CN111139261B - Method for reducing polyphenol oxidase content of wheat grains by using gene editing - Google Patents

Abstract

The invention discloses a method for reducing polyphenol oxidase content of wheat grains by using gene editing. According to the invention, through the design of the specificity guide single-chain gRNA, an expression vector capable of realizing in vivo editing of the PPO gene is constructed, and the wheat PPO gene can be knocked out. In practical application, through a gene conversion process, the silencing of endosperm specific expression PPO gene at the genome level can be realized, so that the content of polyphenol oxidase in wheat grains is reduced, the browning of flour is reduced, and the whiteness of the flour is improved. Provides a powerful tool for improving the whiteness of the wheat flour and improving the processing quality of the wheat by utilizing a genetic engineering technology.

Description

Method for reducing polyphenol oxidase content of wheat grains by using gene editing

Technical Field

The invention relates to a method for reducing polyphenol oxidase content of wheat grains by using gene editing, belonging to the field of gene engineering.

Background

Polyphenol Oxidase (PPO) in wheat endosperm easily causes dough browning, thereby influencing the whiteness of flour products. The PPO activity in wheat flour only accounts for about 3% of the whole grains, but can account for 50-70% of the color browning of flour and flour products in the processing and storage processes. The color of the flour of many wheat varieties in China can not meet the processing requirement, and flour production and food processing enterprises adopt a mode of adding a whitening agent into the flour to increase the whiteness of the product. Benzoyl Peroxide (BPO) is an oxidizing agent and is currently the most widely used flour whitener. The decomposition products of BPO, namely benzoic acid and phenol, need to be detoxified in the liver, and chronic poisoning can be caused by long-term consumption of flour and products thereof with excessive BPO. Therefore, people have long expected that the flour product without the whitening agent comes out, the cultivation of the wheat variety with low PPO will have important influence on the processing technology of the flour product, and the food without the whitening agent will have huge market space and industrialization prospect. Therefore, the cultivation of wheat varieties with low PPO activity is an important way for improving the color and luster properties of the flour products.

The PPO activity of wheat grains is mainly regulated and controlled by genes positioned on a second homologous group and is respectively positioned on 2AL and 2DL, wherein the QTL on 2AL has a larger effect and a contribution rate of 37.9-50.0%; the QTL contribution rate on 2DL is 25.0-29.1% (D.J.Sun, Z.H.He, X.C.Xia, L.P.Zhang, C.F.Morris, R.Appels, W.J.Ma, H.Wang.A novel STS marker for polyphenolic oxidase activity in branched straw breaking, 2005,16: 209. 218). All coding sequences of wheat grain PPO genes on 2A and 2D chromosomes are cloned by using an electronic cloning technology, and the functions of the genes are preliminarily verified by using model plants. Ppo-A1, Ppo-D1 each contained a 1731bp reading frame (X.Y.He, Z.H.He, L.P.Zhang, D.J.Sun, C.F.Morris, E.P.Furerst, and X.C.Xia.Allelic. evolution of polyphenolic oxidases (PPO) localized on chromosomes 2A and 2D and depth of functional markers for the PPO genes in common wheat heat, Theor l Gene, 2007,115:47-58.), which not only showed their homology but also provided the facility for synchronizing the PPO genes silenced on 2A and 2D using RNAi technology.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a method for reducing the polyphenol oxidase content of wheat grains by using gene editing. The method disclosed by the invention has the advantages that the expression level of the PPO gene in the endosperm is reduced through the gene transformation process by constructing the expression vector pBUE411-PPO with the function of knocking out the PPO gene.

A pBUE411-PPO binary vector with a function of knocking out PPO genes comprises an expression box A and an expression box B;

the expression cassette A specifically comprises the following elements from upstream to downstream in sequence: TaU3 promoter A from wheat, sgRNA of the PPO gene and terminator A;

the expression cassette B specifically comprises the following elements from upstream to downstream in sequence: the promoter B, the maize Cas9 coding sequence, and the terminator B.

Further, the sequence of sgRNA of the PPO gene is shown as SEQ 10;

the maize Cas9 coding sequence is shown in SEQ 13;

the sequence of the promoter A is shown as SEQ 1;

the promoter B sequence is shown as SEQ 2;

the terminator A sequence is shown as SEQ 3;

the terminator B sequence is shown as SEQ 4.

Further, the expression cassette A can be specifically shown as a sequence SEQ 5;

the expression cassette B can be specifically shown as a sequence SEQ 6;

the expression cassette A is located at the upstream of the expression cassette B, and the expression cassette A and the expression cassette B are connected in series.

Further, the pBUE411-PPO binary vector also contains a resistance marker gene, such as a bar gene.

Further, the nucleotide sequence of the pBUE411-PPO binary vector is shown as SEQ 7.

A method for constructing the pBUE411-PPO binary vector comprises the following steps:

s11, designing sgRNAs according to targeted PPO genes, wherein the oligonucleotides of the sgRNAs are PPO-gR1-TaU3-F and PPO-gR1-TaU3-R correspondingly, namely the sequence of PPO-gR1-TaU3-F is shown as SEQ8, and the sequence of PPO-gR1-TaU3-R is shown as SEQ 9;

s12, phosphorylating and annealing 2 oligonucleotides of the sgRNA synthesized in the step I to form a double chain, and obtaining the double-chain sgRNA with a sticky end;

s13, utilizing restriction enzyme BsaI to cut the pBUE411 vector, and recovering vector fragments containing the promoter A, the terminator A, the promoter B and the terminator B;

s14, the pBUE411 vector fragment recovered in the step S13 and the double-chain sgRNA are subjected to ligation reaction through T4 ligase to obtain a final pBUE411-PPO binary vector.

Further, the PPO gene may be GENBANK ACCESSION NUMBER: EF070147.1(VERSION ABK 62801.1).

A transgenic cell line or recombinant strain containing the pBUE411-PPO binary vector also belongs to the protection scope of the invention.

The pBUE411-PPO binary vector can be applied to reducing the expression level of PPO genes in wheat endosperm.

The pBUE411-PPO binary vector is applied to the improvement of the color of wheat flour.

The pBUE411-PPO binary vector is applied to cultivation of transgenic wheat; the flour whiteness of the transgenic wheat is increased compared to pre-transgenic wheat.

The invention also protects a DNA segment I, wherein the DNA segment I consists of guide sgRNA of PPO gene, and the sequence of the DNA segment I can be specifically shown as a sequence SEQ 10.

A method for reducing the polyphenol oxidase content of wheat grains by using gene editing comprises the following steps:

s1, constructing a pBUE411-PPO binary vector;

s2, transforming the pBUE411-PPO binary vector obtained in the step S1 into common wheat through agrobacterium tumefaciens to obtain transgenic wheat.

Has the advantages that:

according to the invention, through the design of the specificity guide single-chain gRNA, an expression vector capable of realizing in vivo editing of the PPO gene is constructed, and the wheat PPO gene can be knocked out. In practical application, through a gene conversion process, the silencing of endosperm specific expression PPO gene at the genome level can be realized, so that the content of polyphenol oxidase in wheat grains is reduced, the browning of flour is reduced, and the whiteness of the flour is improved. The invention provides a powerful tool for improving the whiteness of wheat flour and improving the processing quality of wheat by utilizing a genetic engineering technology.

Drawings

FIG. 1 the coding region of the PPO gene.

FIG. 2 restriction electrophoresis of the vector BsaI pBUE 411.

FIG. 3 sequencing results of recombinant vector pBUE411-PPO of targeted PPO gene CRISPR/Cas 9.

FIG. 4Bar test paper strip detection of transgenic wheat plants.

FIG. 5 deduced amino acid sequence of wild-type wheat PPO gene.

FIG. 6 deduced amino acid sequence of mutated wheat PPO gene.

FIG. 7 is a nucleotide sequence alignment chart of wild-type and mutant PPO genes.

FIG. 8 alignment of deduced amino acid sequences of wild-type and mutant PPO genes.

FIG. 9 is a dot diagram of gene editing sites of the PPO of the high-tom test transgenic lines.

FIG. 10 shows the relative expression of the PPO genes of the grain of the gene editing strain and the grain of the control plant.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. High-fidelity enzyme required for PCR amplification, T4 ligase required for DNA fragment ligation, a gel recovery kit required for enzyme fragment recovery, and a plasmid extraction kit are purchased from Dalibao biology. The plasmid extraction kit is purchased from Tiangen organisms, the inorganic salt required for preparing the culture medium is purchased from the national medicine group, and the vitamins, antibiotics and hormones are purchased from Sigma company. The plant CRISPR/Cas9 gene editing vector is pBUE411, contains a wheat U3 promoter TaU3 for starting sgRNA, and Cas9p simulates the characteristic that gramineous plant genes have high GC content at the 5' end and is a designed and synthesized plant optimized codon gene. The E.coli strain used in this laboratory was E.coli DH5 alpha.

