CN111269934B - Method for improving phosphorus utilization rate of wheat by using gene editing - Google Patents

Abstract

The invention discloses a method for improving the utilization rate of phosphorus in wheat by using gene editing. The invention constructs an expression vector capable of in vivo editing of the LPR2 gene by specifically guiding the design of the single-stranded sgRNA, and can knock out the wheat LPR2 gene, thereby realizing the silencing of the LPR2 gene at the genome level, improving the utilization rate of phosphorus in wheat, and cultivating transgenic wheat.

Description

A method for improving phosphorus utilization in wheat using gene editing

technical field

The invention relates to a method for improving the utilization rate of phosphorus in wheat by using gene editing, and relates to the field of gene editing.

Background technique

Phosphorus is one of the macroelements necessary for plant growth and development. It is the main component of plant nucleic acid, nucleoprotein, phospholipid and adenosine triphosphate, etc. It also plays an important role in energy metabolism, photosynthesis, respiration, regulation of enzyme activity, redox reaction, signal transduction and carbon metabolism. The lack of phosphorus will affect the formation of nuclear protein, inhibit cell division and proliferation, reduce protein synthesis, hinder the transportation of sugar, and eventually lead to fewer branches of plants, slender stems and roots, dwarf plants, flowers and fruits easily fall off and yield reduction, etc. . The growth and output of plants are significantly dependent on the availability of inorganic phosphorus (Pi), however, the content of available phosphorus in most soils is very low, only 1-10 μmol/L. Although organic phosphorus in soil can be as high as 80%, it cannot be absorbed by plants, and organic phosphorus must be converted into inorganic phosphorus before it can be absorbed and utilized (Schachtman et al., 1998; Holford, 1997). In agricultural production, the method of adding phosphate fertilizer is often used to ensure the normal growth and development of crops, but the global easily exploitable phosphate rock resources are facing the crisis of disappearing in this century (Gilbert, 2009; Vance et al., 2003) .

Wheat is an important food crop in my country. How to cultivate low-fat, high-yield and environment-friendly wheat varieties is also a great challenge for wheat breeders. In the long-term evolution, plants have formed a mechanism to cope with the severe lack of available phosphorus in agricultural natural ecosystems. Therefore, fully exploiting the genes of efficient phosphorus utilization in wheat has important guiding significance for improving molecular breeding with low phosphorus efficiency and environmental friendly and sustainable yield increase.

Under low phosphorus stress, the growth of the main root of Arabidopsis thaliana was inhibited, but in the mutant Arabidopsis thaliana lacking LPR1 (Low Phosphate Root1) function, the growth of the main root was normal. LPR1 encodes multicopperoxidases, which control the growth of taproots by regulating the expression levels of SCR (Scarecrow) and SHR (Short-root). Svistoonoff et al., 2007). Homologous genes of these genes can be found in rice, maize, wheat, concanavali, and white lupin, suggesting that root architecture changes in response to low phosphorus stress in plants may have similar regulatory pathways.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies of the above-mentioned prior art, and provides a method for improving the utilization rate of phosphorus in wheat by using gene editing. The method of the present invention constructs the expression vector pBUE411-LPR2 with the function of knocking out the LPR2 gene, and reduces the expression level of the LPR2 gene through the process of gene transformation.

A method for improving the utilization of phosphorus in wheat by gene editing, comprising the following steps: reducing or losing the function of the wheat LPR2 gene, thereby inhibiting the expression and/or activity of the protein encoded by the wheat LPR2 gene, thereby improving the phosphorus utilization rate of the wheat.

Further, the method for reducing or losing the function of the wheat LPR2 gene described in the above method is to construct a pBUE411-LPR2 binary vector with the function of knocking out the LPR2 gene.

The above-mentioned pBUE411-LPR2 binary vector with the function of knocking out the LPR2 gene includes an expression cassette A and an expression cassette B; the expression cassette A specifically includes the following elements in sequence from upstream to downstream: the TaU3 promoter A from wheat, so The sgRNA of the LPR2 gene and the terminator A; the expression cassette B specifically includes the following elements from upstream to downstream: the promoter B, the maize Cas9 coding sequence, and the terminator B.

Further, the sgRNA sequence of the LPR2 gene is shown in SEQ10; the maize Cas9 coding sequence is shown in SEQ13; the sequence of the promoter A is shown in SEQ3; the sequence of the promoter B is shown in SEQ4; The sequence of the terminator A is shown in SEQ5; the sequence of the terminator B is shown in SEQ6.

Further, the sequence of the expression cassette A is specifically shown in SEQ7; the sequence of the expression cassette B is specifically shown in SEQ8; the expression cassette A is located upstream of the expression cassette B, and the expression cassette A and the expression cassette B are connected in series with each other .

Further, the pBUE411-LPR2 binary vector also contains a resistance marker gene, such as bar gene; the nucleotide sequence of the pBUE411-LPR2 binary vector is shown in SEQ9.

Further, the construction method of the above-mentioned pBUE411-LPR2 binary vector comprises the following steps:

S11 designs sgRNA targeting the LPR2 gene. The oligonucleotides of the sgRNA correspond to LPR2-gR1-TaU3-F and LPR2-gR1-TaU3-R, that is, the LPR2-gR1-TaU3-F sequence is shown in SEQ1, and the LPR2- The sequence of gR1-TaU3-R is shown in SEQ2;

S12 phosphorylates and anneals the two oligos of the synthesized sgRNA to form double strands;

S13 utilizes restriction endonuclease BsaI to digest the pBUE411 vector, and recovers the vector fragment containing the promoter A, the terminator A, the promoter B, and the terminator B;

S14 The recovered pBUE411 vector fragment and the double-stranded sgRNA with sticky ends were ligated by T4 ligase to obtain the final pBUE411-LPR2 binary vector.

Transformants containing the pBUE411-LPR2 binary vector also belong to the protection scope of the present invention.

The application of the pBUE411-LPR2 binary vector in improving the utilization rate of phosphorus in wheat.

The application of the pBUE411-LPR2 binary vector in the cultivation of transgenic wheat; compared with the pre-transgenic wheat, the phosphorus utilization rate of the transgenic wheat is improved.

The present invention also protects the following DNA fragment I:

The DNA fragment I is composed of the guide sgRNA of the LPR2 gene, and its sequence can be specifically shown in the sequence SEQ10.

