgacct tcaaagagga catccagaag gcccaggtgt 11640

cgggccaggg cgactccctg cacgagcaca tcgccaacct ggcgggctcc cccgcgatca 11700

agaagggcat cctgcagacc gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc 11760

acaagccgga gaacatcgtg atcgagatgg cccgcgagaa ccagaccacg cagaagggcc 11820

agaagaacag ccgcgagcgc atgaagcgca tcgaggaagg catcaaggag ctgggctcgc 11880

agatcctgaa ggagcacccc gtggagaaca cccagctgca gaacgagaag ctgtacctgt 11940

actacctgca gaacggccgc gacatgtacg tggaccagga gctggacatc aaccgcctgt 12000

ccgactacga cgtggaccac atcgtgcccc agagcttcct gaaggacgac tcgatcgaca 12060

acaaggtgct gacccgcagc gacaagaacc gcggcaagag cgacaacgtg ccgtcggagg 12120

aagtggtgaa gaagatgaag aactactggc gccagctgct gaacgccaag ctgatcacgc 12180

agcgcaagtt cgacaacctg accaaggccg agcgcggtgg cctgtcggag ctggacaagg 12240

cgggcttcat caagcgccag ctggtggaga cccgccagat cacgaagcac gtggcgcaga 12300

tcctggactc ccgcatgaac acgaagtacg acgagaacga caagctgatc cgcgaggtga 12360

aggtgatcac cctgaagtcc aagctggtca gcgacttccg caaggacttc cagttctaca 12420

aggtgcgcga gatcaacaac taccaccacg cccacgacgc gtacctgaac gccgtggtgg 12480

gcaccgcgct gatcaagaag taccccaagc tggagagcga gttcgtgtac ggcgactaca 12540

aggtgtacga cgtgcgcaag atgatcgcca agtcggagca ggagatcggc aaggccaccg 12600

cgaagtactt cttctactcc aacatcatga acttcttcaa gaccgagatc acgctggcca 12660

acggcgagat ccgcaagcgc ccgctgatcg agaccaacgg cgagacgggc gagatcgtgt 12720

gggacaaggg ccgcgacttc gcgaccgtgc gcaaggtgct gagcatgccc caggtgaaca 12780

tcgtgaagaa gaccgaggtg cagacgggcg gcttctccaa ggagagcatc ctgccgaagc 12840

gcaactcgga caagctgatc gcccgcaaga aggactggga ccccaagaag tacggcggct 12900

tcgactcccc gaccgtggcc tacagcgtgc tggtggtggc gaaggtggag aagggcaagt 12960

ccaagaagct gaagagcgtg aaggagctgc tgggcatcac catcatggag cgcagctcgt 13020

tcgagaagaa ccccatcgac ttcctggagg ccaagggcta caaagaggtg aagaaggacc 13080

tgatcatcaa gctgccgaag tactcgctgt tcgagctgga gaacggccgc aagcgcatgc 13140

tggcctccgc gggcgagctg cagaagggca acgagctggc cctgcccagc aagtacgtga 13200

acttcctgta cctggcgtcc cactacgaga agctgaaggg ctcgccggag gacaacgagc 13260

agaagcagct gttcgtggag cagcacaagc actacctgga cgagatcatc gagcagatct 13320

cggagttctc caagcgcgtg atcctggccg acgcgaacct ggacaaggtg ctgagcgcct 13380

acaacaagca ccgcgacaag cccatccgcg agcaggcgga gaacatcatc cacctgttca 13440

ccctgacgaa cctgggcgcc ccggccgcgt tcaagtactt cgacaccacg atcgaccgca 13500

agcgctacac ctccacgaaa gaggtgctgg acgcgaccct gatccaccag agcatcaccg 13560

gcctgtacga gacgcgcatc gacctgagcc agctgggcgg cgactcccgc gcggacccga 13620

agaagaagcg caaggtgtaa gaattaattc ggatccattg gtgaccagct cgaatttccc 13680

cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 13740

gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 13800

catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 13860

cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 13920

tatgttacta gatcggggag ctccatggag tcaaagattc aaatagagga cctaacagaa 13980

ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag 14040

aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat 14100

acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac 14160

ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa 14220

ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct 14280

gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 14340

gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 14400

gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 14460

ttggagagaa cacgggggac ttctagaccc caaacacacg ctcggacgca tattacacat 14520

gttcatacac ttaatactcg ctgttttgaa ttgatgtttt aggaatatat atgtagaaaa 14580

gacgactgta gcttatattc acaggtcgtg atatgattca attagcttcc gactcattca 14640

tccaaatacc gagtcgccaa aattcaaact agactcgtta aatgaatgaa tgatgcggta 14700

gacaaattgg atcattgatt ctctttgatt ataaggtaca gtcgtccttt ctctcttttg 14760

tattccaatt ttcttgatta atctttcctg cacaaaaaca tgcttgatcc actaagtgac 14820

atatatgctg ccttcgtata tatagttctg gtaaaattaa cattttgggt ttatctttat 14880

ttaaggcatc gccatggggg gcgcgccagc tcgaatttcc ccgatcgttc aaacatttgg 14940

caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 15000

ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 15060

tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 15120

tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 15180

gcttatccga gaattcccat gggtcgacgt aaagcctgta gaagaggttt ctagcgaacg 15240

acacgagttt gagcctcatg aagcttcgtt gaacaacgga aactcgactt gccttccgca 15300

caatacatca tttcttctta gctttttttc ttcttcttcg ttcatacagt ttttttttgt 15360

ttatcagctt acattttctt gaaccgtagc tttcgttttc ttctttttaa ctttccattc 15420

ggagtttttg tatcttgttt catagtttgt cccaggatta gaatgattag gcatcgaacc 15480