Reagents such as T4 ligase and DNA Marker DL2000 were purchased from Takara; fastpfu, 2 × Taq Mix, DH5 α, etc. were purchased from Beijing Quanji; restriction enzyme BsaI-HF was purchased from NEB; the primers used were synthesized by Qingdao Optimaki Biotechnology Limited and have the following sequences:

TABLE 1 primers used in this study

The wheat variety JW1 is a new germplasm with good tissue culture capability self-bred by crops of agricultural academy of Shandong province, and can be obtained by the public from the research of crops of agricultural academy of Shandong province

Plasmid pBUE411 is shown as sequence 7 (17430bp) in the sequence table and is provided by Sunyu task group of Chinese agriculture university; the public is available from the university of Chinese agriculture.

Experimental example 1 construction of expression vector

1. Design of sgRNA targeting PPO

The sequence of the TaPPO gene is amplified in a wheat variety JW1, the amplified sequence is shown in figure 1, the underlined sequence is a gRNA sequence of the targeted PPO gene, the three-digit sequence after the underlined part is marked as a PAM sequence, and the sequence is 100 percent similar to the gene sequence of wheat in an NCBI database. The gene has a total length of 1734 basic groups, 1 suitable targeting site is searched for in a coding region of a PPO gene through a website CRISPRDIRect (https:// crispr. dbcls. jp /), a 20bp sequence fragment is found in front of a PAM structure and is set as a target sequence, and an sgRNA sequence is selected as GAAGAAGACGCTGCTGTTCC.

Annealing of sgRNA and formation of double strands

The 2 oligos of sgRNA of PPO gene are synthesized by Qingdao catalpi biotechnology limited, wherein the oligonucleotides of sgRNA correspond to PPO-gR1-TaU3-F and PPO-gR1-TaU3-R (Table 1), and the underlined base parts in the primer are matched with the sequence in pBUE411 vector. Respectively phosphorylating 2 oligos of the sgRNA, and directly annealing to form double chains, wherein the system is as follows: PPO-gR1-TaU3-F (10. mu.M): 4 μ L, PPO-gR1-TaU3-R (10 uM): 4 μ L,10 XT 4 PNK buffer:1.5 μ L, PNK:1 μ L, ATP:1 μ L, ddH2O make up to 15 μ L. The program was run in a PCR instrument under the following conditions: 30min at 37 ℃; 95 ℃ for 5 min; ramp to 25 ℃ at5 ℃/min (0.08 ℃/s). Or boiling in boiling water for 5min, and naturally cooling to room temperature.

Linearization of the pBUE411 vector

Digesting the pBUE411 vector for 3h by using BsaI endonuclease, recovering a linear vector fragment after electrophoresis, recovering a vector fragment containing the promoter A, the terminator A, the promoter B and the terminator B, and digesting an electrophoresis diagram of the vector fragment by using BsaI, wherein 1 is that pBUE411 is digested by using BsaI; 2 is DL2000 Marker; 3 is a 1kb Marker.

Ligation of the pBUE411 vector with sgRNA

The connection reaction system is as follows: 5 × ligation Buffer 2 μ L, annealed oligo duplex 4 μ L, pBUE411/BsaI 2ul (100ng), T4 strain 1 μ L, ddH2O to make up 10 μ L, ligation reaction was performed at 25 ℃ for 30 min.

5. Transformation of

The ligation product is transformed into Escherichia coli, the recombinant plasmid is transformed into DH5 alpha competent cells, then the cells are cultured on an LB (kan) plate until clones grow out, and the single clone is selected for carrying out bacteria liquid PCR identification and sequencing, wherein the primers are pBUE411-F and pBUE 411-R. The sequencing result (as shown in fig. 3) detects the target sequence of the sgRNA, and simultaneously detects TaU3 promoter sequences at the upstream of the target sequence, and the sequencing result shows that the sgRNA expression cassette is successfully constructed and successfully assembled into a pBUE411 binary expression vector, which proves that the construction of the CRISPR/Cas9 gene editing vector of PPO is successful.

Experimental example 2 transformation of Gene editing vector into common wheat

1. Preparing a culture medium: see Kan Wang (ed.), Agrobacterium Protocals: Volume 1, Methods in Molecular Biology, vol.1223DOI10.007/978-1-4939-

2. And (3) agrobacterium transformation: taking wheat ears pollinated for about 15 days, taking grains and stripping embryos. The day before the test, the agrobacterium suspension is shaken, cultured at 160r and 28 ℃ for 24 hours, after the ear is prepared, the agrobacterium suspension is prepared, 1ml of the bacterial liquid is taken into a 1.5ml centrifuge tube, and 1.4ul of acetosyringone (0.1M) is added and mixed evenly. Adding prepared bacterial liquid for infection for 5 minutes, placing on a co-culture medium, and performing dark culture at 23 ℃ for 3 days. After co-culture, the cells were placed on a resting medium for dark culture for 5 days at 25 ℃. The calli were transferred to selection medium 1. The petri dish was sealed with a sealing film and cultured in a 25.5 degree incubator for 2 weeks in the dark. The calli were cut and transferred to selection medium 2.

The petri dish was sealed with a sealing film and cultured in a 25.5 degree incubator for 2 weeks in the dark. After 2 weeks of callus excision screening, resistant calli were transferred to regeneration medium. The resistant calli typically have green buds or dots, or have beautiful beige globular structures.

Pasty and brown callus did not transfer. The proliferated callus may be cut into smaller callus. The patches from the same callus should be re-laid on the same line. Note the direction of the callus, e.g., green bud and green dot up.

The culture dish is sealed and put into a 25-degree incubator for 2 weeks under illumination (16 h). After 2 weeks of regeneration, healthy growing plantlets were transferred to new resistant regeneration pods. When the seedlings grow to a certain size, sampling detection can be carried out.

3. Mutant detection of transgenic seedlings

3.1T0 generation transgenic line positive identification and mutant screening

As the T-DNA region of the vector used for the transformation comprises three expression frames of cas9, bar and gRNA, the expression detection of the bar gene is very mature, and a commercial detection reagent strip is provided, and a sample can be judged whether to carry the bar gene within 10 minutes after coarse grinding by using the reagent strip, so that the method is faster and more efficient than PCR amplification. Therefore, the non-transgenic plant is used as a negative control, a bar gene detection kit Quickstix is used for detecting the transgenic wheat T0 plant, and the specific operation of screening the positive transgenic seedling is as follows:

taking 3-5cm of transgenic seedling, placing into a 1.5m centrifuge tube, grinding with a grinding rod, adding 500 μ L Buffer, inserting Bar test strip, standing for 5min, recording data according to the number of bands displayed by the test strip, wherein 2 bands represent positive, and 1 band represents negative. The kit was purchased from maitsen technologies ltd.

The results are shown in fig. 4, samples 1-10 are T0 regenerated seedlings, 11 is a positive control, 12 is a negative control, wherein two bands appear in samples 1,3,4,5,6,7,8,10, and the results are consistent with the results of the positive control 11, and the samples are positive seedlings; 2,9, and numbers 1-10 consistent with negative controls, represent ePO-01, -02, -03, -04, -05, 06, -07, -08, -09, -10, respectively.

3.2 specific amplification and mutant identification of PSY gene of transgenic plant generation T0

Whether the target gene editing is successful or not needs sequencing for identification, and the Hi-TOM gene editing site detection kit produced by Tianjinnuo grass genesis company is adopted in the experiment. The kit automatically completes the high-throughput library building process by a PCR method, and directly analyzes the variation information of multiple samples and multiple sites by using Hi-TOM online software. Specific primers (Primer-F and Primer-R in the table 1) on two sides of the target sequence are used for amplifying PPO, and amplified products are sent to norops for sequencing after being subjected to library building. The wild type nucleotide sequence of the wheat PPO gene of the target gene is shown as SEQ11, the deduced amino acid sequence is shown as figure 5, the nucleotide sequence of the mutant of the target gene is shown as SEQ12, the deduced amino acid sequence is shown as figure 6, after alignment, the nucleotide of ePO-2 at +887 is reduced by 2 bases GT, TGA appears at +1113 of the mutant sequence and becomes a stop codon, which causes the early termination of protein translation, the alignment result of the nucleotide sequences is shown as figure 7, the part marked with bold underline is the part where the wild type and the mutant are different, the alignment result of the amino acid sequences is shown as figure 8, and the part marked with light gray color is the part where the wild type and the mutant are different.