Beneficial effects:

The invention constructs an expression vector capable of in vivo editing of the LPR2 gene by specifically guiding the design of the single-stranded sgRNA, and can knock out the wheat LPR2 gene. The LPR2 gene is located on chromosome 4 4A, 4B and 4D in wheat and named TaLPR2-4A, TaLPR2-4B and TaLPR2-4D, respectively. The full length of its genome sequence on 4A, 4B and 4D is 1972, 1957 and 1940 bases respectively, and these three partial homologous genes are composed of 3 exons and 2 introns. TaLPR2-4A, TaLPR2-4B and TaLPR2-4D contain open reading frames of 1761, 1761 and 1746 bases, respectively, encoding a polypeptide chain containing 587, 587 and 582 amino acid residues, respectively. Based on these gene information, we cloned the TaLPR2-4A gene in Fielder using the method of homologous cloning, and submitted it to NCBI with the GenBank number of MK676068. The similarity of the coding regions between the three partially homologous genes is very high, which provides a theoretical basis for the simultaneous silencing of the TaLPR2 gene at the three sites by gene editing technology.

In practical applications, the silencing of the LPR2 gene at the genome level can be achieved through a gene transformation process, which is a powerful tool for improving the utilization of phosphorus in wheat by genetic engineering technology.

Description of drawings

Figure 1 The coding region of the LPR2 gene.

Figure 2. The electrophoresis of BsaI digestion of pBUE411 vector.

Figure 3 Sequencing results of the CRISPR/Cas9 recombinant vector pBUE411-LPR2 targeting the LPR2 gene.

Figure 4. Bar test strips for detection of transgenic wheat plants.

Figure 5 Deduced amino acid sequence of wild-type wheat LPR2 gene.

Figure 6 Deduced amino acid sequence of mutant wheat LPR2 gene.

Figure 7 is an alignment diagram of the CDS sequences of the wild-type and mutant LPR2 gene.

Figure 8 Alignment of the deduced amino acid sequences of wild-type and mutant LPR2 genes.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged. The high-fidelity enzyme required for PCR amplification, the T4 ligase required for DNA fragment ligation, the gel recovery kit required for the recovery of enzyme-cut fragments, and the plasmid extraction kit were all purchased from Dalian Bao Biological Company. The plasmid extraction kit was purchased from Tiangen Biology, the inorganic salts required for the preparation of the medium were purchased from Sinopharm Group, and the vitamins, antibiotics and hormones were purchased from Sigma Company. The plant CRISPR/Cas9 gene editing vector is pBUE411, which contains the wheat U3 promoter TaU3 to initiate sgRNA. Cas9p simulates the characteristics of grasses with 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 is E. coli DH5α.

T4 ligase, DNA Marker DL2000 and other reagents were purchased from Takara Company; FastPfu, 2*Taq Mix, DH5α, etc. were purchased from Beijing Quanshijin Company; restriction endonuclease BsaI-HF was purchased from NEB Company; the primers used were from Qingdao Synthesized by Qingke Zixi Biotechnology Co., Ltd. The sequence is as follows:

Table 1 Primers used in this study

Wheat variety Jw1 is a new germplasm with good tissue culture ability selected by the Crop Institute of Shandong Academy of Agricultural Sciences, and the public can obtain it from Crop Research Institute of Shandong Academy of Agricultural Sciences.

Plasmid pBUE411 (17430bp) was provided by Sun Qixin's research group of China Agricultural University; the public can obtain it from China Agricultural University.

Experimental Example 1 Construction of Expression Vector

1. Design of sgRNA targeting LPR2

The sequence of the LPR2 gene was amplified in the wheat variety Fielder. The amplified sequence is shown in Figure 1. The underlined sequence is the sgRNA sequence targeting the LPR2 gene. The gene sequence of wheat is 100% similar. The full length of the gene is 1761 bases. Through the website CRISPRdirect (http://crispr.dbcls.jp/), a suitable targeting site was found in the coding region of the LPR2 gene, and a 20bp sequence fragment was found before the PAM structure. The target sequence, sgRNA sequence was selected as CGGAGCCGACGACGTGGAAG.

2. sgRNA annealing and duplex formation

The two oligos of the sgRNA of the LPR2 gene were synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd. The oligonucleotides of the sgRNA correspond to LPR2-gR1-TaU3-F and LPR2-gR1-TaU3-R (Table 1) , the underlined bases in the primers match the sequences in the pBUE411 vector. The two oligos of the sgRNA were phosphorylated and directly annealed to form double strands. The system was: LPR2-gR1-TaU3-F (10μM): 4μL, LPR2-gR1-TaU3-R (10uM): 4μL, 10×T4 PNKbuffer : 1.5 μL, PNK: 1 μL, ATP: 1 μL, ddH ₂ O to make up 15 μL. The program was run in the PCR machine under the following conditions: 37°C, 30 min; 95°C, 5 min; ramp to 25°C at 5°C/min (0.08°C/s). Or boil in boiling water for 5 minutes, then cool to room temperature naturally.

3. Linearization of pBUE411 vector

The pBUE411 vector was digested with BsaI endonuclease 3h as shown in Figure 2, 1 is pBUE411 digested with BsaI; 2 is DL2000 Marker; 3 is 1kb Marker, after electrophoresis, the linear vector fragment was recovered, and the recovery containing the promoter A, The terminator A, the promoter B, the vector fragment of the terminator B;

4. Ligation reaction of pBUE411 vector and sgRNA

The ligation reaction system was as follows: 5×ligation Buffer 2 μL, annealed oligo duplex 4 μL, pBUE411/BsaI 2ul (100 ng), T4 ligase 1 μL, ddH ₂ O supplemented 10 μL, and ligation reaction was performed at 25°C for 30 min.

5. Conversion Verification

The ligation product was transformed into Escherichia coli, and the recombinant plasmid was transformed into DH5α competent cells, and then cultured on LB (kan) plates until the clones grew, single clones were picked for bacterial liquid PCR identification and sequencing, and the primer was pBUE411-F and pBUE411-R. The sequencing results (as shown in Figure 3) detected the target sequence of the sgRNA, and the TaU3 promoter sequence was detected upstream of the target sequence. The sequencing results showed that the sgRNA expression cassette was successfully constructed and successfully assembled into the pBUE411 binary expression vector, proving that TaLPR2 The CRISPR/Cas9 gene editing vector was successfully constructed.