ttcaagaatt tgattgaata aaacatcttc attcttaaga tatgaagata atcttcaaaa 15540

ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag aacaatagta 15600

tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca tcgcttagat 15660

aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgc gcttcaaggt 15720

gcacatgggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt 15780

gaaaaagtgg caccgagtcg gtgctttttt tttttgcaaa attttccaga tcgatttctt 15840

cttcctctgt tcttcggcgt tcaatttctg ggtttttctc ttcgttttct gtaactgaaa 15900

cctaaaattt gacctaaaaa aaatctcaaa taatatgatt cagtggtttt gtacttttca 15960

gttagttgag ttttgcagtt ccgatgagat aaaccaataa ctttgcttag atctaattca 16020

ttccgttaca cctctgatgg agatggaagg ttcttaataa tgatgccatt ttttgggtaa 16080

taattttgaa ttagaatcaa gggtataaga ttcataatta acatcactta agcaaagttc 16140

gtaatatacg accacaggat ataatttttg gtaccaatta aactatcagt gtttgacagg 16200

atatattggc gggtaaacct aagagaaaag agcgttta 16238

<210> 5

<211> 16549

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag 60

gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa 120

gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc 180

cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc 240

ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact 300

agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 360

gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct 420

gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca 480

ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg 540

cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc 600

tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga 660

ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg 720

ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg 780

cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg 840

ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag 900

tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg 960

acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 1020

cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg 1080

gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg 1140

tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 1200

ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 1260

gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 1320

gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 1380

ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 1440

gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 1500

attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 1560

caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 1620

cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 1680

attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 1740

acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 1800

tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 1860

gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 1920

tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa 1980

gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 2040

cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 2100

tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc 2160

taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc 2220

ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 2280

acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 2340

acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 2400

cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 2460

acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 2520

caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 2580

cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 2640

cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 2700

gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 2760

cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 2820

ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 2880

cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 2940

ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 3000

ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 3060

cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 3120

cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 3180

cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 3240

atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 3300

tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 3360

ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 3420

atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 3480

aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 3540

tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 3600

gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 3660

tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 3720

agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 3780

tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 3840

agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 3900

gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 3960

tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 4020

gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 4080

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 4140

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 4200

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4260

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4320

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4380

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4440

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4500

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4560

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4620

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 4680

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 4740

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 4800

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 4860

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 4920

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 4980

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta 5040

ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc 5100

cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg 5160

gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa 5220

gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa 5280

gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt 5340

aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa 5400

gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc 5460

gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg 5520

ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct 5580

ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca 5640

tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt 5700

catttttaaa tataggtttt cattttctcc caccagctta tataccttag caggagacat 5760

tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat 5820

tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa 5880

gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa 5940

taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg 6000

gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa 6060

catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc 6120

cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc 6180

cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg 6240

ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac 6300

aacttaataa cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg 6360

ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt 6420

attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc 6480

ctataggaac cctaattccc ttatctggga actactcaca cattattatg gagaaactcg 6540

agcttgtcga tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg 6600

ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc 6660

gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg 6720

tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag 6780

agttggtcaa gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc 6840

ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag 6900

aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg 6960

accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg 7020

aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg 7080

acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca 7140

atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg 7200

ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc 7260

ggttgtagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca 7320

cggcgggaga tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga 7380

atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg 7440

gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga 7500

gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga 7560

aacttctcga cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga 7620

tctgcgaaag ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct 7680

ctccaaatga aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt 7740

gcgtcatccc ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt 7800