First round PCR reaction System: mu.L of pBUE411-PPO transgenic wheat plant leaf DNA was used as a template, and 2 XTAQA Master Mix 10. mu.L, Primer-F and Primer-R (Table 1) (10. mu.M) in the kit were each 0.5. mu.L, and nucleic-free Water was supplemented to 20. mu.L. The PCR reaction conditions are as follows: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30s, renaturation at 65 ℃ for 30s, and extension at 72 ℃ for 20s for 33 cycles; finally, extension is carried out for 5min at 72 ℃. After the PCR is finished, 5 mu L of agarose gel electrophoresis is taken to detect the PCR product, so that the existence of the target product is ensured and the specificity is good.

Then, a second round of PCR reaction was performed, in which 12. mu.L of Hi-TOM Mix in the kit was added to make up the volume to 20. mu.L using the first round of PCR product as a template, and 1. mu.L of nucleic-free Water was added. PCR reaction procedure: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and extension at 72 ℃ for 25s for 33 cycles; finally, extension is carried out for 5min at 72 ℃.

And (5) constructing a library of the amplification product and sending the library to a Nordheim source for sequencing. As shown in FIG. 9 (samples 1-12 are different transgenic lines, M is DL2000 marker, indicating a base change at the mutation site), where exactly 2 bases GT, the nucleotide of ePO-2 at +887 was reduced by 2 bases GT by alignment, TGA appeared at +1113 of the mutated sequence, becoming a stop codon, causing premature termination of protein translation.

3.3 analysis of the expression level of PPO Gene in Gene-editing Strain

The total RNA extraction kit (Tiangen, Beijing, China) of the RNAprep Pure plant is used for extracting the total RNA of grains of transgenic plants and control plants in different development periods (7, 14, 21 and 28DPA), and the relative expression quantity of the PPO genes of each transgenic plant line in different development periods of the grains is detected. Based on the conservation and difference among the PPO homologous gene cDNA sequences of wheat grains, a conservative primer capable of amplifying A, B, D genome homologous genes simultaneously is designed and used for qRT-PCR detection. The common wheat beta-actin gene is used as reference gene. qRT-PCR reaction System: 2 XSSYBR Green Mix 5. mu.L, Primer-F and Primer-R (Table 1) (10. mu.M) each 0.5. mu.L, 50ng cDNA, nucleic-free Water make up volume to 10. mu.L. Reaction procedure: denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 15s, renaturation at 60 ℃ for 20s, and elongation at 72 ℃ for 20s for 40 cycles. 3 biological replicates were selected for each transgenic line, 3 technical replicates per sample. The results showed that the expression level of PPO gene in the gene-editing line was significantly reduced as shown in fig. 10 (×) indicating P <0.05 and WT indicating wild type.

TABLE 2 primer sequences (II)

3.4PPO Activity assay

Weighing 0.25g of flour sample, adding 3mL of 50mmol/L MOPS buffer solution with pH6.5 by using catechol as a substrate, carrying out shaking extraction for 30min, then adding 1mL of 120mmol/L catechol, carrying out shaking reaction for 20min, centrifuging for 10min at 10000g/min, taking the buffer solution and the catechol as a control, and taking the supernatant to measure the light absorption value at 405 nm. 3 biological replicates were assayed per transgenic line and three replicates per sample were tested. The results show that compared with the non-transgenic control wheat (13.16min-1g-1), the polyphenol oxidase (PPO activity) in the grains of the gene editing line is obviously reduced (8.72min-1 g-1).

SEQUENCE LISTING

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gaaaaagtgg caccgagtcg gtgctttttt ttttcgtttt gcattgagtt ttctccgtcg 660