Experimental Example 2 Transformation of common wheat with gene editing vector

1. Medium preparation: see the literature Kan Wang (ed.), Agrobacterium Protocals: Volume 1, Methods in Molecular Biology, vol.1223DOI10.007/978-1-4939-1695-5_15, SpringScience+Businedd Media New York 2015

2. Agrobacterium transformation: Take the wheat ears that have been pollinated for about 15 days, take the grains and peel the embryos. The day before the test, the Agrobacterium suspension was shaken at 160 r and cultured at 28°C for 24 hours. After the ear was ready, the preparation of the Agrobacterium suspension was started. ) and mix well. After adding the prepared bacterial solution to infect for 5 minutes, it was placed on the co-cultivation medium and cultivated in the dark at 23 degrees for 3 days. After co-cultivation, they were placed on rest medium for 5 days in the dark at 25°C. The callus was transferred to selection medium 1. Seal the petri dishes with parafilm and incubate in the dark for 2 weeks in a 25.5 degree incubator. The callus was cut and transferred to selection medium 2.

Seal the petri dishes with parafilm and incubate in the dark for 2 weeks in a 25.5 degree incubator. Two weeks after callus cutting selection, callus showing resistance were transferred to regeneration medium. Resistant callus generally has green shoots or green spots, or has a beautiful beige globular structure.

Mushy and brown calluses do not transfer. The callus with hyperplasia can be cut into smaller callus. Small pieces from the same callus should be placed on the same line again. Note the direction of the callus, for example, the green shoots and green dots are facing up.

The petri dish was sealed and placed in a 25°C incubator for 2 weeks under light (16h). After 2 weeks of regeneration, healthy growing seedlings were transferred to new resistant regeneration cassettes. When the seedlings grow to a certain size, they can be sampled for testing.

3. Mutant detection of transgenic seedlings

3.1 Positive identification of T0 transgenic lines and screening of mutants

Since the T-DNA region of the vector used for this transformation has three expression cassettes, cas9, bar and gRNA, the expression detection of bar gene is very mature, and there are commercial detection reagent strips. It can be judged whether it has the bar gene within 10 minutes, which is faster and more efficient than PCR amplification. Therefore, we used the non-transgenic plants as the negative control, and used the bar gene detection kit quickstix ^TM to detect the transgenic wheat T0 plants. The specific operations are as follows:

Take 3-5cm transgenic seedlings, put them into a 1.5m centrifuge tube, grind with a grinding rod, add 500 μL of Buffer, insert a Bar test strip, and let it stand for 5 minutes. Record the data according to the number of bands displayed by the test strip. Bands represent negative. The above kits were purchased from Madison Technology Co., Ltd.

The results of screening positive transgenic seedlings are shown in Figure 4. Samples 1-8 are regenerated seedlings of T0 generation, 9 is a positive control, and 10 is a negative control. Two bands appear in samples 1, 3, 4, 5, 6, 7, and 8. , which is consistent with the result of positive control 9, which is a positive seedling; 1-8 represent LPR2-01, -02, -03, -04, -05, 06, -07, -08, respectively.

3.2 Specific amplification of DA2 gene in T0 generation transgenic plants and identification of mutants

Whether the target gene editing is successful needs to be identified by sequencing. In this experiment, the Hi-TOM gene editing site detection kit produced by Tianjin Nuohezhiyuan Co., Ltd. was used. The kit automatically completes the high-throughput library construction process by PCR method, and directly analyzes the variation information of multiple samples and multiple sites with Hi-TOM online software. LPR2 was amplified using specific primers (Primer-F and Primer-R in Table 1) on both sides of the targeting sequence, and the amplified products were sent to Nuovogen for sequencing after library construction. The wild-type nucleotide sequence of the target gene wheat LPR2 gene is shown in SEQ11, the deduced amino acid sequence is shown in Figure 5, the target gene nucleotide sequence of the mutant is shown in SEQ12, and the deduced amino acid sequence is shown in Figure 6 As shown, after the alignment, the nucleotide at +954 of LPR2-2 was deleted by 4 bases CCAC (the first part indicated by the underline and arrow), and TGA appeared at +1087 of the mutated sequence (the underline and arrow indicated Refers to the second part), which becomes a stop codon, resulting in premature termination of protein translation. The alignment results of the nucleotide sequences are shown in Figure 7. The parts marked with bold underlines and arrows are wild type and mutants with The difference part, the amino acid sequence alignment result is shown in Fig. 8, the part marked with underline and arrow is the part with difference between the wild type and the mutant.

The first round of PCR reaction system: 1 μL of leaf DNA of wheat plants transfected with pBUE411-LPR2 gene was used as template, 10 μL of 2×Taq Master Mix in the kit, 0.5 μL of Primer-F and Primer-R (Table 1) (10 μM) each , Nuclease-free Water to make up the volume to 20μL. PCR reaction conditions were as follows: denaturation at 94°C for 2 min; denaturation at 94°C for 30s, renaturation at 65°C for 30s, extension at 72°C for 20s, a total of 33 cycles; and a final extension at 72°C for 5 min. After the PCR, take 5 μL of agarose gel electrophoresis to detect the PCR product to ensure that the target product exists and has good specificity. After the second round of PCR reaction, 12 μL of Hi-TOM Mix in the kit was added, 1 μL of the first round PCR product was used as the template, and Nuclease-free Water was used to make up the volume to 20 μL. PCR reaction program: denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, renaturation at 57 °C for 30 s, extension at 72 °C for 25 s, a total of 33 cycles; and a final extension at 72 °C for 5 min.

4 Phenotypic identification of low phosphorus treatment in gene editing material and control material

Place the non-transgenic Fielder material and gene editing material TaLPR-6 wheat seeds in a petri dish, add an appropriate amount of water, and germinate in a germination box for 15-18 hours. The uniform seedlings are transferred to the culture box to start the nutrient solution culture. The cultivation conditions of wheat seedlings were temperature (21±1)℃, humidity 50%～60%, light intensity 150～200μmol/(cm2·s). The nutrient solution was changed every 3 days, and the seedling stage traits were investigated after 18 days of continuous culture. The Hoagland nutrient solution was used for hydroponics, and the KH ₂ PO ₄ concentration was 200 μmol/L under the control condition and ₅ μmol/L under the low phosphorus condition _. Six replicates were set for each treatment, and each replicate contained about 20 wheat seedlings.

In the control experiment CK, the average seedling lengths of the non-transgenic Fielder material and the gene-edited material TaLPR-6 were 41.32 cm and 41.54 cm, respectively (Table 2), and there was no significant difference in statistical tests; in the low-phosphorus treatment experiment, the non-transgenic The average seedling lengths of the Fielder material and the gene editing material TaLPR-6 were 37.77 cm and 40.07 cm, respectively (Table 2), and the P-value of the statistical test was less than 2.2e-16, reaching a significant level of difference.

Table 2. Seedling length of non-transgenic Fielder material and gene-edited material TaLPR-6 after low phosphorus treatment.