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 7860

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 7920

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 7980

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 8040

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat 8100

caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg 8160

ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct 8220

ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag 8280

tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa 8340

gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga 8400

gtgtcgtgct ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg 8460

cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 8520

gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 8580

ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 8640

acagctatga catgattacg aattgagctt ggcactggcc gtcgttttac aacgtcgtga 8700

ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 8760

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 8820

tggcgaatgc tagagcagct tgagcttgga tcagattgtc gtttcccgcc ttcagtttag 8880

cttcatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 8940

acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 9000

ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 9060

ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 9120

agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 9180

tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 9240

tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 9300

gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 9360

ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 9420

tcttgaccat ggtagatctg actagtgatt acaaggacga tgatgacaag aaagactata 9480

aagatgacga tgataagcat atggacaaga agtacagcat cggcctggac atcggcacga 9540

actcggtggg ctgggcggtg atcacggacg agtacaaggt gccctccaag aagttcaagg 9600

tgctgggcaa caccgaccgc cactcgatca agaagaacct gatcggcgcc ctgctgttcg 9660

actccggcga gaccgccgag gcgacgcgcc tgaagcgcac cgcgcgtcgc cgctacacgc 9720

gtcgcaagaa ccgcatctgc tacctgcagg agatcttcag caacgagatg gccaaggtgg 9780

acgactcgtt cttccaccgc ctggaggagt ccttcctggt ggaggaagac aagaagcacg 9840

agcgccaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccga 9900

cgatctacca cctgcgcaag aagctggtgg acagcaccga caaggcggac ctgcgcctga 9960

tctacctggc cctggcgcac atgatcaagt tccgcggcca cttcctgatc gagggcgacc 10020

tgaaccccga caactcggac gtggacaagc tgttcatcca gctggtgcag acctacaacc 10080

agctgttcga ggagaacccg atcaacgcct ccggcgtgga cgccaaggcg atcctgagcg 10140

cgcgcctgtc caagagccgt cgcctggaga acctgatcgc ccagctgccc ggcgagaaga 10200

agaacggcct gttcggcaac ctgatcgcgc tgtcgctggg cctgacgccg aacttcaagt 10260

ccaacttcga cctggccgag gacgcgaagc tgcagctgag caaggacacc tacgacgacg 10320

acctggacaa cctgctggcc cagatcggcg accagtacgc ggacctgttc ctggccgcga 10380

agaacctgtc ggacgccatc ctgctgtccg acatcctgcg cgtgaacacc gagatcacga 10440

aggcccccct gtcggcgtcc atgatcaagc gctacgacga gcaccaccag gacctgaccc 10500

tgctgaaggc gctggtgcgc cagcagctgc cggagaagta caaggagatc ttcttcgacc 10560

agagcaagaa cggctacgcc ggctacatcg acggcggcgc gtcgcaagag gagttctaca 10620

agttcatcaa gcccatcctg gagaagatgg acggcacgga ggagctgctg gtgaagctga 10680

accgcgagga cctgctgcgc aagcagcgca ccttcgacaa cggcagcatc ccccaccaga 10740

tccacctggg cgagctgcac gccatcctgc gtcgccaaga ggacttctac ccgttcctga 10800

aggacaaccg cgagaagatc gagaagatcc tgacgttccg catcccctac tacgtgggcc 10860

cgctggcccg cggcaacagc cgcttcgcgt ggatgacccg caagtcggag gagaccatca 10920

cgccctggaa cttcgaggaa gtggtggaca agggcgccag cgcgcagtcg ttcatcgagc 10980

gcatgaccaa cttcgacaag aacctgccca acgagaaggt gctgccgaag cactccctgc 11040

tgtacgagta cttcaccgtg tacaacgagc tgacgaaggt gaagtacgtg accgagggca 11100

tgcgcaagcc cgccttcctg agcggcgagc agaagaaggc gatcgtggac ctgctgttca 11160

agaccaaccg caaggtgacg gtgaagcagc tgaaagagga ctacttcaag aagatcgagt 11220

gcttcgacag cgtggagatc tcgggcgtgg aggaccgctt caacgccagc ctgggcacct 11280

accacgacct gctgaagatc atcaaggaca aggacttcct ggacaacgag gagaacgagg 11340

acatcctgga ggacatcgtg ctgaccctga cgctgttcga ggaccgcgag atgatcgagg 11400

agcgcctgaa gacgtacgcc cacctgttcg acgacaaggt gatgaagcag ctgaagcgtc 11460

gccgctacac cggctggggc cgcctgagcc gcaagctgat caacggcatc cgcgacaagc 11520

agtccggcaa gaccatcctg gacttcctga agagcgacgg cttcgcgaac cgcaacttca 11580

tgcagctgat ccacgacgac tcgctgacct tcaaagagga catccagaag gcccaggtgt 11640

cgggccaggg cgactccctg cacgagcaca tcgccaacct ggcgggctcc cccgcgatca 11700

agaagggcat cctgcagacc gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc 11760