catgtttgca gttttatttt ccgttttgca ttgaaatttc tccgtctcat gtttgcagcg 720

tgttcaaaaa gtacgcagct gtatttcact tatttacggc gccacatttt catgccgttt 780

gtgccaacta tcccgagcta gtgaatacag cttggcttca cacaacactg gtgacccgct 840

gacctgctcg tacctcgtac cgtcgtacgg cacagcattt ggaattaaag ggtgtgatcg 900

atactgcttg ctgct 915

<210> 6

<211> 6799

<212> DNA

<213> Artificial sequence

<400> 6

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380

agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440

gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500

ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560

gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620

aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680

catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740

ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800

ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860

gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920

atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980

ttacttctgc agccctaggc ctactagatg gattacaagg accacgacgg ggattacaag 2040

gaccacgaca ttgattacaa ggatgatgat gacaagatgg ctccgaagaa gaagaggaag 2100

gttggcatcc acggggtgcc agctgctgac aagaagtact cgatcggcct cgatattggg 2160

actaactctg ttggctgggc cgtgatcacc gacgagtaca aggtgccctc aaagaagttc 2220

aaggtcctgg gcaacaccga tcggcattcc atcaagaaga atctcattgg cgctctcctg 2280

ttcgacagcg gcgagacggc tgaggctacg cggctcaagc gcaccgcccg caggcggtac 2340

acgcgcagga agaatcgcat ctgctacctg caggagattt tctccaacga gatggcgaag 2400

gttgacgatt ctttcttcca caggctggag gagtcattcc tcgtggagga ggataagaag 2460

cacgagcggc atccaatctt cggcaacatt gtcgacgagg ttgcctacca cgagaagtac 2520

cctacgatct accatctgcg gaagaagctc gtggactcca cagataaggc ggacctccgc 2580

ctgatctacc tcgctctggc ccacatgatt aagttcaggg gccatttcct gatcgagggg 2640

gatctcaacc cggacaatag cgatgttgac aagctgttca tccagctcgt gcagacgtac 2700

aaccagctct tcgaggagaa ccccattaat gcgtcaggcg tcgacgcgaa ggctatcctg 2760

tccgctaggc tctcgaagtc tcggcgcctc gagaacctga tcgcccagct gccgggcgag 2820

aagaagaacg gcctgttcgg gaatctcatt gcgctcagcc tggggctcac gcccaacttc 2880

aagtcgaatt tcgatctcgc tgaggacgcc aagctgcagc tctccaagga cacatacgac 2940

gatgacctgg ataacctcct ggcccagatc ggcgatcagt acgcggacct gttcctcgct 3000

gccaagaatc tgtcggacgc catcctcctg tctgatattc tcagggtgaa caccgagatt 3060

acgaaggctc cgctctcagc ctccatgatc aagcgctacg acgagcacca tcaggatctg 3120

accctcctga aggcgctggt caggcagcag ctccccgaga agtacaagga gatcttcttc 3180

gatcagtcga agaacggcta cgctgggtac attgacggcg gggcctctca ggaggagttc 3240

tacaagttca tcaagccgat tctggagaag atggacggca cggaggagct gctggtgaag 3300

ctcaatcgcg aggacctcct gaggaagcag cggacattcg ataacggcag catcccacac 3360

cagattcatc tcggggagct gcacgctatc ctgaggaggc aggaggactt ctaccctttc 3420

ctcaaggata accgcgagaa gatcgagaag attctgactt tcaggatccc gtactacgtc 3480

ggcccactcg ctaggggcaa ctcccgcttc gcttggatga cccgcaagtc agaggagacg 3540

atcacgccgt ggaacttcga ggaggtggtc gacaagggcg ctagcgctca gtcgttcatc 3600

gagaggatga cgaatttcga caagaacctg ccaaatgaga aggtgctccc taagcactcg 3660

ctcctgtacg agtacttcac agtctacaac gagctgacta aggtgaagta tgtgaccgag 3720

ggcatgagga agccggcttt cctgtctggg gagcagaaga aggccatcgt ggacctcctg 3780

ttcaagacca accggaaggt cacggttaag cagctcaagg aggactactt caagaagatt 3840

gagtgcttcg attcggtcga gatctctggc gttgaggacc gcttcaacgc ctccctgggg 3900

acctaccacg atctcctgaa gatcattaag gataaggact tcctggacaa cgaggagaat 3960

gaggatatcc tcgaggacat tgtgctgaca ctcactctgt tcgaggaccg ggagatgatc 4020

gaggagcgcc tgaagactta cgcccatctc ttcgatgaca aggtcatgaa gcagctcaag 4080

aggaggaggt acaccggctg ggggaggctg agcaggaagc tcatcaacgg cattcgggac 4140

aagcagtccg ggaagacgat cctcgacttc ctgaagagcg atggcttcgc gaaccgcaat 4200

ttcatgcagc tgattcacga tgacagcctc acattcaagg aggatatcca gaaggctcag 4260

gtgagcggcc agggggactc gctgcacgag catatcgcga acctcgctgg ctcgccagct 4320

atcaagaagg ggattctgca gaccgtgaag gttgtggacg agctggtgaa ggtcatgggc 4380

aggcacaagc ctgagaacat cgtcattgag atggcccggg agaatcagac cacgcagaag 4440

ggccagaaga actcacgcga gaggatgaag aggatcgagg agggcattaa ggagctgggg 4500

tcccagatcc tcaaggagca cccggtggag aacacgcagc tgcagaatga gaagctctac 4560

ctgtactacc tccagaatgg ccgcgatatg tatgtggacc aggagctgga tattaacagg 4620

ctcagcgatt acgacgtcga tcatatcgtt ccacagtcat tcctgaagga tgactccatt 4680

gacaacaagg tcctcaccag gtcggacaag aaccggggca agtctgataa tgttccttca 4740

gaggaggtcg ttaagaagat gaagaactac tggcgccagc tcctgaatgc caagctgatc 4800

acgcagcgga agttcgataa cctcacaaag gctgagaggg gcgggctctc tgagctggac 4860

aaggcgggct tcatcaagag gcagctggtc gagacacggc agatcactaa gcacgttgcg 4920

cagattctcg actcacggat gaacactaag tacgatgaga atgacaagct gatccgcgag 4980

gtgaaggtca tcaccctgaa gtcaaagctc gtctccgact tcaggaagga tttccagttc 5040

tacaaggttc gggagatcaa caattaccac catgcccatg acgcgtacct gaacgcggtg 5100

gtcggcacag ctctgatcaa gaagtaccca aagctcgaga gcgagttcgt gtacggggac 5160

tacaaggttt acgatgtgag gaagatgatc gccaagtcgg agcaggagat tggcaaggct 5220

accgccaagt acttcttcta ctctaacatt atgaatttct tcaagacaga gatcactctg 5280

gccaatggcg agatccggaa gcgccccctc atcgagacga acggcgagac gggggagatc 5340

gtgtgggaca agggcaggga tttcgcgacc gtcaggaagg ttctctccat gccacaagtg 5400

aatatcgtca agaagacaga ggtccagact ggcgggttct ctaaggagtc aattctgcct 5460

aagcggaaca gcgacaagct catcgcccgc aagaaggact gggatccgaa gaagtacggc 5520

gggttcgaca gccccactgt ggcctactcg gtcctggttg tggcgaaggt tgagaagggc 5580

aagtccaaga agctcaagag cgtgaaggag ctgctgggga tcacgattat ggagcgctcc 5640

agcttcgaga agaacccgat cgatttcctg gaggcgaagg gctacaagga ggtgaagaag 5700

gacctgatca ttaagctccc caagtactca ctcttcgagc tggagaacgg caggaagcgg 5760

atgctggctt ccgctggcga gctgcagaag gggaacgagc tggctctgcc gtccaagtat 5820

gtgaacttcc tctacctggc ctcccactac gagaagctca agggcagccc cgaggacaac 5880

gagcagaagc agctgttcgt cgagcagcac aagcattacc tcgacgagat cattgagcag 5940

atttccgagt tctccaagcg cgtgatcctg gccgacgcga atctggataa ggtcctctcc 6000

gcgtacaaca agcaccgcga caagccaatc agggagcagg ctgagaatat cattcatctc 6060

ttcaccctga cgaacctcgg cgcccctgct gctttcaagt acttcgacac aactatcgat 6120

cgcaagaggt acacaagcac taaggaggtc ctggacgcga ccctcatcca ccagtcgatt 6180

accggcctct acgagacgcg catcgacctg tctcagctcg ggggcgacaa gcggccagcg 6240

gcgacgaaga aggcggggca ggcgaagaag aagaagtgag ctcagagctt tcgttcgtat 6300

catcggtttc gacaacgttc gtcaagttca atgcatcagt ttcattgcgc acacaccaga 6360

atcctactga gtttgagtat tatggcattg ggaaaactgt ttttcttgta ccatttgttg 6420

tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga aatggaaatg 6480

gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt aatattattt 6540

gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg caaacatttt 6600

gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac cgaagttaat atgaggagta 6660

aaacacttgt agttgtacca ttatgcttat tcactaggca acaaatatat tttcagacct 6720

agaaaagctg caaatgttac tgaatacaag tatgtcctct tgtgttttag acatttatga 6780

actttccttt atgtaattt 6799

<210> 7

<211> 16230

<212> DNA

<213> Artificial sequence

<400> 7

gtaaacgctc ttttctctta ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt 60

aaactgaagg cgggaaacga caatctgatc caagctcaag ctgctctagc attcgccatt 120

caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 180

ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc 240

acgacgttgt aaaacgacgg ccagtgccaa gcttcatgaa tccaaaccac acggagttca 300

aattcccaca gattaaggct cgtccgtcgc acaaggtaat gtgtgaatat tatatctgtc 360

gtgcaaaatt gcctggcctg cacaattgct gttatagttg gcggcaggga gagttttaac 420

attgactagc gtgctgataa tttgtgagaa ataataattg acaagtagat actgacattt 480

gagaagagct tctgaactgt tattagtaac aaaaatggaa agctgatgca cggaaaaagg 540

aaagaaaaag ccatactttt ttttaggtag gaaaagaaaa agccatacga gactgatgtc 600

tctcagatgg gccgggatct gtctatctag caggcagcag cccaccaacc tcacgggcca 660

gcaattacga gtccttctaa aagctcccgc cgaggggcgc tggcgctgct gtgcagcagc 720

acgtctaaca ttagtcccac ctcgccagtt tacagggagc agaaccagct tataagcgga 780

ggcgcggcac caagaagcgg cggaagaaga cgctgctgtt ccgttttaga gctagaaata 840

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 900

ttttttttcg ttttgcattg agttttctcc gtcgcatgtt tgcagtttta ttttccgttt 960

tgcattgaaa tttctccgtc tcatgtttgc agcgtgttca aaaagtacgc agctgtattt 1020

cacttattta cggcgccaca ttttcatgcc gtttgtgcca actatcccga gctagtgaat 1080

acagcttggc ttcacacaac actggtgacc cgctgacctg ctcgtacctc gtaccgtcgt 1140

acggcacagc atttggaatt aaagggtgtg atcgatactg cttgctgcta agcttgcatg 1200

cctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag ataatgagca ttgcatgtct 1260

aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg cagtttatct 1320

atctttatac atatatttaa actttactct acgaataata taatctatag tactacaata 1380

atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa aggacaattg 1440

agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg ttctcctttt 1500

tttttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac atccatttag 1560

ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt tattctattt 1620

tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa taatttagat 1680

ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa gaaattaaaa 1740

aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta aacgccgtcg 1800

acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag 1860

acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc tccaccgttg 1920

gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg tgagccggca 1980

cggcaggcgg cctcctcctc ctctcacggc acggcagcta cgggggattc ctttcccacc 2040

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 2100

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 2160

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 2220

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 2280

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 2340

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 2400

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 2460

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 2520

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 2580

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 2640

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 2700

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 2760

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 2820

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 2880

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 2940

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 3000

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 3060

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 3120

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 3180

gttacttctg cagccctagg cctactagat ggattacaag gaccacgacg gggattacaa 3240

ggaccacgac attgattaca aggatgatga tgacaagatg gctccgaaga agaagaggaa 3300

ggttggcatc cacggggtgc cagctgctga caagaagtac tcgatcggcc tcgatattgg 3360

gactaactct gttggctggg ccgtgatcac cgacgagtac aaggtgccct caaagaagtt 3420

caaggtcctg ggcaacaccg atcggcattc catcaagaag aatctcattg gcgctctcct 3480

gttcgacagc ggcgagacgg ctgaggctac gcggctcaag cgcaccgccc gcaggcggta 3540

cacgcgcagg aagaatcgca tctgctacct gcaggagatt ttctccaacg agatggcgaa 3600

ggttgacgat tctttcttcc acaggctgga ggagtcattc ctcgtggagg aggataagaa 3660

gcacgagcgg catccaatct tcggcaacat tgtcgacgag gttgcctacc acgagaagta 3720

ccctacgatc taccatctgc ggaagaagct cgtggactcc acagataagg cggacctccg 3780

cctgatctac ctcgctctgg cccacatgat taagttcagg ggccatttcc tgatcgaggg 3840

ggatctcaac ccggacaata gcgatgttga caagctgttc atccagctcg tgcagacgta 3900

caaccagctc ttcgaggaga accccattaa tgcgtcaggc gtcgacgcga aggctatcct 3960

gtccgctagg ctctcgaagt ctcggcgcct cgagaacctg atcgcccagc tgccgggcga 4020

gaagaagaac ggcctgttcg ggaatctcat tgcgctcagc ctggggctca cgcccaactt 4080

caagtcgaat ttcgatctcg ctgaggacgc caagctgcag ctctccaagg acacatacga 4140

cgatgacctg gataacctcc tggcccagat cggcgatcag tacgcggacc tgttcctcgc 4200

tgccaagaat ctgtcggacg ccatcctcct gtctgatatt ctcagggtga acaccgagat 4260

tacgaaggct ccgctctcag cctccatgat caagcgctac gacgagcacc atcaggatct 4320

gaccctcctg aaggcgctgg tcaggcagca gctccccgag aagtacaagg agatcttctt 4380

cgatcagtcg aagaacggct acgctgggta cattgacggc ggggcctctc aggaggagtt 4440

ctacaagttc atcaagccga ttctggagaa gatggacggc acggaggagc tgctggtgaa 4500

gctcaatcgc gaggacctcc tgaggaagca gcggacattc gataacggca gcatcccaca 4560

ccagattcat ctcggggagc tgcacgctat cctgaggagg caggaggact tctacccttt 4620

cctcaaggat aaccgcgaga agatcgagaa gattctgact ttcaggatcc cgtactacgt 4680

cggcccactc gctaggggca actcccgctt cgcttggatg acccgcaagt cagaggagac 4740

gatcacgccg tggaacttcg aggaggtggt cgacaagggc gctagcgctc agtcgttcat 4800

cgagaggatg acgaatttcg acaagaacct gccaaatgag aaggtgctcc ctaagcactc 4860

gctcctgtac gagtacttca cagtctacaa cgagctgact aaggtgaagt atgtgaccga 4920

gggcatgagg aagccggctt tcctgtctgg ggagcagaag aaggccatcg tggacctcct 4980

gttcaagacc aaccggaagg tcacggttaa gcagctcaag gaggactact tcaagaagat 5040

tgagtgcttc gattcggtcg agatctctgg cgttgaggac cgcttcaacg cctccctggg 5100

gacctaccac gatctcctga agatcattaa ggataaggac ttcctggaca acgaggagaa 5160

tgaggatatc ctcgaggaca ttgtgctgac actcactctg ttcgaggacc gggagatgat 5220

cgaggagcgc ctgaagactt acgcccatct cttcgatgac aaggtcatga agcagctcaa 5280

gaggaggagg tacaccggct gggggaggct gagcaggaag ctcatcaacg gcattcggga 5340

caagcagtcc gggaagacga tcctcgactt cctgaagagc gatggcttcg cgaaccgcaa 5400

tttcatgcag ctgattcacg atgacagcct cacattcaag gaggatatcc agaaggctca 5460

ggtgagcggc cagggggact cgctgcacga gcatatcgcg aacctcgctg gctcgccagc 5520

tatcaagaag gggattctgc agaccgtgaa ggttgtggac gagctggtga aggtcatggg 5580

caggcacaag cctgagaaca tcgtcattga gatggcccgg gagaatcaga ccacgcagaa 5640

gggccagaag aactcacgcg agaggatgaa gaggatcgag gagggcatta aggagctggg 5700

gtcccagatc ctcaaggagc acccggtgga gaacacgcag ctgcagaatg agaagctcta 5760

cctgtactac ctccagaatg gccgcgatat gtatgtggac caggagctgg atattaacag 5820

gctcagcgat tacgacgtcg atcatatcgt tccacagtca ttcctgaagg atgactccat 5880

tgacaacaag gtcctcacca ggtcggacaa gaaccggggc aagtctgata atgttccttc 5940

agaggaggtc gttaagaaga tgaagaacta ctggcgccag ctcctgaatg ccaagctgat 6000

cacgcagcgg aagttcgata acctcacaaa ggctgagagg ggcgggctct ctgagctgga 6060

caaggcgggc ttcatcaaga ggcagctggt cgagacacgg cagatcacta agcacgttgc 6120

gcagattctc gactcacgga tgaacactaa gtacgatgag aatgacaagc tgatccgcga 6180

ggtgaaggtc atcaccctga agtcaaagct cgtctccgac ttcaggaagg atttccagtt 6240

ctacaaggtt cgggagatca acaattacca ccatgcccat gacgcgtacc tgaacgcggt 6300

ggtcggcaca gctctgatca agaagtaccc aaagctcgag agcgagttcg tgtacgggga 6360

ctacaaggtt tacgatgtga ggaagatgat cgccaagtcg gagcaggaga ttggcaaggc 6420

taccgccaag tacttcttct actctaacat tatgaatttc ttcaagacag agatcactct 6480

ggccaatggc gagatccgga agcgccccct catcgagacg aacggcgaga cgggggagat 6540

cgtgtgggac aagggcaggg atttcgcgac cgtcaggaag gttctctcca tgccacaagt 6600

gaatatcgtc aagaagacag aggtccagac tggcgggttc tctaaggagt caattctgcc 6660

taagcggaac agcgacaagc tcatcgcccg caagaaggac tgggatccga agaagtacgg 6720

cgggttcgac agccccactg tggcctactc ggtcctggtt gtggcgaagg ttgagaaggg 6780

caagtccaag aagctcaaga gcgtgaagga gctgctgggg atcacgatta tggagcgctc 6840

cagcttcgag aagaacccga tcgatttcct ggaggcgaag ggctacaagg aggtgaagaa 6900

ggacctgatc attaagctcc ccaagtactc actcttcgag ctggagaacg gcaggaagcg 6960

gatgctggct tccgctggcg agctgcagaa ggggaacgag ctggctctgc cgtccaagta 7020

tgtgaacttc ctctacctgg cctcccacta cgagaagctc aagggcagcc ccgaggacaa 7080

cgagcagaag cagctgttcg tcgagcagca caagcattac ctcgacgaga tcattgagca 7140

gatttccgag ttctccaagc gcgtgatcct ggccgacgcg aatctggata aggtcctctc 7200

cgcgtacaac aagcaccgcg acaagccaat cagggagcag gctgagaata tcattcatct 7260

cttcaccctg acgaacctcg gcgcccctgc tgctttcaag tacttcgaca caactatcga 7320

tcgcaagagg tacacaagca ctaaggaggt cctggacgcg accctcatcc accagtcgat 7380

taccggcctc tacgagacgc gcatcgacct gtctcagctc gggggcgaca agcggccagc 7440

ggcgacgaag aaggcggggc aggcgaagaa gaagaagtga gctcagagct ttcgttcgta 7500

tcatcggttt cgacaacgtt cgtcaagttc aatgcatcag tttcattgcg cacacaccag 7560

aatcctactg agtttgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt 7620

gtgcttgtaa tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat 7680

ggatggagaa gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt 7740

tgttttttct cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt 7800

tgttttgagt aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt 7860

aaaacacttg tagttgtacc attatgctta ttcactaggc aacaaatata ttttcagacc 7920

tagaaaagct gcaaatgtta ctgaatacaa gtatgtcctc ttgtgtttta gacatttatg 7980