In the control experiment CK, the average fresh weight of the shoots of the non-transgenic Fielder material and the gene-edited material TaLPR-6 were 695.63 mg and 696.33 mg, respectively (Table 3), and there was no significant difference in the statistical test; in the low phosphorus treatment experiment , the average fresh weight of the shoots of the non-transgenic Fielder material and the gene-edited material TaLPR-6 were 561.03 mg and 690.77 mg, respectively (Table 3), and the P-value of the statistical test was less than 2.2e-16, reaching a significant level of difference.

Table 3. Aboveground fresh weight of non-transgenic Fielder material and gene-edited material TaLPR-6 after low phosphorus treatment.

SEQUENCE LISTING

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<213> Artificial sequences

<400> 8

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 aatggtttttt atagactaat tttttagta 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 ttttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg ctttttttttg 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 gaggagttc 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> 9

<211> 16231

<212> DNA

<213> Artificial sequences

<400> 9

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 cgacggagcc gacgacgtgg aaggttttag agctagaaat 840

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 900

tttttttttc gttttgcatt gagttttctc cgtcgcatgt ttgcagtttt attttccgtt 960

ttgcattgaa atttctccgt ctcatgtttg cagcgtgttc aaaaagtacg cagctgtatt 1020

tcacttattt acggcgccac attttcatgc cgtttgtgcc aactatcccg agctagtgaa 1080

tacagcttgg cttcacacaa cactggtgac ccgctgacct gctcgtacct cgtaccgtcg 1140

tacggcacag catttggaat taaagggtgt gatcgatact gcttgctgct aagcttgcat 1200

gcctgcagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc attgcatgtc 1260

taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt gcagtttatc 1320

tatctttata catatattta aactttactc tacgaataat ataatctata gtactacaat 1380

aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta aaggacaatt 1440

gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt gttctccttt 1500

ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta catccattta 1560

gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt ttattctatt 1620

ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta ataatttaga 1680

tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta agaaattaaa 1740

aaaactaagg aaacatttttt cttgtttcga gtagataatg ccagcctgtt aaacgccgtc 1800

gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc aagcgaagca 1860

gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg ctccaccgtt 1920

ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac gtgagccggc 1980

acggcaggcg gcctcctcct cctctcacgg cacggcagct acgggggatt cctttcccac 2040

cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct ccacaccctc 2100

tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc ccccaaatcc 2160

acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc ccctctctac 2220

cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 2280

tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 2340

cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 2400

ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 2460

tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 2520

catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 2580

ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 2640

atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 2700

taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 2760

tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 2820

agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 2880

tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 2940

tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 3000

ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tattttgatc 3060

ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 3120

tcatacgcta ttttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 3180

tgttacttct gcagccctag gcctactaga tggattacaa ggaccacgac ggggattaca 3240

aggaccacga cattgattac aaggatgatg atgacaagat ggctccgaag aagaagagga 3300

aggttggcat ccacggggtg ccagctgctg acaagaagta ctcgatcggc ctcgatattg 3360

ggactaactc tgttggctgg gccgtgatca ccgacgagta caaggtgccc tcaaagaagt 3420

tcaaggtcct gggcaacacc gatcggcatt ccatcaagaa gaatctcatt ggcgctctcc 3480

tgttcgacag cggcgagacg gctgaggcta cgcggctcaa gcgcaccgcc cgcaggcggt 3540

acacgcgcag gaagaatcgc atctgctacc tgcaggagat tttctccaac gagatggcga 3600

aggttgacga ttctttcttc cacaggctgg aggagtcatt cctcgtggag gaggataaga 3660

agcacgagcg gcatccaatc ttcggcaaca ttgtcgacga ggttgcctac cacgagaagt 3720

accctacgat ctaccatctg cggaagaagc tcgtggactc cacagataag gcggacctcc 3780

gcctgatcta cctcgctctg gcccacatga ttaagttcag gggccatttc ctgatcgagg 3840

gggatctcaa cccggacaat agcgatgttg acaagctgtt catccagctc gtgcagacgt 3900

acaaccagct cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc 3960

tgtccgctag gctctcgaag tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg 4020