acaagccgga gaacatcgtg atcgagatgg cccgcgagaa ccagaccacg cagaagggcc 11820

agaagaacag ccgcgagcgc atgaagcgca tcgaggaagg catcaaggag ctgggctcgc 11880

agatcctgaa ggagcacccc gtggagaaca cccagctgca gaacgagaag ctgtacctgt 11940

actacctgca gaacggccgc gacatgtacg tggaccagga gctggacatc aaccgcctgt 12000

ccgactacga cgtggaccac atcgtgcccc agagcttcct gaaggacgac tcgatcgaca 12060

acaaggtgct gacccgcagc gacaagaacc gcggcaagag cgacaacgtg ccgtcggagg 12120

aagtggtgaa gaagatgaag aactactggc gccagctgct gaacgccaag ctgatcacgc 12180

agcgcaagtt cgacaacctg accaaggccg agcgcggtgg cctgtcggag ctggacaagg 12240

cgggcttcat caagcgccag ctggtggaga cccgccagat cacgaagcac gtggcgcaga 12300

tcctggactc ccgcatgaac acgaagtacg acgagaacga caagctgatc cgcgaggtga 12360

aggtgatcac cctgaagtcc aagctggtca gcgacttccg caaggacttc cagttctaca 12420

aggtgcgcga gatcaacaac taccaccacg cccacgacgc gtacctgaac gccgtggtgg 12480

gcaccgcgct gatcaagaag taccccaagc tggagagcga gttcgtgtac ggcgactaca 12540

aggtgtacga cgtgcgcaag atgatcgcca agtcggagca ggagatcggc aaggccaccg 12600

cgaagtactt cttctactcc aacatcatga acttcttcaa gaccgagatc acgctggcca 12660

acggcgagat ccgcaagcgc ccgctgatcg agaccaacgg cgagacgggc gagatcgtgt 12720

gggacaaggg ccgcgacttc gcgaccgtgc gcaaggtgct gagcatgccc caggtgaaca 12780

tcgtgaagaa gaccgaggtg cagacgggcg gcttctccaa ggagagcatc ctgccgaagc 12840

gcaactcgga caagctgatc gcccgcaaga aggactggga ccccaagaag tacggcggct 12900

tcgactcccc gaccgtggcc tacagcgtgc tggtggtggc gaaggtggag aagggcaagt 12960

ccaagaagct gaagagcgtg aaggagctgc tgggcatcac catcatggag cgcagctcgt 13020

tcgagaagaa ccccatcgac ttcctggagg ccaagggcta caaagaggtg aagaaggacc 13080

tgatcatcaa gctgccgaag tactcgctgt tcgagctgga gaacggccgc aagcgcatgc 13140

tggcctccgc gggcgagctg cagaagggca acgagctggc cctgcccagc aagtacgtga 13200

acttcctgta cctggcgtcc cactacgaga agctgaaggg ctcgccggag gacaacgagc 13260

agaagcagct gttcgtggag cagcacaagc actacctgga cgagatcatc gagcagatct 13320

cggagttctc caagcgcgtg atcctggccg acgcgaacct ggacaaggtg ctgagcgcct 13380

acaacaagca ccgcgacaag cccatccgcg agcaggcgga gaacatcatc cacctgttca 13440

ccctgacgaa cctgggcgcc ccggccgcgt tcaagtactt cgacaccacg atcgaccgca 13500

agcgctacac ctccacgaaa gaggtgctgg acgcgaccct gatccaccag agcatcaccg 13560

gcctgtacga gacgcgcatc gacctgagcc agctgggcgg cgactcccgc gcggacccga 13620

agaagaagcg caaggtgtaa gaattaattc ggatccattg gtgaccagct cgaatttccc 13680

cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 13740

gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 13800

catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 13860

cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 13920

tatgttacta gatcggggag ctccatggag tcaaagattc aaatagagga cctaacagaa 13980

ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag 14040

aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat 14100

acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac 14160

ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa 14220

ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct 14280

gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 14340

gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 14400

gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 14460

ttggagagaa cacgggggac ttctagaccc caaacacacg ctcggacgca tattacacat 14520

gttcatacac ttaatactcg ctgttttgaa ttgatgtttt aggaatatat atgtagaaaa 14580

gacgactgta gcttatattc acaggtcgtg atatgattca attagcttcc gactcattca 14640

tccaaatacc gagtcgccaa aattcaaact agactcgtta aatgaatgaa tgatgcggta 14700

gacaaattgg atcattgatt ctctttgatt ataaggtaca gtcgtccttt ctctcttttg 14760

tattccaatt ttcttgatta atctttcctg cacaaaaaca tgcttgatcc actaagtgac 14820

atatatgctg ccttcgtata tatagttctg gtaaaattaa cattttgggt ttatctttat 14880

ttaaggcatc gccatggggg gcgcgccagc tcgaatttcc ccgatcgttc aaacatttgg 14940

caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 15000

ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 15060

tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 15120

tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 15180

gcttatccga gaattccgac ttgccttccg cacaatacat catttcttct tagctttttt 15240

tcttcttctt cgttcataca gttttttttt gtttatcagc ttacattttc ttgaaccgta 15300

gctttcgttt tcttcttttt aactttccat tcggagtttt tgtatcttgt ttcatagttt 15360

gtcccaggat tagaatgatt aggcatcgaa ccttcaagaa tttgattgaa taaaacatct 15420

tcattcttaa gatatgaaga taatcttcaa aaggcccctg ggaatctgaa agaagagaag 15480

caggcccatt tatatgggaa agaacaatag tatttcttat ataggcccat ttaagttgaa 15540

aacaatcttc aaaagtccca catcgcttag ataagaaaac gaagctgagt ttatatacag 15600

ctagagtcga agtagtgatt gtggactatg ttcttaatca gttttagagc tagaaatagc 15660

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 15720

ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt cttcggcgtt caatttctgg 15780