aactttcctt tatgtaattt tccagaatcc ttgtcagatt ctaatcattg ctttataatt 8040

atagttatac tcatggattt gtagttgagt atgaaaatat tttttaatgc attttatgac 8100

ttgccaattg attgacaacg aattcgtaat catggtcata gctgtttcct gtgtgaaatt 8160

gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 8220

gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 8280

cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 8340

tgcgtattgg ctagagcagc ttgccaacat ggtggagcac gacactctcg tctactccaa 8400

gaatatcaaa gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt 8460

aatatcggga aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac 8520

agtagaaaag gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt 8580

tcaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt 8640

ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg ataacatggt 8700

ggagcacgac actctcgtct actccaagaa tatcaaagat acagtctcag aagaccaaag 8760

ggctattgag acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc 8820

agctatctgt cacttcatca aaaggacagt agaaaaggaa ggtggcacct acaaatgcca 8880

tcattgcgat aaaggaaagg ctatcgttca agatgcctct gccgacagtg gtcccaaaga 8940

tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 9000

gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 9060

ttcgcaagac cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgaaat 9120

caccagtctc tctctacaaa tctatctctc tcgagtctac catgagccca gaacgacgcc 9180

cggccgacat ccgccgtgcc accgaggcgg acatgccggc ggtctgcacc atcgtcaacc 9240

actacatcga gacaagcacg gtcaacttcc gtaccgagcc gcaggaaccg caggagtgga 9300

cggacgacct cgtccgtctg cgggagcgct atccctggct cgtcgccgag gtggacggcg 9360

aggtcgccgg catcgcctac gcgggcccct ggaaggcacg caacgcctac gactggacgg 9420

ccgagtcgac cgtgtacgtc tccccccgcc accagcggac gggactgggc tccacgctct 9480

acacccacct gctgaagtcc ctggaggcac agggcttcaa gagcgtggtc gctgtcatcg 9540

ggctgcccaa cgacccgagc gtgcgcatgc acgaggcgct cggatatgcc ccccgcggca 9600

tgctgcgggc ggccggcttc aagcacggga actggcatga cgtgggtttc tggcagctgg 9660

acttcagcct gccggtaccg ccccgtccgg tcctgcccgt caccgagatt tgactcgagt 9720

ttctccataa taatgtgtga gtagttccca gataagggaa ttagggttcc tatagggttt 9780

cgctcatgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc 9840

tatcaataaa atttctaatt cctaaaacca aaatccagta ctaaaatcca gatcccccga 9900

attaattcgg cgttaattca gtacattaaa aacgtccgca atgtgttatt aagttgtcta 9960

agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca gctccccgac 10020

cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagtccg ggacggcgtc 10080

agcgggagag ccgttgtaag gcggcagact ttgctcatgt taccgatgct attcggaaga 10140

acggcaacta agctgccggg tttgaaacac ggatgatctc gcggagggta gcatgttgat 10200

tgtaacgatg acagagcgtt gctgcctgtg atcaccgcgg tttcaaaatc ggctccgtcg 10260

atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc tggtatttaa 10320

ggttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta gcttcttggg 10380

gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa tgagaatatc 10440

accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg aaggaatgtc 10500

tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa aaatgacgga 10560

cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga tgctatggct 10620

ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg gctggagcaa 10680

tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag atgaacaaag 10740

ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact ccatcgacat 10800

atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg attacttact 10860

gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc catttaaaga 10920

tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac ttgtcttttc 10980

ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa gtggctttat 11040

tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct gcgtccggtc 11100

gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact tactggggat 11160

caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt tttagtacct 11220

agaatgcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 11280

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 11340

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 11400

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 11460

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 11520

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 11580

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 11640

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 11700

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 11760

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 11820

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 11880

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 11940

tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 12000

tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 12060

ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 12120

ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac 12180

actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct 12240

gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 12300

tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagggt 12360

gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag 12420

attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga 12480

agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc 12540

tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt 12600

taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg 12660

tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcccaatgta 12720

cggctttggg ttcccaatgt acgtgctatc cacaggaaac agaccttttc gacctttttc 12780

ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc 12840

gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc 12900

cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa 12960

cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc 13020

ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc 13080

gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc 13140

gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt 13200

tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt 13260

cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc 13320

taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc 13380

aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat 13440

ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat 13500

gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc 13560

gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact 13620

atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt 13680

gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt tgccgggatt ctttgcggat 13740

tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg 13800

ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat 13860

ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt cctcgggctt gggggttcca 13920

gtgccattgc agggccggca gacaacccag ccgcttacgc ctggccaacc gcccgttcct 13980

ccacacatgg ggcattccac ggcgtcggtg cctggttgtt cttgattttc catgccgcct 14040

cctttagccg ctaaaattca tctactcatt tattcatttg ctcatttact ctggtagctg 14100

cgcgatgtat tcagatagca gctcggtaat ggtcttgcct tggcgtaccg cgtacatctt 14160

cagcttggtg tgatcctccg ccggcaactg aaagttgacc cgcttcatgg ctggcgtgtc 14220

tgccaggctg gccaacgttg cagccttgct gctgcgtgcg ctcggacggc cggcacttag 14280

cgtgtttgtg cttttgctca ttttctcttt acctcattaa ctcaaatgag ttttgattta 14340

atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc cctcgggttc tgattcaaga 14400

acggttgtgc cggcggcggc agtgcctggg tagctcacgc gctgcgtgat acgggactca 14460

agaatgggca gctcgtaccc ggccagcgcc tcggcaacct caccgccgat gcgcgtgcct 14520

ttgatcgccc gcgacacgac aaaggccgct tgtagccttc catccgtgac ctcaatgcgc 14580

tgcttaacca gctccaccag gtcggcggtg gcccatatgt cgtaagggct tggctgcacc 14640

ggaatcagca cgaagtcggc tgccttgatc gcggacacag ccaagtccgc cgcctggggc 14700

gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct tcacgtcgcg gtcaatcgtc 14760