agaagaagaa cggcctgttc gggaatctca ttgcgctcag cctggggctc acgcccaact 4080

tcaagtcgaa tttcgatctc gctgaggacg ccaagctgca gctctccaag gacacatacg 4140

acgatgacct ggataacctc ctggcccaga tcggcgatca gtacgcggac ctgttcctcg 4200

ctgccaagaa tctgtcggac gccatcctcc tgtctgatat tctcagggtg aacaccgaga 4260

ttacgaaggc tccgctctca gcctccatga tcaagcgcta cgacgagcac catcaggatc 4320

tgaccctcct gaaggcgctg gtcaggcagc agctccccga gaagtacaag gagatcttct 4380

tcgatcagtc gaagaacggc tacgctgggt acattgacgg cggggcctct caggaggagt 4440

tctacaagtt catcaagccg attctggaga agatggacgg cacggaggag ctgctggtga 4500

agctcaatcg cgaggacctc ctgaggaagc agcggacatt cgataacggc agcatcccac 4560

accagattca tctcggggag ctgcacgcta tcctgaggag gcaggaggac ttctaccctt 4620

tcctcaagga taaccgcgag aagatcgaga agattctgac tttcaggatc ccgtactacg 4680

tcggcccact cgctagggggc aactcccgct tcgcttggat gacccgcaag tcagaggaga 4740

cgatcacgcc gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca 4800

tcgagaggat gacgaatttc gacaagaacc tgccaaatga gaaggtgctc cctaagcact 4860

cgctcctgta cgagtacttc acagtctaca acgagctgac taaggtgaag tatgtgaccg 4920

agggcatgag gaagccggct ttcctgtctg gggagcagaa gaaggccatc gtggacctcc 4980

tgttcaagac caaccggaag gtcacggtta agcagctcaa ggaggactac ttcaagaaga 5040

ttgagtgctt cgattcggtc gagatctctg gcgttgagga ccgcttcaac gcctccctgg 5100

ggacctacca cgatctcctg aagatcatta aggataagga cttcctggac aacgaggaga 5160

atgaggatat cctcgaggac attgtgctga cactcactct gttcgaggac cgggagatga 5220

tcgaggagcg cctgaagact tacgcccatc tcttcgatga caaggtcatg aagcagctca 5280

agaggaggag gtacaccggc tgggggaggc tgagcaggaa gctcatcaac ggcattcggg 5340

acaagcagtc cgggaagacg atcctcgact tcctgaagag cgatggcttc gcgaaccgca 5400

atttcatgca gctgattcac gatgacagcc tcacattcaa ggaggatatc cagaaggctc 5460

aggtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcgccag 5520

ctatcaagaa ggggattctg cagaccgtga aggttgtgga cgagctggtg aaggtcatgg 5580

gcaggcacaa gcctgagaac atcgtcattg agatggcccg ggagaatcag accacgcaga 5640

agggccagaa gaactcacgc gagaggatga agaggatcga ggagggcatt aaggagctgg 5700

ggtcccagat cctcaaggag cacccggtgg agaacacgca gctgcagaat gagaagctct 5760

acctgtacta cctccagaat ggccgcgata tgtatgtgga ccaggagctg gatattaaca 5820

ggctcagcga ttacgacgtc gatcatatcg ttccacagtc attcctgaag gatgactcca 5880

ttgacaacaa ggtcctcacc aggtcggaca agaaccgggg caagtctgat aatgttcctt 5940

cagaggaggt cgttaagaag atgaagaact actggcgcca gctcctgaat gccaagctga 6000

tcacgcagcg gaagttcgat aacctcacaa aggctgagag gggcgggctc tctgagctgg 6060

acaaggcggg cttcatcaag aggcagctgg tcgagacacg gcagatcact aagcacgttg 6120

cgcagattct cgactcacgg atgaacacta agtacgatga gaatgacaag ctgatccgcg 6180

aggtgaaggt catcaccctg aagtcaaagc tcgtctccga cttcaggaag gatttccagt 6240

tctacaaggt tcgggagatc aacaattacc accatgccca tgacgcgtac ctgaacgcgg 6300

tggtcggcac agctctgatc aagaagtacc caaagctcga gagcgagttc gtgtacgggg 6360

actacaaggt ttacgatgtg aggaagatga tcgccaagtc ggagcaggag attggcaagg 6420

ctaccgccaa gtacttcttc tactctaaca ttatgaattt cttcaagaca gagatcactc 6480

tggccaatgg cgagatccgg aagcgccccc tcatcgagac gaacggcgag acgggggaga 6540

tcgtgtggga caagggcagg gatttcgcga ccgtcaggaa ggttctctcc atgccacaag 6600

tgaatatcgt caagaagaca gaggtccaga ctggcgggtt ctctaaggag tcaattctgc 6660

ctaagcggaa cagcgacaag ctcatcgccc gcaagaagga ctgggatccg aagaagtacg 6720

gcgggttcga cagccccact gtggcctact cggtcctggt tgtggcgaag gttgagaagg 6780

gcaagtccaa gaagctcaag agcgtgaagg agctgctggg gatcacgatt atggagcgct 6840

ccagcttcga gaagaacccg atcgatttcc tggaggcgaa gggctacaag gaggtgaaga 6900

aggacctgat cattaagctc cccaagtact cactcttcga gctggagaac ggcaggaagc 6960

ggatgctggc ttccgctggc gagctgcaga aggggaacga gctggctctg ccgtccaagt 7020

atgtgaactt cctctacctg gcctcccact acgagaagct caagggcagc cccgaggaca 7080

acgagcagaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcattgagc 7140

agatttccga gttctccaag cgcgtgatcc tggccgacgc gaatctggat aaggtcctct 7200

ccgcgtacaa caagcaccgc gacaagccaa tcagggagca ggctgagaat atcattcatc 7260

tcttcaccct gacgaacctc ggcgcccctg ctgctttcaa gtacttcgac acaactatcg 7320

atcgcaagag gtacacaagc actaaggagg tcctggacgc gaccctcatc caccagtcga 7380

ttaccggcct ctacgagacg cgcatcgacc tgtctcagct cgggggcgac aagcggccag 7440

cggcgacgaa gaaggcgggg caggcgaaga agaagaagtg agctcagagc tttcgttcgt 7500

atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc gcacacacca 7560

gaatcctact gagtttgagt attatggcat tgggaaaact gtttttcttg taccatttgt 7620

tgtgcttgta atttactgtg ttttttattc ggttttcgct atcgaactgt gaaatggaaa 7680

tggatggaga agagttaatg aatgatatgg tccttttgtt cattctcaaa ttaatattat 7740

ttgttttttc tcttatttgt tgtgtgttga atttgaaatt ataagagata tgcaaacatt 7800

ttgttttgag taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta atatgaggag 7860

taaaacactt gtagttgtac cattatgctt attcactagg caacaaatat attttcagac 7920

ctagaaaagc tgcaaatgtt actgaataca agtatgtcct cttgtgtttt agacatttat 7980

gaactttcct ttatgtaatt ttccagaatc cttgtcagat tctaatcatt gctttataat 8040

tatagttata ctcatggatt tgtagttgag tatgaaaata ttttttaatg cattttatga 8100

cttgccaatt gattgacaac gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 8160