ggttttctct tcgttttctg taactgaaac ctaaaatttg acctaaaaaa aatctcaaat 15840

aatatgattc agtggttttg tacttttcag ttagttgagt tttgcagttc cgatgagata 15900

aaccaatatt aatccaaact actgcagcct gacagacaaa tgaggatgca aacaatttta 15960

aagtttatct aacgctagct gttttgtttc ttctctctgg tgcaccaacg acggcgtttt 16020

ctcaatcata aagaggcttg ttttacttaa ggccaataat gttgatggat cgaaagaaga 16080

gggcttttaa taaacgagcc cgtttaagct gtaaacgatg tcaaaaacat cccacatcgt 16140

tcagttgaaa atagaagctc tgtttatata ttggtagagt cgactaagag attgtgagcc 16200

ctaatgtgaa caagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 16260

aacttgaaaa agtggcaccg agtcggtgct tttttttgca aaattttcca gatcgatttc 16320

ttcttcctct gttcttcggc gttcaatttc tggggttttc tcttcgtttt ctgtaactga 16380

aacctaaaat ttgacctaaa aaaaatctca aataatatga ttcagtggtt ttgtactttt 16440

cagttagttg agttttgcag ttccgatgag ataaaccaat ggtaccaatt aaactatcag 16500

tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgttta 16549

Claims (2)

1. efficient CRISPR-CAS9 gene editing carrier in a kind of arabidopsis, it is characterised in that: including pCambia-Cas9- AmiR-RDR6 sequence, as shown in sequence table SEQ ID NO.4.

2. the construction method of efficient CRISPR-CAS9 gene editing carrier in a kind of arabidopsis, which is characterized in that method is as follows:

Step 1: the required gRNA of the target gene of selection CRISPR/Caa9 system editor selects specific N₁₉NGG sequence, synthesis Following templa-primer group sequence:

CRIPSR-F:

GAATGGTCTCTATTG N₁₉NGG sequenceGTTTTAGAGCTAGAAATAGCAAGTT

CRIPSR-R:

ATTATTGGTCTCTAAAC N₁₉NGG sequenceCAATCTCTTAGTCGACTCTACCA；

Step 2: using pCBC-DT1T2 sequence as template, sequence is as shown in sequence table SEQ ID NO.1, with templa-primer group CRISPR-F/R carries out PCR amplification, purification and recovery PCR product；

Step 3: using BsaI enzyme digestion PCR product and pEntry-gRNA sequence, shown in sequence table SEQ ID NO.2, returns Target fragment is received, with the recovery product of both T4 DNA ligase connections, obtains pEntry-gRNA-CRIS- target gene plasmid；

Step 4: two enzyme digestion pEntry-gRNA-CRIS- target gene plasmids of KpnI+EcoRI and pCambia-Cas9- are used AmiR-RDR6 sequence, sequence recycle target fragment as shown in sequence table SEQ ID NO.4, with T4DNA ligase connection two Person's recovery product obtains ultimate sequence pCas9-amiR-R6-CRIS- target gene；

Step 5: Agrobacterium infestation method is utilized, pCas9-amiR-R6-CRIS- target gene plasmid is transferred in plant, to plant T1 is collected after maturation for seed, with the Screening of Media T1 containing hygromycin resistance for seed positive transgenic seedling, to T1 for seed After positive transgenic plant seedling is mature, T2 is collected for seed；

Step 6: T2 can be presented particular vane phenotype, utilize this point for the Arabidopsis plant containing transgenosis plasmid in seed The T2 without transgenosis plasmid can be filtered out for plant, the spy of plant appearance after target gene is edited in conjunction with CRISPR/Cas9 Determining phenotype can choose target gene to be edited and be free of the Mutants homozygous of transgenosis plasmid.