gggcggtcga tgccgacaac ggttagcggt tgatcttccc gcacggccgc ccaatcgcgg 14820

gcactgccct ggggatcgga atcgactaac agaacatcgg ccccggcgag ttgcagggcg 14880

cgggctagat gggttgcgat ggtcgtcttg cctgacccgc ctttctggtt aagtacagcg 14940

ataaccttca tgcgttcccc ttgcgtattt gtttatttac tcatcgcatc atatacgcag 15000

cgaccgcatg acgcaagctg ttttactcaa atacacatca cctttttaga cggcggcgct 15060

cggtttcttc agcggccaag ctggccggcc aggccgccag cttggcatca gacaaaccgg 15120

ccaggatttc atgcagccgc acggttgaga cgtgcgcggg cggctcgaac acgtacccgg 15180

ccgcgatcat ctccgcctcg atctcttcgg taatgaaaaa cggttcgtcc tggccgtcct 15240

ggtgcggttt catgcttgtt cctcttggcg ttcattctcg gcggccgcca gggcgtcggc 15300

ctcggtcaat gcgtcctcac ggaaggcacc gcgccgcctg gcctcggtgg gcgtcacttc 15360

ctcgctgcgc tcaagtgcgc ggtacagggt cgagcgatgc acgccaagca gtgcagccgc 15420

ctctttcacg gtgcggcctt cctggtcgat cagctcgcgg gcgtgcgcga tctgtgccgg 15480

ggtgagggta gggcgggggc caaacttcac gcctcgggcc ttggcggcct cgcgcccgct 15540

ccgggtgcgg tcgatgatta gggaacgctc gaactcggca atgccggcga acacggtcaa 15600

caccatgcgg ccggccggcg tggtggtgtc ggcccacggc tctgccaggc tacgcaggcc 15660

cgcgccggcc tcctggatgc gctcggcaat gtccagtagg tcgcgggtgc tgcgggccag 15720

gcggtctagc ctggtcactg tcacaacgtc gccagggcgt aggtggtcaa gcatcctggc 15780

cagctccggg cggtcgcgcc tggtgccggt gatcttctcg gaaaacagct tggtgcagcc 15840

ggccgcgtgc agttcggccc gttggttggt caagtcctgg tcgtcggtgc tgacgcgggc 15900

atagcccagc aggccagcgg cggcgctctt gttcatggcg taatgtctcc ggttctagtc 15960

gcaagtattc tactttatgc gactaaaaca cgcgacaaga aaacgccagg aaaagggcag 16020

ggcggcagcc tgtcgcgtaa cttaggactt gtgcgacatg tcgttttcag aagacggctg 16080

cactgaacgt cagaagccga ctgcactata gcagcggagg ggttggatca aagtactttg 16140

atcccgaggg gaaccctgtg gttggcatgc acatacaaat ggacgaacgg ataaaccttt 16200

tcacgccctt ttaaatatcc gttattctaa 16230

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<400> 8

ggcggaagaa gacgctgctg ttcc 24

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence

<400> 9

aaacggaaca gcagcgtctt cttct 25

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

gaagaagacg ctgctgttcc 20

<210> 11

<211> 1734

<212> DNA

<213> sequence

<400> 11

atggagagca gtcgcatgcc actgagtgcc acccctcgca tgccatgcag cctccaaacc 60

cttgcgcgcc gcaaccttct ccgtggcctt cacctccgga aggacgcgag gcagccacgg 120

cgtctctcag tctcatgcga ggcgaccggc ggctgccgcg tcgaccgccg tgaggtgctc 180

ctcggtctcg gcggcgccgc ggctgccggt ctggccacgg acaaaggtcg aggcgcgatc 240

gccgcgccca tccaggcccc ggacctccgc aactgccaaa cgcccgagct cccgaacacg 300

ccgccggaca ccaactgctg cccgacgccc ggcaccggca tcaccgactt cgtgctgccg 360

cccgtctcct cgccgctccg cgtgcgtccg gcagcgcacc tggtggacgc ggggtacctg 420

gccaagtacg agagggccgt ggcgctcatg aagcagctgc ccgccgacga cccgcgcagc 480

ttcgagcagc agtggcacgt gcactgcgcc tactgcgacg ccgcctacga ccaggtcggg 540

ttcccggacc tggagctcca gatacacaac tgctggctct tcttcccatg gcacaggttc 600

tacctctact tccacgagag gatcctcggc aagctcatcg gcgacgacac cttcgcgctg 660

cccttctgga actgggacgc gccggccggc atgacgctgc cggccatcta cgccgacagg 720

tcgtcgccgc tctacgacga gaggcgcgac cccgcgcacc agccgccggt gctggtcgac 780

cttgactcca gtgggtccga caccaatatc ccaagagacc agcagatcga cgagaacctc 840

aagatcatgt accgccagat gatttcgaac gcgaagaaga cgctgctgtt cctgggacag 900

ccgtaccgcg ccggcgacca gccggacccg ggcgccggct ccctggagaa cgtgccgcac 960

ggcacggtcc acgtctggac tggcgaccca aggcagccca acttggcgga catgggcaac 1020

ttcttctcgg cggcgcgcga ccccatcttc ttcgcgcacc acggcaacat cgaccgcctg 1080

tggcacgtct ggcgcggcct ccgcccgagc aacactgact tcaccgaccc cgactggctc 1140

gacgccgcct tcctcttcta cgacgaggag gcccgccccg tgcgcgtgcg cgtccgggac 1200

tgcctcgacc cggccgcgct gcggtacatg taccaggacg tcggcctgcc gtggctcaac 1260

gccaggccgg ccaaggcgtc cggcgggacg ccggcgcccg ccaccaccgg taccctccct 1320

gccaccctgg acagcaccat acgggtgacc gtgacgaggc ccagggtgtc gaggagccgc 1380

cgggaaaagg acgaggagga ggaggtgctg gtcgtggagg ggatcgagat cgccgaccat 1440

ttcaacaagt tcatcaagtt cgacgtgctg gtgaacgagc ccgagggagg ggtggacggc 1500

acgccggcga cggcgacggg gtactgcgcc gggagcttcg cgcacacgcc gcacatggtc 1560

cggcccgagg agacgaggaa ggggtcggtc aagacggtgg cgaggttcgg cgtgtgcgac 1620

ctgatggacg acatcggagc ggacggcgac cagacggtgg tcgtgtcgct cgtacccagg 1680

tgcggcggtg agctggtcac cgtaggcggc gtcagcatca gctacctcaa gtga 1734

<210> 12

<211> 1732

<212> DNA

<213> sequence

<400> 12

atggagagca gtcgcatgcc actgagtgcc acccctcgca tgccatgcag cctccaaacc 60

cttgcgcgcc gcaaccttct ccgtggcctt cacctccgga aggacgcgag gcagccacgg 120

cgtctctcag tctcatgcga ggcgaccggc ggctgccgcg tcgaccgccg tgaggtgctc 180

ctcggtctcg gcggcgccgc ggctgccggt ctggccacgg acaaaggtcg aggcgcgatc 240

gccgcgccca tccaggcccc ggacctccgc aactgccaaa cgcccgagct cccgaacacg 300

ccgccggaca ccaactgctg cccgacgccc ggcaccggca tcaccgactt cgtgctgccg 360

cccgtctcct cgccgctccg cgtgcgtccg gcagcgcacc tggtggacgc ggggtacctg 420

gccaagtacg agagggccgt ggcgctcatg aagcagctgc ccgccgacga cccgcgcagc 480

ttcgagcagc agtggcacgt gcactgcgcc tactgcgacg ccgcctacga ccaggtcggg 540

ttcccggacc tggagctcca gatacacaac tgctggctct tcttcccatg gcacaggttc 600

tacctctact tccacgagag gatcctcggc aagctcatcg gcgacgacac cttcgcgctg 660

cccttctgga actgggacgc gccggccggc atgacgctgc cggccatcta cgccgacagg 720

tcgtcgccgc tctacgacga gaggcgcgac cccgcgcacc agccgccggt gctggtcgac 780

cttgactcca gtgggtccga caccaatatc ccaagagacc agcagatcga cgagaacctc 840

aagatcatgt accgccagat gatttcgaac gcgaagaaga cgctgcttcc tgggacagcc 900

gtaccgcgcc ggcgaccagc cggacccggg cgccggctcc ctggagaacg tgccgcacgg 960

cacggtccac gtctggactg gcgacccaag gcagcccaac ttggcggaca tgggcaactt 1020

cttctcggcg gcgcgcgacc ccatcttctt cgcgcaccac ggcaacatcg accgcctgtg 1080

gcacgtctgg cgcggcctcc gcccgagcaa cactgacttc accgaccccg actggctcga 1140

cgccgccttc ctcttctacg acgaggaggc ccgccccgtg cgcgtgcgcg tccgggactg 1200

cctcgacccg gccgcgctgc ggtacatgta ccaggacgtc ggcctgccgt ggctcaacgc 1260

caggccggcc aaggcgtccg gcgggacgcc ggcgcccgcc accaccggta ccctccctgc 1320

caccctggac agcaccatac gggtgaccgt gacgaggccc agggtgtcga ggagccgccg 1380

ggaaaaggac gaggaggagg aggtgctggt cgtggagggg atcgagatcg ccgaccattt 1440

caacaagttc atcaagttcg acgtgctggt gaacgagccc gagggagggg tggacggcac 1500

gccggcgacg gcgacggggt actgcgccgg gagcttcgcg cacacgccgc acatggtccg 1560

gcccgaggag acgaggaagg ggtcggtcaa gacggtggcg aggttcggcg tgtgcgacct 1620

gatggacgac atcggagcgg acggcgacca gacggtggtc gtgtcgctcg tacccaggtg 1680

cggcggtgag ctggtcaccg taggcggcgt cagcatcagc tacctcaagt ga 1732

<210> 13

<211> 4107

<212> DNA

<213> sequence

<400> 13

atggacaaga agtactcgat cggcctcgat attgggacta actctgttgg ctgggccgtg 60

atcaccgacg agtacaaggt gccctcaaag aagttcaagg tcctgggcaa caccgatcgg 120

cattccatca agaagaatct cattggcgct ctcctgttcg acagcggcga gacggctgag 180

gctacgcggc tcaagcgcac cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc 240

tacctgcagg agattttctc caacgagatg gcgaaggttg acgattcttt cttccacagg 300

ctggaggagt cattcctcgt ggaggaggat aagaagcacg agcggcatcc aatcttcggc 360

aacattgtcg acgaggttgc ctaccacgag aagtacccta cgatctacca tctgcggaag 420

aagctcgtgg actccacaga taaggcggac ctccgcctga tctacctcgc tctggcccac 480

atgattaagt tcaggggcca tttcctgatc gagggggatc tcaacccgga caatagcgat 540

gttgacaagc tgttcatcca gctcgtgcag acgtacaacc agctcttcga ggagaacccc 600

attaatgcgt caggcgtcga cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg 660

cgcctcgaga acctgatcgc ccagctgccg ggcgagaaga agaacggcct gttcgggaat 720

ctcattgcgc tcagcctggg gctcacgccc aacttcaagt cgaatttcga tctcgctgag 780

gacgccaagc tgcagctctc caaggacaca tacgacgatg acctggataa cctcctggcc 840

cagatcggcg atcagtacgc ggacctgttc ctcgctgcca agaatctgtc ggacgccatc 900

ctcctgtctg atattctcag ggtgaacacc gagattacga aggctccgct ctcagcctcc 960

atgatcaagc gctacgacga gcaccatcag gatctgaccc tcctgaaggc gctggtcagg 1020

cagcagctcc ccgagaagta caaggagatc ttcttcgatc agtcgaagaa cggctacgct 1080

gggtacattg acggcggggc ctctcaggag gagttctaca agttcatcaa gccgattctg 1140

gagaagatgg acggcacgga ggagctgctg gtgaagctca atcgcgagga cctcctgagg 1200

aagcagcgga cattcgataa cggcagcatc ccacaccaga ttcatctcgg ggagctgcac 1260

gctatcctga ggaggcagga ggacttctac cctttcctca aggataaccg cgagaagatc 1320

gagaagattc tgactttcag gatcccgtac tacgtcggcc cactcgctag gggcaactcc 1380

cgcttcgctt ggatgacccg caagtcagag gagacgatca cgccgtggaa cttcgaggag 1440

gtggtcgaca agggcgctag cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag 1500