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 8220

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 8280

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 8340

ttgcgtattg gctagagcag cttgccaaca tggtggagca cgacactctc gtctactcca 8400

agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 8460

taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 8520

cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 8580

ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 8640

tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gataacatgg 8700

tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaaa 8760

gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc 8820

cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc 8880

atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag 8940

atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 9000

agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 9060

cttcgcaaga ccttcctcta tataaggaag ttcatttcat ttggagagga cacgctgaaa 9120

tcaccagtct ctctctacaa atctatctct ctcgagtcta ccatgagccc agaacgacgc 9180

ccggccgaca tccgccgtgc caccgaggcg gacatgccgg cggtctgcac catcgtcaac 9240

cactacatcg agacaagcac ggtcaacttc cgtaccgagc cgcaggaacc gcaggagtgg 9300

acggacgacc tcgtccgtct gcgggagcgc tatccctggc tcgtcgccga ggtggacggc 9360

gaggtcgccg gcatcgccta cgcgggcccc tggaaggcac gcaacgccta cgactggacg 9420

gccgagtcga ccgtgtacgt ctccccccgc caccagcgga cgggactggg ctccacgctc 9480

tacacccacc tgctgaagtc cctggaggca cagggcttca agagcgtggt cgctgtcatc 9540

gggctgccca acgacccgag cgtgcgcatg cacgaggcgc tcggatatgc cccccgcggc 9600

atgctgcggg cggccggctt caagcacggg aactggcatg acgtgggttt ctggcagctg 9660

gacttcagcc tgccggtacc gccccgtccg gtcctgcccg tcaccgagat ttgactcgag 9720

tttctccata ataatgtgtg agtagttccc agataaggga attagggttc ctatagggtt 9780

tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 9840

ctatcaataa aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatcccccg 9900

aattaattcg gcgttaattc agtacattaa aaacgtccgc aatgtgttat taagttgtct 9960

aagcgtcaat ttgtttacac cacaatatat cctgccacca gccagccaac agctccccga 10020

ccggcagctc ggcacaaaat caccactcga tacaggcagc ccatcagtcc gggacggcgt 10080

cagcgggaga gccgttgtaa ggcggcagac tttgctcatg ttaccgatgc tattcggaag 10140

aacggcaact aagctgccgg gtttgaaaca cggatgatct cgcggagggt agcatgttga 10200

ttgtaacgat gacagagcgt tgctgcctgt gatcaccgcg gtttcaaaat cggctccgtc 10260

gatactatgt tatacgccaa ctttgaaaac aactttgaaa aagctgtttt ctggtattta 10320

aggttttaga atgcaaggaa cagtgaattg gagttcgtct tgttataatt agcttcttgg 10380

ggtatcttta aatactgtag aaaagaggaa ggaaataata aatggctaaa atgagaatat 10440

caccggaatt gaaaaaactg atcgaaaaat accgctgcgt aaaagatacg gaaggaatgt 10500

ctcctgctaa ggtatataag ctggtgggag aaaatgaaaa cctatattta aaaatgacgg 10560

acagccggta taaagggacc acctatgatg tggaacggga aaaggacatg atgctatggc 10620

tggaaggaaa gctgcctgtt ccaaaggtcc tgcactttga acggcatgat ggctggagca 10680

atctgctcat gagtgaggcc gatggcgtcc tttgctcgga agagtatgaa gatgaacaaa 10740

gccctgaaaa gattatcgag ctgtatgcgg agtgcatcag gctctttcac tccatcgaca 10800

tatcggattg tccctatacg aatagcttag acagccgctt agccgaattg gattacttac 10860

tgaataacga tctggccgat gtggattgcg aaaactggga agaagacact ccatttaaag 10920

atccgcgcga gctgtatgat ttttttaaaga cggaaaagcc cgaagaggaa cttgtctttt 10980

cccacggcga cctgggagac agcaacatct ttgtgaaaga tggcaaagta agtggcttta 11040

ttgatcttgg gagaagcggc agggcggaca agtggtatga cattgccttc tgcgtccggt 11100

cgatcaggga ggatatcggg gaagaacagt atgtcgagct atttttttgac ttactgggga 11160

tcaagcctga ttgggagaaa ataaaatatt atattttact ggatgaattg ttttagtacc 11220

tagaatgcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 11280

tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 11340

aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 11400

tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 11460

agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 11520

taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 11580

caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 11640

agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 11700

aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 11760

gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 11820

tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 11880

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 11940

ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 12000

ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 12060

aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 12120

accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 12180

cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 12240

tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 12300

ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcaggg 12360

tgccttgatg tgggcgccgg cggtcgagtg gcgacggcgc ggcttgtccg cgccctggta 12420

gattgcctgg ccgtaggcca gccatttttg agcggccagc ggccgcgata ggccgacgcg 12480

aagcggcggg gcgtagggag cgcagcgacc gaagggtagg cgctttttgc agctcttcgg 12540

ctgtgcgctg gccagacagt tatgcacagg ccaggcgggt tttaagagtt ttaataagtt 12600

ttaaagagtt ttaggcggaa aaatcgcctt ttttctcttt tatatcagtc acttacatgt 12660

gtgaccggtt cccaatgtac ggctttgggt tcccaatgta cgggttccgg ttcccaatgt 12720

acggctttgg gttcccaatg tacgtgctat ccacaggaaa cagacctttt cgaccttttt 12780

cccctgctag ggcaatttgc cctagcatct gctccgtaca ttaggaaccg gcggatgctt 12840

cgccctcgat caggttgcgg tagcgcatga ctaggatcgg gccagcctgc cccgcctcct 12900

ccttcaaatc gtactccggc aggtcatttg acccgatcag cttgcgcacg gtgaaacaga 12960

acttcttgaa ctctccggcg ctgccactgc gttcgtagat cgtcttgaac aaccatctgg 13020

cttctgcctt gcctgcggcg cggcgtgcca ggcggtagag aaaacggccg atgccgggat 13080

cgatcaaaaa gtaatcgggg tgaaccgtca gcacgtccgg gttcttgcct tctgtgatct 13140

cgcggtacat ccaatcagct agctcgatct cgatgtactc cggccgcccg gtttcgctct 13200

ttacgatctt gtagcggcta atcaaggctt caccctcgga taccgtcacc aggcggccgt 13260

tcttggcctt cttcgtacgc tgcatggcaa cgtgcgtggt gtttaaccga atgcaggttt 13320

ctaccaggtc gtctttctgc tttccgccat cggctcgccg gcagaacttg agtacgtccg 13380

caacgtgtgg acggaacacg cggccgggct tgtctccctt cccttcccgg tatcggttca 13440

tggattcggt tagatgggaa accgccatca gtaccaggtc gtaatcccac acactggcca 13500

tgccggccgg ccctgcggaa acctctacgt gcccgtctgg aagctcgtag cggatcacct 13560

cgccagctcg tcggtcacgc ttcgacagac ggaaaacggc cacgtccatg atgctgcgac 13620

tatcgcgggt gcccacgtca tagagcatcg gaacgaaaaa atctggttgc tcgtcgccct 13680

tgggcggctt cctaatcgac ggcgcaccgg ctgccggcgg ttgccgggat tctttgcgga 13740

ttcgatcagc ggccgcttgc cacgattcac cggggcgtgc ttctgcctcg atgcgttgcc 13800

gctgggcggc ctgcgcggcc ttcaacttct ccaccaggtc atcacccagc gccgcgccga 13860

tttgtaccgg gccggatggt ttgcgaccgc tcacgccgat tcctcgggct tgggggttcc 13920

agtgccattg cagggccggc agacaaccca gccgcttacg cctggccaac cgcccgttcc 13980

tccacacatg gggcattcca cggcgtcggt gcctggttgt tcttgatttt ccatgccgcc 14040

tcctttagcc gctaaaattc atctactcat ttattcattt gctcatttac tctggtagct 14100

gcgcgatgta ttcagatagc agctcggtaa tggtcttgcc ttggcgtacc gcgtacatct 14160

tcagcttggt gtgatcctcc gccggcaact gaaagttgac ccgcttcatg gctggcgtgt 14220

ctgccaggct ggccaacgtt gcagccttgc tgctgcgtgc gctcggacgg ccggcactta 14280

gcgtgtttgt gcttttgctc attttctctt tacctcatta actcaaatga gttttgattt 14340

aatttcagcg gccagcgcct ggacctcgcg ggcagcgtcg ccctcgggtt ctgattcaag 14400

aacggttgtg ccggcggcgg cagtgcctgg gtagctcacg cgctgcgtga tacgggactc 14460

aagaatgggc agctcgtacc cggccagcgc ctcggcaacc tcaccgccga tgcgcgtgcc 14520

tttgatcgcc cgcgacacga caaaggccgc ttgtagcctt ccatccgtga cctcaatgcg 14580

ctgcttaacc agctccacca ggtcggcggt ggcccatatg tcgtaagggc ttggctgcac 14640

cggaatcagc acgaagtcgg ctgccttgat cgcggacaca gccaagtccg ccgcctgggg 14700

cgctccgtcg atcactacga agtcgcgccg gccgatggcc ttcacgtcgc ggtcaatcgt 14760

cgggcggtcg atgccgacaa cggttagcgg ttgatcttcc cgcacggccg cccaatcgcg 14820

ggcactgccc tggggatcgg aatcgactaa cagaacatcg gccccggcga gttgcagggc 14880

gcgggctaga tgggttgcga tggtcgtctt gcctgacccg cctttctggt taagtacagc 14940

gataaccttc atgcgttccc cttgcgtatt tgtttattta ctcatcgcat catatacgca 15000

gcgaccgcat gacgcaagct gttttactca aatacacatc acctttttag acggcggcgc 15060

tcggtttctt cagcggccaa gctggccggc caggccgcca gcttggcatc agacaaaccg 15120

gccaggattt catgcagccg cacggttgag acgtgcgcgg gcggctcgaa cacgtacccg 15180

gccgcgatca tctccgcctc gatctcttcg gtaatgaaaa acggttcgtc ctggccgtcc 15240

tggtgcggtt tcatgcttgt tcctcttggc gttcattctc ggcggccgcc agggcgtcgg 15300

cctcggtcaa tgcgtcctca cggaaggcac cgcgccgcct ggcctcggtg ggcgtcactt 15360

cctcgctgcg ctcaagtgcg cggtacaggg tcgagcgatg cacgccaagc agtgcagccg 15420

cctctttcac ggtgcggcct tcctggtcga tcagctcgcg ggcgtgcgcg atctgtgccg 15480

gggtgagggt agggcggggg ccaaacttca cgcctcgggc cttggcggcc tcgcgcccgc 15540

tccgggtgcg gtcgatgatt agggaacgct cgaactcggc aatgccggcg aacacggtca 15600

acaccatgcg gccggccggc gtggtggtgt cggcccacgg ctctgccagg ctacgcaggc 15660

ccgcgccggc ctcctggatg cgctcggcaa tgtccagtag gtcgcgggtg ctgcgggcca 15720

ggcggtctag cctggtcact gtcacaacgt cgccagggcg taggtggtca agcatcctgg 15780

ccagctccgg gcggtcgcgc ctggtgccgg tgatcttctc ggaaaacagc ttggtgcagc 15840

cggccgcgtg cagttcggcc cgttggttgg tcaagtcctg gtcgtcggtg ctgacgcggg 15900

catagcccag caggccagcg gcggcgctct tgttcatggc gtaatgtctc cggttctagt 15960

cgcaagtatt ctactttatg cgactaaaac acgcgacaag aaaacgccag gaaaagggca 16020

gggcggcagc ctgtcgcgta acttaggact tgtgcgacat gtcgttttca gaagacggct 16080

gcactgaacg tcagaagccg actgcactat agcagcggag gggttggatc aaagtacttt 16140

gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt 16200

ttcacgccct tttaaatatc cgttattcta a 16231

<210> 10

<211> 20

<212> DNA

<213> Artificial sequences

<400> 10

cggagccgac gacgtggaag 20

<210> 11

<211> 1761

<212> DNA

<213> Artificial sequences

<400> 11

atgccggacg gaatgctcgg caaggcggcg ctcgccgtcc ttctcgtgct cgccggcctt 60

gcggccggga ccaggccgcc cccgccgccg gtgtcggagg acacgctgga gaaggtcgcc 120

ggctcgctgg aaatgtacgt cgacaggctc ccccagatgc ccaaggtacg cggctactcc 180

gtggaacgcg gccgcgccac gccggtgcac ctcgccgtcg ggatgtacca aaagaaatgg 240

aaattccacc gcgacctgcc ggcgaccacc gtgttcgtgt tcggagagtc ggcggagagc 300

gccacgttcc ccgggccgac catcgaggcg ctgcaagggg tccctctgtc ggtgacgtgg 360

gagaaccacc tgccggagaa ccacatcctt ccgtgggacc ccaccgtgcc cgtcgccatc 420

cccaggcacg gcggcgtccc gaccgtcgtc cacctccacg gcggcgtcca cccgccgcag 480

tccgacgcca gcgccttcgc atggttcacc gccggcttcc gcgagaccgg cgccaagtgg 540

acgacgccga cgtacgtgta cccgaacgtg cagtcccccg gtgtcctctg gtaccacgac 600

cacgccctcg gcctcacgcg cgccaacctc ctcgccggcc tcctcggcgc ctacgtcatc 660

cgcaatccgg cggtggagtg gccgctcggc ctcccttgcg gcgacgagtt cgaccgcgtg 720

ctcgtgctcg ccgaccgcag cttctatgcc aacggctccc tctacatgaa ctccaccggc 780

gacaacccgc acgtccaccc tcagtggcag cctgagtact tcggcgacgt catcaccgtc 840

aacggcaagg cgtggccgtt ccttcccgtc gcccggcgcc gctaccgctt ccgcgtcatc 900

aacgccagca acgcgcgcta cttcaacctc tcgctgagca acggcctccc cttccacgtc 960

gtcggctccg acaccagcta cctggcccgg ccggtcgccg ccacccacgt cgtcgtcggc 1020

gtgtccgagt cgtttgacgt cgtcatcgac ttctccgacg acgagtccaa cacgcccgag 1080

gtggagctgg tgaacacggc gccgtacccg ttccccgacg gcaacgcgcc gggccgcctc 1140

tccagcaagg tgatgaagtt cgtcatcgaa ccagcgaaga caagggacca ctccagggtg 1200

ccggcgaggc tgctggagta ccacgcgaag gtcgccgagg aagaggcgac aagaaagcga 1260