aacctgccaa atgagaaggt gctccctaag cactcgctcc tgtacgagta cttcacagtc 1560

tacaacgagc tgactaaggt gaagtatgtg accgagggca tgaggaagcc ggctttcctg 1620

tctggggagc agaagaaggc catcgtggac ctcctgttca agaccaaccg gaaggtcacg 1680

gttaagcagc tcaaggagga ctacttcaag aagattgagt gcttcgattc ggtcgagatc 1740

tctggcgttg aggaccgctt caacgcctcc ctggggacct accacgatct cctgaagatc 1800

attaaggata aggacttcct ggacaacgag gagaatgagg atatcctcga ggacattgtg 1860

ctgacactca ctctgttcga ggaccgggag atgatcgagg agcgcctgaa gacttacgcc 1920

catctcttcg atgacaaggt catgaagcag ctcaagagga ggaggtacac cggctggggg 1980

aggctgagca ggaagctcat caacggcatt cgggacaagc agtccgggaa gacgatcctc 2040

gacttcctga agagcgatgg cttcgcgaac cgcaatttca tgcagctgat tcacgatgac 2100

agcctcacat tcaaggagga tatccagaag gctcaggtga gcggccaggg ggactcgctg 2160

cacgagcata tcgcgaacct cgctggctcg ccagctatca agaaggggat tctgcagacc 2220

gtgaaggttg tggacgagct ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc 2280

attgagatgg cccgggagaa tcagaccacg cagaagggcc agaagaactc acgcgagagg 2340

atgaagagga tcgaggaggg cattaaggag ctggggtccc agatcctcaa ggagcacccg 2400

gtggagaaca cgcagctgca gaatgagaag ctctacctgt actacctcca gaatggccgc 2460

gatatgtatg tggaccagga gctggatatt aacaggctca gcgattacga cgtcgatcat 2520

atcgttccac agtcattcct gaaggatgac tccattgaca acaaggtcct caccaggtcg 2580

gacaagaacc ggggcaagtc tgataatgtt ccttcagagg aggtcgttaa gaagatgaag 2640

aactactggc gccagctcct gaatgccaag ctgatcacgc agcggaagtt cgataacctc 2700

acaaaggctg agaggggcgg gctctctgag ctggacaagg cgggcttcat caagaggcag 2760

ctggtcgaga cacggcagat cactaagcac gttgcgcaga ttctcgactc acggatgaac 2820

actaagtacg atgagaatga caagctgatc cgcgaggtga aggtcatcac cctgaagtca 2880

aagctcgtct ccgacttcag gaaggatttc cagttctaca aggttcggga gatcaacaat 2940

taccaccatg cccatgacgc gtacctgaac gcggtggtcg gcacagctct gatcaagaag 3000

tacccaaagc tcgagagcga gttcgtgtac ggggactaca aggtttacga tgtgaggaag 3060

atgatcgcca agtcggagca ggagattggc aaggctaccg ccaagtactt cttctactct 3120

aacattatga atttcttcaa gacagagatc actctggcca atggcgagat ccggaagcgc 3180

cccctcatcg agacgaacgg cgagacgggg gagatcgtgt gggacaaggg cagggatttc 3240

gcgaccgtca ggaaggttct ctccatgcca caagtgaata tcgtcaagaa gacagaggtc 3300

cagactggcg ggttctctaa ggagtcaatt ctgcctaagc ggaacagcga caagctcatc 3360

gcccgcaaga aggactggga tccgaagaag tacggcgggt tcgacagccc cactgtggcc 3420

tactcggtcc tggttgtggc gaaggttgag aagggcaagt ccaagaagct caagagcgtg 3480

aaggagctgc tggggatcac gattatggag cgctccagct tcgagaagaa cccgatcgat 3540

ttcctggagg cgaagggcta caaggaggtg aagaaggacc tgatcattaa gctccccaag 3600

tactcactct tcgagctgga gaacggcagg aagcggatgc tggcttccgc tggcgagctg 3660

cagaagggga acgagctggc tctgccgtcc aagtatgtga acttcctcta cctggcctcc 3720

cactacgaga agctcaaggg cagccccgag gacaacgagc agaagcagct gttcgtcgag 3780

cagcacaagc attacctcga cgagatcatt gagcagattt ccgagttctc caagcgcgtg 3840

atcctggccg acgcgaatct ggataaggtc ctctccgcgt acaacaagca ccgcgacaag 3900

ccaatcaggg agcaggctga gaatatcatt catctcttca ccctgacgaa cctcggcgcc 3960

cctgctgctt tcaagtactt cgacacaact atcgatcgca agaggtacac aagcactaag 4020

gaggtcctgg acgcgaccct catccaccag tcgattaccg gcctctacga gacgcgcatc 4080

gacctgtctc agctcggggg cgactga 4107

Claims (8)

1. A pBUE411-PPO binary vector with a function of knocking out PPO genes is characterized by comprising an expression cassette A and an expression cassette B;

the expression cassette A specifically comprises the following elements from upstream to downstream in sequence: TaU3 promoter A from wheat, sgRNA of the PPO gene, terminator A;

the expression cassette B specifically comprises the following elements from upstream to downstream in sequence: promoter B, maize Cas9 coding sequence, terminator B;

the sequence of sgRNA of the PPO gene is shown as SEQ ID NO. 10;

the maize Cas9 coding sequence is shown as SEQ ID NO. 13;

the sequence of the promoter A is shown as SEQ ID NO. 1;

the promoter B sequence is shown as SEQ ID NO. 2;

the terminator A sequence is shown as SEQ ID NO. 3;

the terminator B sequence is shown in SEQ ID NO. 4;

the pBUE411-PPO binary vector contains a resistance marker gene, and the marker gene is a bar gene;

the nucleotide sequence of the pBUE411-PPO binary vector is shown in SEQ ID NO. 7.

2. The pBUE411-PPO binary vector of claim 1, wherein the expression cassette A can be specifically represented by a sequence SEQ ID No. 5;

the expression cassette B can be specifically shown as a sequence SEQ ID NO. 6;

the expression cassette A is located at the upstream of the expression cassette B, and the expression cassette A and the expression cassette B are connected in series.

3. The method of constructing the pBUE411-PPO binary vector according to claim 1 or 2, characterized in that it comprises the following steps:

s11: the sgRNA is designed according to a targeted PPO gene, and the oligonucleotides of the sgRNA correspond to PPO-gR1-TaU3-F and PPO-gR1-TaU3-R, namely the sequence of PPO-gR1-TaU3-F is shown in SEQ ID No.8, and the sequence of PPO-gR1-TaU3-R is shown in SEQ ID No. 9;

s12: phosphorylating and annealing 2 oligonucleotides of the sgRNA synthesized in step S11 to form a double strand, and obtaining a double strand sgRNA with a sticky end;

s13: utilizing restriction enzyme BsaI to cut the pBUE411 vector, and recovering vector fragments containing the promoter A, the terminator A, the promoter B and the terminator B;

s14: and (3) performing a ligation reaction on the pBUE411 vector fragment recovered in the step S13 and the double-stranded sgRNA obtained in the step S12 by using T4 ligase to obtain a final pBUE411-PPO binary vector.

4. The method for constructing the pBUE411-PPO binary vector according to claim 3, wherein the PPO gene is GENBANK ACCESS NUMBER: EF 070147.1.

5. Use of the pBUE411-PPO binary vector according to any one of claims 1-3 for reducing the expression level of PPO gene in wheat endosperm.

6. Use of the binary vector pBUE411-PPO as defined in any one of claims 1 to 3 for improving the color of wheat flour.

7. Use of the pBUE411-PPO binary vector of any one of claims 1-3 in breeding transgenic wheat.

8. A method for reducing the polyphenol oxidase content of wheat grains by using gene editing is characterized by comprising the following steps:

s1 constructing the pBUE411-PPO binary vector as described in any one of claims 1-3;

s2, transforming the pBUE411-PPO binary vector obtained in the step S1 into common wheat through agrobacterium tumefaciens to obtain transgenic wheat.

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