tacatcgtga tgtacgagta cgacgacggc gcgacgggcg gcccgacgca cctgtacatc 1320

aacgggaagc ggctggagga cccggcgacg gagacgccgc gcgtgggggc gacggaggtg 1380

tgggaggtgg tcaacctcac gccggacgac cacccgctcc acctccatct ggccacgttc 1440

caggcggtgc gcgcccgggg gctggtcggg cttgacgagt tcaggcgctg catggccagg 1500

ctcaacgacg cggccaggtg caacgtgagc cagcacgccg ctggggacgc ggtgggcgtg 1560

ccggagcacg agaggacgtg gaagaacgtg gggaagatgg cgccggggca cgtgacgacg 1620

gcggtggtca agttcctgat ggtggacacc ggcgaggact acccgttcga cgccacggcc 1680

gagcctggct acgtctatca ctgtcacatt ttggatcatg aggacaatgc catgattcgc 1740

ccgctaaagc tcatcagata a 1761

<210> 12

<211> 1757

<212> DNA

<213> Artificial sequences

<400> 12

atgccggacg gaatgctcgg caaggcggcg ctcgccgtcc ttctcgtgct cgccggcctt 60

gcggccggga ccaggccgcc cccgccgccg gtgtcggagg acacgctgga gaaggtcgcc 120

ggctcgctgg aaatgtacgt cgacaggctc ccccagatgc ccaaggtacg cggctactcc 180

gtggaacgcg gccgcgccac gccggtgcac ctcgccgtcg ggatgtacca aaagaaatgg 240

aaattccacc gcgacctgcc ggcgaccacc gtgttcgtgt tcggagagtc ggcggagagc 300

gccacgttcc ccgggccgac catcgaggcg ctgcaagggg tccctctgtc ggtgacgtgg 360

gagaaccacc tgccggagaa ccacatcctt ccgtgggacc ccaccgtgcc cgtcgccatc 420

cccaggcacg gcggcgtccc gaccgtcgtc cacctccacg gcggcgtcca cccgccgcag 480

tccgacgcca gcgccttcgc atggttcacc gccggcttcc gcgagaccgg cgccaagtgg 540

acgacgccga cgtacgtgta cccgaacgtg cagtcccccg gtgtcctctg gtaccacgac 600

cacgccctcg gcctcacgcg cgccaacctc ctcgccggcc tcctcggcgc ctacgtcatc 660

cgcaatccgg cggtggagtg gccgctcggc ctcccttgcg gcgacgagtt cgaccgcgtg 720

ctcgtgctcg ccgaccgcag cttctatgcc aacggctccc tctacatgaa ctccaccggc 780

gacaacccgc acgtccaccc tcagtggcag cctgagtact tcggcgacgt catcaccgtc 840

aacggcaagg cgtggccgtt ccttcccgtc gcccggcgcc gctaccgctt ccgcgtcatc 900

aacgccagca acgcgcgcta cttcaacctc tcgctgagca acggcctccc cttgtcgtcg 960

gctccgacac cagctacctg gcccggccgg tcgccgccac ccacgtcgtc gtcggcgtgt 1020

ccgagtcgtt tgacgtcgtc atcgacttct ccgacgacga gtccaacacg cccgaggtgg 1080

agctggtgaa cacggcgccg tacccgttcc ccgacggcaa cgcgccgggc cgcctctcca 1140

gcaaggtgat gaagttcgtc atcgaaccag cgaagacaag ggaccactcc agggtgccgg 1200

cgaggctgct ggagtaccac gcgaaggtcg ccgaggaaga ggcgacaaga aagcgataca 1260

tcgtgatgta cgagtacgac gacggcgcga cgggcggccc gacgcacctg tacatcaacg 1320

ggaagcggct ggaggacccg gcgacggaga cgccgcgcgt gggggcgacg gaggtgtggg 1380

aggtggtcaa cctcacgccg gacgaccacc cgctccacct ccatctggcc acgttccagg 1440

cggtgcgcgc ccggggggctg gtcgggcttg acgagttcag gcgctgcatg gccaggctca 1500

acgacgcggc caggtgcaac gtgagccagc acgccgctgg ggacgcggtg ggcgtgccgg 1560

agcacgagag gacgtggaag aacgtgggga agatggcgcc ggggcacgtg acgacggcgg 1620

tggtcaagtt cctgatggtg gacaccggcg aggactaccc gttcgacgcc acggccgagc 1680

ctggctacgt ctatcactgt cacatttttgg atcatgagga caatgccatg attcgcccgc 1740

taaagctcat cagataa 1757

<210> 13

<211> 4107

<212> DNA

<213> Artificial sequences

<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 (7)

1. A pBUE411-LPR2 binary vector with the function of knocking out wheat LPR2 gene 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: the TaU3 promoter A from wheat, the sgRNA of the LPR2 gene and the terminator A; the sequence of the promoter A is shown as SEQ ID NO: 3 is shown in the specification; the DNA sequence of sgRNA of the LPR2 gene is shown in SEQ ID NO: 10 is shown in the figure;

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, wherein the sequence of the promoter B is shown in SEQ ID NO: 4 is shown in the specification; the maize Cas9 coding sequence is set forth in SEQ ID NO: 13 is shown in the figure; the terminator A sequence is shown as SEQ ID NO: 5 is shown in the specification; the terminator B sequence is shown as SEQ ID NO: and 6.

2. The pBUE411-LPR2 binary vector of claim 1, wherein said expression cassette a sequence is specifically set forth in SEQ ID NO: 7 is shown in the specification;

the sequence of the expression box B is specifically shown in SEQ ID NO: 8 is shown in the specification;

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 pBUE411-LPR2 binary vector of claim 2, further comprising a resistance marker gene; the nucleotide sequence of the pBUE411-LPR2 binary vector is shown as SEQ ID NO: shown at 9.

4. The method for constructing the binary vector pBUE411-LPR2 according to claim 1, comprising the steps of:

s11 designs sgrnas targeting LPR2 gene, and primers corresponding to oligonucleotides of the sgrnas are as set forth in SEQ ID NOs: 1, LPR2-gR1-TaU3-F shown in SEQ ID NO: 2 LPR2-gR1-TaU 3-R;

s12 phosphorylates and anneals 2 oligos of the synthesized sgRNA to form double chains;

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 recovered pBUE411 carrier fragment and double-stranded sgRNA with sticky ends are connected by T4 ligase to obtain the final pBUE411-LPR2 binary carrier.

5. The use of the pBUE411-LPR2 binary vector of any one of claims 1-4 to increase phosphorus utilization in wheat.

6. Use of the pBUE411-LPR2 binary vector of claims 1-4 in breeding transgenic wheat.

7. A method for improving phosphorus utilization rate of wheat by using gene editing is characterized by comprising the following steps: the function of the wheat LPR2 gene is reduced or lost, and the expression and/or activity of the protein coded by the wheat LPR2 gene is inhibited, so that the phosphorus utilization rate of the wheat is improved;

the method for reducing or losing the function of wheat LPR2 gene is to construct pBUE411-LPR2 binary vector as described in any one of claims 1-5.

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